Methods and compositions for using extracellular particles to detect pathology-related pathways

Abstract

Disclosed are methods of detecting or quantifying a pathology -related or pathology-protective-pathway in a subject comprising combining a first ligand with a sample obtained from the subject comprising an extracellular particle, wherein the first ligand binds to a cell-type specific marker on the extracellular particle; combining a second ligand with the sample, wherein the second ligand binds to a pathology-related or pathology-protective pathway molecule on the extracellular particle; combining a donor bead to the sample, wherein the donor bead binds to the first ligand or second ligand; combining an acceptor bead to the sample, wherein the acceptor bead binds to the first ligand or second ligand, wherein the donor bead and acceptor bead do not bind to the same ligand; triggering the donor bead to activate the acceptor bead to produce a signal; wherein the amount, presence, or absence of a signal indicates the amount, presence, or absence of a pathological condition in the subject.

Description

ATTORNEY DOCKET NO.37759.0498P1 METHODS AND COMPOSITIONS FOR USING EXTRACELLULAR PARTICLES TO DETECT PATHOLOGY-RELATED PATHWAYS STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support under BX004332-01A1 awarded by Veterans Affairs and R01AG0662 awarded by the National Institutes of Health The government has certain rights in the invention. BACKGROUND

[0002] A method is needed to detect biomarkers from specific cell types, for example, in the peripheral system for neurodegenerative disease. Conventional methods first use immunoprecipitation to purify cell specific extracellular particles, such as neuron or Astrocyte extracellular particles, then run ELISA, western-blot or use Flow cytometry to measure the target protein biomarkers. The limitations of these methods were obvious: including low and variable yields, highly variable platelet fragment impurities, difficulty to scale and do high- throughout, and the use of Biotinylated antibodies in purifying cell source specific extracellular particles, but followed by subsequent ELISA assays for the biomarkers that also use biotinylated antibodies to detect, which may cause some artificial results in detecting signal from residual biotinylated antibody for the cell source, not the analyte. BRIEF SUMMARY

[0003] Disclosed are methods of detecting or quantifying a pathology-related or pathology- protective-pathway in a subject comprising combining a first ligand with a sample obtained from the subject comprising an extracellular particle, wherein the first ligand binds to a cell-type specific marker on the extracellular particle; combining a second ligand with the sample, wherein the second ligand binds to a pathology-related or pathology-protective pathway molecule on the extracellular particle; combining a donor bead to the sample, wherein the donor bead binds to the first ligand or second ligand; combining an acceptor bead to the sample, wherein the acceptor bead binds to the first ligand or second ligand, wherein the donor bead and acceptor bead do not bind to the same ligand; triggering the donor bead to activate the acceptor bead to produce a signal; wherein the amount, presence, or absence of a signal indicates the amount, presence, or absence of an altered, normal, or pathological condition in the subject.

[0004] Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows, and in part will be understood from the description, orATTORNEY DOCKET NO.37759.0498P1 may be learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. BRIEF DESCRIPTION OF THE DRAWINGS

[0005] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed method and compositions and together with the description, serve to explain the principles of the disclosed method and compositions.

[0006] FIG.1 is a schematic of an example of the disclosed methods implemented with streptavidin donor beads binding a biotinylated antibody to “X” (a cell source specific marker) defining a target particle also coated with an antibody or other reagent specific for “Y”(an index for a pathological or protective pathway), that binds acceptor beads which emit light in proportion to pathway marker only when excited by singlet oxygen from the donor bead coupled to the same particle.

[0007] FIGs.2A-2C show total C3 (FIG.2A) and activated C3 (CD21ligand) (FIG.2B) elevation on plasma astrocyte-derived particles detected using biotinylated antibody to an astrocyte-specific marker (bio-GLAST) and further increase 3 months after traumatic brain injury (TBI), normalized to a ratio to total astrocyte EV signal (bioGLAST paired with CD81)(FIG.2C) from rT1 tau pathology accumulating human Tau transgenic mice. CD21 is an active recombinant fragment of complement receptor CR2 that recognizes convertase- cleaved active C3 (iC3b, C3d, etc) but not total C3 or C3a.

[0008] FIG.3 depicts potential age-dependent rise in Astrocyte-derived (bioGlast) signal for total Complement C3 in PS19 mutant p301S tau transgenic mice where elevated CNS complement C3 occurs with tauopathy and aging.

[0009] FIG.4 shows the loss of glutamate transporter EAAT2 on plasma astrocyte-derived EV in normotensive WKY / FAD positive mice, p=0.056, which becomes significant when normalized by astrocyte EV CD81 levels to correct for differences in astrocyte particle numbers and clearance. Loss of astrocyte EAAT2 is a known cause of excitatory synapse loss in FAD model mice. The antibiotic Ceftriazone and novel therapeutics aim to increase CNS EAAT2.

[0010] FIGs.5A and 5B show the loss of GluR1,2 excitatory synapse marker in L1CAM neuronal sourced plasma particles (FIG.5A) and similar loss of NCAM neuron sourced particles tagged with C1q (FIG.5B) in aged stroke prone spontaneously hypertensive (SHRSP) rats. C1qATTORNEY DOCKET NO.37759.0498P1 targets synapses for removal. Excitatory synapse loss is a major factor in cognitive disorders and there have not been methods to assess this.

[0011] FIG.6 shows the very high (r=0.925) correlation between levels for plasma particles from excitatory synapses labeled with biotinylated anti-NCAM1 and rabbit C1q detected by anti-rabbit coated acceptor beads on one 96-well plate (FIG.6) versus particles from the same rat plasma labeled with biotinylated antibody to GluR1 / GluR2 and 6-histidine tagged recombinant CD21 binding to nickel chelate coated acceptor beads on a second plate.

[0012] FIG.7 depicts the trend for a reduced ExPath signal from astrocyte-derived plasma particles positive for Glast combined with the perivascular astrocyte process marker AQP4 in amyloid bearing normotensive WKY rats positive for the FAD transgenes which cause amyloid deposits and retraction of perivascular astrocyte endfeet that release astrocyte EV into blood. In FIG.7, no normalizaztion was performed in order to show reduced signal reflecting particles bearing the astrocyte endfoot marker, AQP4, a major water channel that controls edema / swelling.

[0013] FIG.8 shows significantly elevated ExPath signal from endothelial (CD144+) EV labeled with inflammatory activation marker VCAM1 detected in plasma from chronically hypertensive SHRSP rats on WKY background compared with age-matched control background strain normotensive WKY rats. Chronically hypertensive rats have vascular inflammation and elevated VCAM1. Again, this experiment was not normalized. SHRP Rats have multiple spontaneous genetic differences from the normotensive parent strain WKY that cause chronic hypertension and endothelial activation / inflammation indexed by elevated VCAM1.

[0014] FIG.9 depicts ROC curve analysis for plasma Biotinylated CD144 and VCAM1 signal and shows the high sensitivity and specificity, for distinguishing plasma from hypertensive SHRSP vs WKY rats. Endothelial inflammation plays important roles in many cardiovascular, CNS, renal and pulmonary diseases.

[0015] FIG.10 shows VCAM1 is significantly elevated per endothelial plasma EV in SHRSP hypertensive rats expressing FAD transgenes and AD pathology (SHRSP / FAD+) compared with SHRSP without FAD transgenes. The BioCD144-VCAM1 / BioCD144 is signal normalized with BioCD144-CD81 and this ratio shows a significant, ~50% increase with AD pathology. The use of ratios to separate groups corrects for possible differences in particle numbers and also produces meaningful data without requiring standard curves and absolute (unitless) values to compare across assays. The increased vascular activation in SHRSP vs WKY is further increased in SHRSP FAD rats which have familial AD mutant transgenes that cause plaques and elevated vascular amyloid that causes vascular inflammation and interacts with FDA-approvedATTORNEY DOCKET NO.37759.0498P1 antibody treatments to cause serious adverse events, including reported deaths from bleeds.

[0016] FIGs.11A and 11B illustrates human dementia versus normal control subjects. FIG. 11A shows the brain amyloid PET imaging values (SUVR) are elevated in many of the dementia cases (Dem) consistent with AD and low in some but not all normal cognition (NC) controls so that with the n=24, the ROC AUC is 0.72 for discriminating dementia cases. FIG.11B shows an ExPath ratio for plasma astrocytic (bioGlast) EV signal for activated C3 (CD21) normalized by plasma astrocytic signal for total C3 in plasma samples from the same 24 patients produces a ROC AUC of 0.807 for distinguishing Dem from NC. This assay measures the astrocyte EV levels of convertase-cleaved C3 product normalized to its substrate C3 precursor, a measure of CNS complement pathway activation that is implicated in synapse loss.

[0017] FIG.12 shows normotensive (WKY background) and spontaneously hypertensive stroke prone rat models originally developed from WKY. Co-integrated familial Alzheimer disease (FAD) transgenes (APP / PS1) have been bred into WKY or SHRSP strains and in some experiments also bred in human ApoE3 or ApoE4 knockin genes replacing rat ApoE. ApoE4 is the most prevalent and important risk gene for late onset AD. The use of these rat models reduce the variation found in diverse human populations and FAD genes ensure the presence of AD pathology allowing significant differences to be seen with pathology confirmation and only small sample numbers. Plasma biomarkers can be developed with access to brain tissue taken at the time plasma was sampled. This permits confirmation of biomarker relevance to brain pathology and pathways that lack imaging biomarkers to confirm their status in human cohort or clinical trial studies.

[0018] FIGS.13A and 13B show assays to detect biomarkers. FIG.13A depicts conventional assays to purify plasma EV, followed by multi-step immuno-affinity purification of source- specific EV and sample extraction with repeated centrifugation steps and losses before extraction, centrifugation and conventional ELISAs using multiple wash steps and technician time. FIG 13B shows an example of the ExPath assays that require no purifications or wash steps avoiding variable losses and technician time so it can be automated and scaled for high throughput routine clinical use. The AlphaLISA platform is already automated, and its reagents are commercially available.

[0019] FIGS.14A-14C show MCI Clinical trial ExPath CNS active Complement C3 assays demonstrate that (800 mg / day) curcumin treatment reduced plasma levels in the group randomized to curcumin compared with placebo controls in 40 MCI patients after 6 months (FIG.14B) and levels remained lower combining post 9 or 12 months treatment when patients continued on curcumin (e.g. with or without added yoga in the last 6 months of the trial)ATTORNEY DOCKET NO.37759.0498P1 (FIG.14C). Consistent with reports using conventional assays (see FIG 13A), age-matched AD compared to cognitively normal control plasma showed higher levels of active C3 in astrocyte- derived (Glast labeled) EV (FIG.14A). Here assays used Glast-CD81 normalization.

[0020] FIGS.15A and 15B apply ExPath assays to heparan plasma from a different population of Long Covid Patients with NeuroCovid symptoms (NS) (compared to mainly respiratory symptom Long Covid with no NeuroCovid symptoms (Non NS) and show elevation of active C3 in both FIG.15A, Astrocyte (GLAST1+) and FIG.15B, endothelial (CD144+) EVs. This is consistent with CNS complement activation in Neurocovid patients and proteomic and other extensive evidence for endothelial complement activation in Long Covid.

[0021] FIG.16 illustrates microinfarcts in 15 mos. old SHRSPFAD rat. Representative micrographs show microinfarcts in cortical (left) and subcortical region of 18 months old SHRSP. While subcortical infarcts are more common in the subcortical area, FAD increases cortical microinfarcts (left) in SHRSP rats to be quantified along with small vessel disease (SVD, labeled by Col-IV). Asterisks indicate areas with enlarged perivascular space and the arrow shows a microinfarct in the white matter of external capsule. These examples document that the rat SHRSP models capture human vascular pathology present in many dementia cases with or without Alzheimer pathology that may impact treatment outcomes with drugs targeting only AD pathology (found alone in a minority of aging patients).

[0022] FIGS.17A-17E show a plasma assay for inflammatory and degenerative changes in endothelial (CD144)-derived EVs in response to hypertension, FAD, peripheral s.c. injection of Covid S1 Spike, or oral C5aR1 antagonists. Endothelia. Plasma assay for inflammatory and degenerative changes in endothelial (CD144) -derived EV. Plasma EC CD144 EV from 15-17 mo old FAD+ or FAD- rats reveal SHRSP increases VCAM1 vs WKY (FIG.17A) that were reduced by 5 day / wk C5aR1 antagonist (PMX205) for 8 wks in SHRSP-FAD at (10mg / Kg) (FIG.17B). One s.c injection of 250 ug / kg HEK recombinant glycosylated S1 spike (no LPS) or vehicle into sex and age-matched FAD rats (n=10) increased endothelial active C3 / total C3 normalized to total endothelial EV CD816wks post-S1 spike (FIG.17C). Similarly, peripheral S1 spike injection in human ApoE-FAD rats increased endothelial EV Col6a1, a non -fibrillar collagen matrix protein implicated in fibrotic matrix remodeling in injured vessel walls and tissues (FIG.17D). Assays for EV use ExPath and CD81 normalization are described in the methods. FIG.17E shows that S1 Spike protein increased endothelial Abeta detected with rabbit recombinant (H31L21) on endothelial EV, a potential marker for vascular abeta found in Cerebral amyloid angiopathy (CAA) that is elevated in ApoE4 carriers. Both ApoE4 and CAA are known to increase FDA-approved anti-Abeta antibody treatment risks for vascularATTORNEY DOCKET NO.37759.0498P1 inflammation and bleeds and COVID exposure also causes micro-angiopathy that is persistent in Long Covid.

[0023] FIGS.18A-18C show astrocyte EV C3 treatment and Spike Effects. Active C3 (iC3b, C3d) detected by CR2 = CD21 with 6HIS tag, anti-6HIS acceptor beads or rabbit anti- total C3 as in ExPath methods (FIG.18A) 15-17 mo old SHRSP-FAD plasma reveals elevation compared to other strains that was reduced by oral PMX205, a C5aR1 antagonist, intervention (Fig.18B).18A-!8B experiments used rat models without human ApoE. A third experiment using FAD+ rats with human ApoE (EFAD) shows that a single S1 spike (s.c.) injection increased 6 wks post-spike. This shows Glast-CD21 / C3 ExPath plasma assays detect elevated astrocyte-derived EV complement activation in the rat models that is reduced by complement C5aR1 receptor-directed treatment that is currently in clinical trials. Covid S1 spike increases this index of CNS active C3 in rat models carrying human ApoE and FAD transgenes (collectively EFAD) (FIG.18C).

[0024] FIGS.19A-19E show confirmation of S1 spike-increased active complement in SHRSPFAD models with rat instead of human ApoE. With S1 spike, SHRSP-FAD with more vascular pathology had higher endothelial EV active C3 (FIG.19A) while active C3 in astrocyte EV was still elevated by spike (FIG.19B). In normotensive rats, only FAD rats showed a spike effect on CSF levels of C5a by ELISA (FIG.19C), a measure already elevated in SHRSP FAD rats where Spike only trended to increase CSF C5a. A significant correlation between CSF C5a and active C3 was observed in plasma endothelial EVs (r(36)=0.543, p-0.002). Similarly, CSF C5a correlated with ExPath EV VCAM1 (r(34)=0.519, p=0.001). This is consistent with C3 activation upstream of C3 and CNS vascular inflammation. ptau217 (an Abeta-sensitive tau phosphorylation site) was also measured by ELISA in TBS cortical extracts; S1 Spike also increased ptau217 in Tg - normotensive rats and FAD further increased this. (FIG.19D). Less clear but significant Spike effects were observed in SHRSP vs WKY in frontal cortex (FIG. 19E).

[0025] FIGS.20A-20I show S1 spike s.c. injection increaseed pathology in E4FADSHRSP rat models. FIGS.20A-20D show spike effects on FAD coronal sections stained for Abeta 6E10 (20A), ptau217 (FIG.20B), activated complement C3 (C3d) (20C), C5 terminal attack complex (C5b-9) (FIG.20D). Although all groups (WKYE3FAD, WKYE4FAD, SHRE3FAD and SHRE4FAD) with or without S1spike injection were stained in blinded random order at the same time, quantification of whole section scans at 1x show all the spike groups had significantly increased Abeta, ptau217, C3d and C5b-9 (FIG20I). FIG.20A shows Abeta-ir in plaque and vasculature at 10X while FIG.20B shows tau-ir dystrophic neurites (asterisks)ATTORNEY DOCKET NO.37759.0498P1 around plaques and in neurons. C3d labeling for membrane bound persistent active C3 was primarily in vessel associated cells, including the microvasculature (FIG 20C) while antibody to C5b-9 membrane attack complex labeled mainly neurons and vessels cells (FIG 20D). Prominent perivascular microglia and perivascular macrophage (inset) and nodule or plaque labeling with Iba1 (FIG.20E) consistent with Long Covid neuropathology. Anti- Myeloperoxidase (MPO) a neutrophil marjer (FIG.20F, arrows) labeled neutrophils lining vessels (arrows) as in NeuroCovid and neurons (as in AD). Similarly, H&E showed increased leukocyte adhesion along vessels (yellow arrows) with spike (FIG.20G) vs no spike (FIG.20H). These figures validate animal model pathology resembling Long Covid and the relevance of the results with ExPath EV assays to report on CNS pathology.

[0026] FIG.21 is a schematic to appreciate the overall approach of the experiments of Example 6 (Covid spike experiments), except here only 45 days was used and it was not compared with planned live virus infection..

[0027] FIGS.22A-22D show the Attentional Set Shift Task (ASST) is a validated measure of executive function in rodents. FAD- (FIG.22 A, FIG.22B) and Spike 1-independent effects and interactions (FIG.22C, 22D) were observed. S1-treated FAD+ rats, but not FAD- rats, struggled to identify the correct digging medium when odorants were switched (FIG.22C, CD2), confirmed with new odorants and media (FIG.22D, ID1). FAD-treated rats were also impaired in reversal tasks, such as learning the correct medium or odor after a switch in the correct choice. These findings show that ASST detects spike effects interacting with FAD, consistent with cognitive deficits accelerated by Covid in patients with pre-existing AD pathology which is present in ~30% of patients over 65 years of age. FIG.22 illustrates FAD effects on executive function measured as reversal learning using the Attentional Set Shift Task (ASST). Fig 22 demonstrates executive deficits with reversal paradigms in our models, consistent with dementia patients

[0028] FIGS.23A-B shows the results of cases of patients diagnosed with mild cognitive impairment (MCI). ApoE3 / E3 MCI patients often do not have Alzheimer’s Disease (AD) and don’t decline while ApoE3 / E4 patients are more likely to have AD and decline to dementia. Active C3 (FIG 23A) and the C5b-9 membrane attack complex (FIG23B) on astrocyte-derived extracellular vesicles (ADEV) were increased in the MCI E4 carriers that are more likely to have AD and more likely to decline. This illustrates the potential predictive value of ExPath assays for CNS complement activation in human subjects and is consistent with complement-mediated synapse loss as a driver of circuit disruption and cognitive decline.

[0029] FIGS.24A-24C show data with and without curcumin treatment. FIG.24A showsATTORNEY DOCKET NO.37759.0498P1 baseline, MCI patients randomized to curcumin turned out to have higher levels of ADEV active C3 in plasma EV by the disclosed methods. FIG.24B shows results after 6 mos of curcumin trreatment (visit 5). The curcumin treated MCI patients had lower levels than the placebo controls. FIG.24C shows results at 9 and 12 months of patients continuing on curcumin with or without additional yoga had lower CNS ADEV active C3.

[0030] FIGS.25A-25C shows ExPath assays for plasma ADEV Complement C5b-9 in clinical trial. FIG.25A shows the group randomized to treatment were elevated at baseline relative to placebo assigned group. FIG.25B shows the curcumin treated group had significantly lower CNS ADEV C5b-9 membrane attack complex (MAC) after 9 and 12 mos treatment. Fig.25C shows treatment was very effective in lowering CNS complement activation in patients carrying ApoE4, the Major AD risk gene, and most likely to have AD pathology and risk of dementia.

[0031] FIGS.26A-B compare age and sex-matched health controls (HC) with Long Covid patients. Fig.26A shows a highly significant reduction in active C3 tagged ADEV normalized to CD81 in Long Covid (LC) patients compared to HC patients. Fig 26B shows similar reduction in ADEV active C3 normalized to total C3 and CD81 which was elevated in LC with Neuro- Covid compared to LC with no neuro symptoms. This result was unexpected and may reflect more active phagocytosis and elimination by activated microglia or macrophages expressing increased receptors for active C3 opsonized EV, consistent with LC human brain autopsy results for CD11b, CD11c and other complement receptors on Iba1 or CD68 labeled microglia in LC.

[0032] FIG.26 shows ExPath plasma assays distinguish healthy control vs human long covid plasma. Plasma is from n=10 sex and age-matched Healthy Controls (HC) vs n=40 patients diagnosed with Long Covid viral nucleocapsid positive sera and persistent symptoms for at least 3 months. ExPath assays for active C3 (CD21-detected) on astrocyte derived EV (BioGlast EV) normalized to total astrocyte EV ( BioGlast-CD81). Lower plasma levels of the BioGlastCD21 / BioGlastCD81 ratio in Long Covid patients could reflect more aggressive clearance of active C3 tagged EV from plasma (by activated innate immune cells bearing complement receptors or receptors like CR1 on RBC where C3b was shown elevated in Covid patients using flow cytometry.

[0033] FIGS.27A-27B depict ROC analysis showing plasma ExPath BioGlastCD21 / BioGlastCD81 (FIG.27A) discriminated Long Covid patients from healthy controls (with an AUC=0.94, p<0.0001) and outperforming the CD21 / C3 ratio (AUC=0.708,p=0.0445) (FIG. 27B).

[0034] FIGS.28A-B show elevated plasma endothelial EV active C3 / total C3 assayed as CD144-CD21 / CD144-total C3 discriminated Long Covid patients (n=20) with neurologicalATTORNEY DOCKET NO.37759.0498P1 symptoms / brain fog from Long Covid patients with no Neurological symptoms (non–NS).

[0035] FIGS.29A-29B show an ROC analysis showing the endothelial EV active C3 / total C3 ratio alone (FIG.29A) had AUC =0.75, p=0.0078 for NS (Neuro-covid) vs. non-NS plasma; normalization to CD81 reduced this (AUC=0.6947, p.04) (FIG.29B). Sequentially combining CD144-CD21 normalized to CD81 with AUC =0.94 identified Long Covid LC vs Healthy Control (HC) and then CD144-CD21 / total C3 was used to identify LC with neurological symptoms (discriminates Long NeuroCovid form LC with no neuro symptoms).

[0036] FIG.30 shows active C3 / total C3 on ADEV had uneven variance comparing Long Covid NS vs Non-NS samples, so square root was used to show a significant elevation in Long Covid with NS (normalizing to CD81).

[0037] FIG.31 shows an ROC analysis showing ADEV active C3 / total C3 in Long Covid only trended to distinguish Long Covid with NS (AUC=0.697, p=0.0699).

[0038] FIGS.32A and 32B show a brain aging pathway monitored in plasma: CNS complement activation that is implicated in normal brain aging is detected in older rat plasma bio-Glast+ astrocyte-derived EV (ADEV). FIG.32A shows active C3 detected using CD21 (CR2) is significantly increased in older 18-21 month (ms) old normal aging WKY strain of rats. FIG.32B shows a signal for CD81, an exosome-enriched marker present on Astrocyte EV was also elevated in the same samples from aging WKY rats.

[0039] FIGS.33A-33B show brain aging monitored in plasma normalized: increased active C3 / total C3 ratio on ADEV from WKY rats without AD or hypertensive pathology (FIG.33A) as well as the same ratio normalized to total ADEV CD81 (FIG.33B). This is consistent with reports showing neuroinflammation and CNS complement activation are elevated by normal aging in the absence of AD pathology in both mice and humans at the level of C1q-C3. Very few plasma biomarkers report on normal brain aging or the effects of prevention programs.

[0040] FIGS.34A-34C depict additional ExPath plasma assays showing GluR2 / 3+ excitatory synapse derived EV loss in hypertensive SHRSP rats with AD pathology (SHRSPFAD+) with different assay configurations than in FIG.5. BioGluR2 / 3 EV tagged with active C3 detected by CD21 (FIG.34A) and BioGluR2 / 3 tagged with neuronal marker NCAM1 normalized by BioGlur2 / 3 tagged with alternative neuronal marker L1CAM (FIG 34B) were all reduced. Fig.34C shows the same result with BioGluR2 / 3- CD21 normalized another way. The results all show deficits consistent with loss of CNS excitatory neuronal synaptic sources in models with FAD transgenes and humans with MCI or AD consistent with our results in FIG.5.

[0041] FIG.35 shows that the significant increase in endothelial VCAM1 in SHRSP shown in Fig 8 is still maintained after normalization to total endothelial EV assessed by CD144-CD81ATTORNEY DOCKET NO.37759.0498P1 ExPath.

[0042] FIG.36 shows that treating SHRSP-FAD+ with PMX205, an antagonist to C5aR1 (the major receptor for complement C5a), reduces the elevated endothelial activation marker VCAM1. This shows the treatment effects shown pooled FAD+ and FADneg rats in Fig 17 are also significant in the FAD+ group alone where FAD+ elevates endothelial VCAM1.

[0043] FIGS.37A-B combine a measure of C3 activation in SHRSP with AD pathology (FAD+) normalized to a measure of total plasma ADEV ( GLAST-CD81) showing a elevation with FAD (FIG.37A) and a response to treatment (FIG.37B). GlastCD21toC3 / BioGlastCD81 is elevated by FAD+ and reduced by PMX205. This illustrates surrogate biomarker potential for CNS inflammation within the animal studies complementing evidence of surrogate biomarker potential in MCI patients treated with curcumin. DETAILED DESCRIPTION

[0044] The disclosed method and compositions may be understood more readily by reference to the following detailed description of particular embodiments and the Example included therein and to the Figures and their previous and following description.

[0045] It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0046] Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a peptide or protein is disclosed and discussed and a number of modifications that can be made to a number of molecules including the amino acids are discussed, each and every combination and permutation of the peptide and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of theATTORNEY DOCKET NO.37759.0498P1 combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed. A. Definitions

[0047] It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only; it is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0048] It must be noted that as used herein and in the appended claims, the singular forms "a ", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a ligand" includes a plurality of such ligands, reference to "the ligand” is a reference to one or more ligands and equivalents thereof known to those skilled in the art, and so forth.

[0049] The word “or” as used herein means any one member of a particular list and also includes any combination of members of that list.

[0050] As used herein, “sample” is meant to mean a specimen from a subject; a tissue or organ from an animal; a cell (either within a subject, taken directly from a subject, or a cell maintained in culture or from a cultured cell line); a cell lysate (or lysate fraction) or cell extract; or a solution containing one or more molecules derived from a cell or cellular material, which is assayed as described herein. A sample may also be any body fluid or excretion (for example, but not limited to, blood, urine, cerebrospinal fluid, stool, saliva, tears, bile) that contains cells or cell components.

[0051] As used herein, “subject” refers to the object from which a sample can be obtained, e.g. an animal. Thus, the subject of the disclosed methods can be a vertebrate, such as a mammal. For example, the subject can be a human. The term does not denote a particular age or sex.ATTORNEY DOCKET NO.37759.0498P1 Subject can be used interchangeably with “individual” or “patient”.

[0052] Expath, as used throughout, stands for extracellular vesicle pathway which can include exosomes and pathology pathways. One reagent for exosomes detects the extracellular vesicle cell source and one or more additional reagents detect markers for pathways (Path), typically reporting on pathological or normal functional and protective pathways and / or a marker for normalization to take account of particle numbers with the cell source or exosome marker or precursor / product measures of pathway activity.

[0053] Ranges may be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and / or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. Finally, it should be understood that all of the individual values and sub-ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. The foregoing applies regardless of whether in particular cases some or all of these embodiments are explicitly disclosed.

[0054] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described. Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of publications are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0055] Throughout the description and claims of this specification, the word “comprise” andATTORNEY DOCKET NO.37759.0498P1 variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. In particular, in methods stated as comprising one or more steps or operations it is specifically contemplated that each step comprises what is listed (unless that step includes a limiting term such as “consisting of”), meaning that each step is not intended to exclude, for example, other additives, components, integers or steps that are not listed in the step. B. Methods

[0056] Disclosed are methods of detecting or quantifying a pathology-related or pathology- protective-pathway in a subject comprising combining a first ligand with a sample obtained from the subject comprising an extracellular particle, wherein the first ligand binds to a cell-type specific marker on the extracellular particle; combining a second ligand with the sample, wherein the second ligand binds to a pathology-related or pathology-protective pathway molecule on the extracellular particle; combining a donor bead to the sample, wherein the donor bead binds to the first ligand or second ligand; combining an acceptor bead to the sample, wherein the acceptor bead binds to the first ligand or second ligand, wherein the donor bead and acceptor bead do not bind to the same ligand; triggering the donor bead to activate the acceptor bead to produce a signal; wherein the amount, presence, or absence of a signal indicates the amount, presence, or absence of a pathological condition in the subject.

[0057] In some aspects, methods of detecting or quantifying a pathology-related or pathology- protective pathway can also refer to detecting or quantifying a pathology-related or pathology- protective conditions. In some aspects, the presence of a pathology-related or pathology- protective pathway can be indicative of the presence of a pathology-related or pathology- protective condition.

[0058] In some aspects, a sample obtained from the subject can be cerebrospinal fluid, synovial fluid, blood, plasma, serum, saliva, urine, ocular fluid (e.g. s tears), aqueous or vitreous humor or tissue extracts. In some aspects, samples can also be obtained from cell culture media.

[0059] In some aspects, the extracellular particles can include, but is not limited to an ectosome, exosome, microparticle, bleb, cell debris, or apoptotic body derived from any cell type. In some aspects, extracellular particles can include vesicles ranging from 10 to 1,000 nm. In some aspects, extracellular particles can exclude individual molecules and macromolecules. In some aspects, extracellular particles can be purified from a sample by removing intact cells from the sample, typically by centrifugation, filtration, and / or sedimentation.

[0060] In some aspects, the pathology-related and pathology-protective conditions can be, but are not limited to, pathologies of any aging or disease-related pathway. In some aspects,ATTORNEY DOCKET NO.37759.0498P1 examples include, but are not limited to, aging-related deficits in lysosomal and autophagic clearance mechanisms indexed by lysosomal associated membrane proteins (Lamps) and autophagy markers (LC3b including LC3bII, p62 and beclin-1). In some aspects, autophagic clearance of damaged proteins and organelles can be increased by caloric restriction, exercise or drug regimens that benefit aging but excessive autophagy can cause tissue damage so the disclosed assays to monitor cell source autophagy markers are needed. Similarly, it is protective to increase tissue-specific oxidative damage protective enzymes including Trx1 and GPX4 which can be induced by FDA-approved drugs (for example, dimethyl formamide, DMF for multiple sclerosis) or supplements (curcumin, pterostilbene, resveratrol) that induce Nrf2, but there is currently no method to monitor levels in specific organs like brain or heart and ExPath assays for Nrf2-induced protective antioxidant enzymes can meet this need. Another example is senescent cell or SASP markers that index harmful senescent cell numbers including but not limited to DPP4, PLD3, ICAM-1 and beta 2 microglobulin. In some aspects, senescent cells are targets of new senolytic therapies that kill senescent cells now in clinical trials for age-related deficits and chronic diseases of aging and ExPath assays for cell-source specific senescent markers could be used to monitor tissue-specific target engagement. 1. First ligand and cell-type specific marker

[0061] In some aspects, a first ligand and cell-type specific marker combination can provide indices of cell-type specific protective responses.

[0062] In some aspects, the first ligand can be any molecule that can bind to a cell-type specific marker. In some aspects, the first ligand can be, but is not limited to, a protein, nucleic acid, compound, or fragment thereof. For example, the first ligand can be an antibody, antibody fragment, aptamer, receptor or active receptor fragment.

[0063] The disclosed methods can be used on any pathology-related or pathology protective pathways including, but not limited to, neurological-related (e.g. brain, nerve) pathways, cardiovascular (e.g., heart, vessels), and cancer-related pathways. In some aspects, pathology related conditions include, but are not limited to, chronic endothelial inflammation in atherosclerotic plaques that can lead to heart attacks and strokes, chronic inflammation in the gastrointestinal tract that ultimately leads to ulcers, colitis or inflammatory bowel disease, chronic inflammation associated with adipose macrophages and obesity that leads to metabolic syndrome and type II diabetes and other organ specific chronic inflammatory conditions including but not limited to myocarditis, pancreatitis and glomerulonephritis). In some aspects, examples of pathology protective pathways include, but are not limited to, increases in complement inhibitory molecules (including CD59, DAF, CR1, C4bp, MCP, MAP-1, C1, FactorATTORNEY DOCKET NO.37759.0498P1 I, NPTX2, vitronectin, clusterin and SRPX2), glucose transporters (Glut1-4), trophic factor receptors (TrkA, IGFR1), incretin receptors, phagocytic receptors (CD11c, scavenger receptors), anti-inflammatory receptors (for TGFbeta, IL-10, IL-4) and heat shock proteins. In some aspects, pathology protective pathways can increase, fail to increase, or be reduced in response to pathological challenge.

[0064] In some aspects, the presence of a pathology-related pathway can be indicative of the presence of a pathology-related condition. For example, the presence of a cancer related pathway can be indicative of the presence of cancer in a subject. Thus, the disclosed methods are also methods of detecting the presence of a pathology-related condition.

[0065] In some aspects, the cell-type specific marker is heavily enriched.

[0066] The disclosed methods can use cell-type specific markers from any cell type. In some aspects, a cell-type specific marker can be any marker expressed only in a specific cell type or expressed in much higher quantities in one cell type compared to another. In some aspects, cell- type specific markers can be, but are not limited to, astrocyte specific, neuron specific, oligodendrocyte specific, microglia specific, or vascular endothelial cell specific markers.

[0067] In some aspects, when assessing brain or neurological conditions, brain-derived cells can be used in the disclosed methods. Encased in a skull and sensitive to injury, the brain can be difficult to biopsy and contains multiple types of cells (neurons, astrocytes, oligodendrocytes, microglia, vascular endothelial cells and their associated pericytes). The cell types include specialized types of neurons using different neurotransmitters (glutamate, GABA, acetylcholine, dopamine, serotonin, etc). In some aspects, each of these cells can release extracellular particles (exosomes, ectosomes or “blebs” of membrane) into extracellular fluids that can reach bodily fluids, such as, cerebrospinal fluid, blood or urine, and reflect on the status of health, aging or disease when used in the disclosed methods.

[0068] In some aspects, combining a first ligand with a sample obtained from the subject comprising an extracellular particle allows the first ligand to bind to the cell-type specific marker on the extracellular particle. In some aspects, combining can be incubating or mixing everything together for any length of time. In some aspects, the combining can be for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some aspects, the presence of a cell-type specific marker in high abundance on the extracellular particle can result in incubation (or mixing) times that are lower than those cell-type specific markers in low abundance on the extracellular particle. For example, cell-type specific markers in high abundance can be combined with a first ligand for 1, 2, 3, 4, 5, or 6 hours while cell-type specific markers in low abundance can be combined with a first ligand for 7, 8, 9, 10, 11, 12, 13,ATTORNEY DOCKET NO.37759.0498P1 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. i. Example Brain-derived Cell Types

[0069] In some aspects, the disclosed methods can be used to detect or quantify a pathology- related or pathology-protective pathway in a subject using extracellular particles derived from many different cell types. For example, extracellular particles derived from brain cells can be used in determining brain-related or neurological pathways and conditions. a. Astrocytes

[0070] Astrocyte specific markers, which can be targets for the first ligand, and can be enriched on and / or specific to astrocytes (and thus present on astrocyte derived extracellular particles), include, but are not limited to, EAAT1 / GLAST, EAAT2 / GLT-1, Kir4.1, AQP4 and GFAP. b. Oligodendrocytes

[0071] Oligodendrocyte specific markers, which can be targets for the first ligand, and can be enriched on and / or specific to oligodendrocytes (and thus present on oligodendrocyte derived extracellular particles), can be, but are not limited to, MOG, NG2, PDGFRA, MAG, PLP1 and MBP. c. Microglia

[0072] Microglia specific markers, which can be targets for the first ligand, and can be enriched on and / or specific to microglia (and thus present on microglia derived extracellular particles), can be, but are not limited to, TMEM119, P2RY12 or Siglec H. In some aspects, microglia enriched, but not microglia specific markers can include, but are not limited to, TSPO, CD11b, CD11c, C3aR1,TREM2 and TyroBP which can also be used as activation state- dependent pathway markers. d. Neurons

[0073] Neuron specific markers, which can be targets for the first ligand, and can be enriched on and / or specific to neurons (and thus present on neuron derived extracellular particles), can be, but are not limited to, L1CAM and NCAM1, synaptic markers (SNAP25, neurligin 1, neurexin 2, synaptotagmins 1 and 4) excitatory (glutamatergic) synapse selective (AMPA rceceptors, (GluR1-4 and GluR2,3) NPTXR, NPTX1, VGLUT1, VGLUT2), inhibitory (Gabaergic) neuron markers (SLC32a1,GAT1,GAT3, GABAAR, GABABR1, GABABR2), dopaminergic neuronal markers (SLC6A3 / DAT, GIRK2, SLC6A2 / Net1), GM1 gangliosides upregulated by beta amyloid and cholinergic markers ( GT1aalpha and GQ1balpha gangliosides, VaChT, AChE). 2. Second ligand and pathology-related or pathology-protective pathway molecules

[0074] In some aspects, the second ligand can be any molecule that can bind to a pathology-ATTORNEY DOCKET NO.37759.0498P1 related or pathology-protective pathway molecule. In some aspects, the second ligand can be, but is not limited to, a protein, nucleic acid, compound, or fragment thereof. For example, the second ligand can be an antibody, antibody fragment, aptamer, lectin, receptor or receptor fragment. For example, in some aspects, a second ligand can be a recombinant CD21 with high affinity binding to C3 activation products. Thus, in some aspects, the second ligand can be a pathology-related or pathology-protective pathway molecule-specific antibody.

[0075] In some aspects, the pathology-related or pathology-protective pathway molecule- specific antibody is an antibody that specifically binds VCAM1, ICAM1, Pecam1, complement activation marker-specific (C1q, Bb, Masp1 / 2 and / or C3 and its activation products (C3b, ic3b, C3d), C4 (C4A, C4B, C4b, C4d)), CD59, cancer stem cell (CD133, CD44), cancer invasive (MMP-14),CEA, checkpoint specific (PD-L1,GPNMB, CTLA-4, CD20 and CD52), metabolic (Glut1), EAAT2.LAT-1, MDR1, proteinopathy-specific (Abeta, p-tau (181, 231), tau oligomer, PrPsc, p-129SaSyn, aSyn aggregate), PrPc. Thus, a pathology-related or pathology-protective pathway molecule can be, but is not limited to, VCAM1, ICAM1, Pecam1, complement activation marker-specific (C1q, Bb, Masp1 / 2 and / or C3 and its activation products (C3b, ic3b, C3d), C4 (C4A, C4B, C4b, C4d)), CD59, cancer stem cell (CD133, CD44), cancer invasive (MMP-14),CEA, checkpoint specific (PD-L1,GPNMB, CTLA-4, CD20 and CD52), metabolic (Glut1), EAAT2.LAT-1, MDR1, proteinopathy specific (Abeta, p-tau (181, 231), tau oligomer, PrPsc, p-129SaSyn, aSyn aggregate), or PrPc.

[0076] In some aspects, pathology-related or pathology-protective pathway molecules are also cell type specific. For example, certain cell types may express specific molecules at higher or lower levels depending on the state of the disease / pathology. Some examples of different cell types and their pathology-related or pathology-protective pathway molecules are listed below.

[0077] In some aspects, combining a second ligand with a sample allows the second ligand to bind to the pathology-related or pathology-protective pathway molecules on the extracellular particles in the sample. In some aspects, combining can be incubating or mixing everything together for any length of time. In some aspects, combining can be incubating or mixing everything together for any length of time. In some aspects, the combining can be for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some aspects, the presence of a pathology-related or pathology-protective pathway molecule in high abundance on the extracellular particle can result in incubation (or mixing) times that are lower than those pathology-related or pathology-protective pathway molecules in low abundance on the extracellular particle. For example, the pathology-related or pathology-protective pathway molecules in high abundance can be combined with a second ligand for 1, 2, 3, 4, 5, or 6 hoursATTORNEY DOCKET NO.37759.0498P1 while pathology-related or pathology-protective pathway molecules in low abundance can be combined with a second ligand for 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours.

[0078] Methods that use any of these cell type specific extracellular particles alone or combined with a second cell type specific extracellular particle assay, (such as combining astrocyte markers and microglial markers) can permit characterization of functional status. i. Astrocytes

[0079] In some aspects, targets (e.g. pathology-related or pathology-protective pathway molecules) on astrocytes for a second ligand to bind which reflect disease altered processes include, but are not limited to, activation markers such as HepaCam, GFAP, CD44, Cav1, Cap1, decorin, HLA-A, HLA-B, ITGB1 and ITGB6, all surface markers which can be elevated by neuroinflammation in astrocytes. These pathology-related or pathology-protective pathway molecules can be increased in Alzheimer Disease (AD), Parkinson’s Disease, stroke, and other less common CNS diseases such as chronic traumatic encephalopathy, and frontal temporal dementia.

[0080] In some aspects, other pathology-related or pathology-protective pathway molecules in astrocytes can be, but are not limited to, VCAM1, CAV1, GABA reuptake transporters (GAT1, GAT3), Cd49f / IGA6, a potassium reuptake channel (Kir4.1), metabolic markers (GLUT1, MCT4) and lipoprotein receptors (e.g. LRP1 and Reln). In some aspects, each of these molecules can be increased or reduced in disease states.

[0081] In some aspects, other pathology-related or pathology-protective pathway molecules on astrocytes, specifically for complement activation pathways, can be, but are not limited to, Bb, Masp1 / 2 and / or C3 and its activation products (C3b, ic3b, C3d), C4 (C4A, C4B, C4b, C4d). In some aspects, these molecules are increased in Alzheimer’s Disease and other tauopathies as well as multiple sclerosis, traumatic brain injury, schizophrenia and stroke. ii. Oligodendrocytes

[0082] In some aspects, targets (e.g. pathology-related or pathology-protective pathway molecules) on oligodendrocytes for a second ligand to bind, which reflect disease altered processes include, but are not limited to, MAG and PLP1 (their ratio can be reduced in vascular dementia).

[0083] In some aspects, other pathology-related or pathology-protective pathway molecules on oligodendrocytes, specifically for complement activation pathways, can be, but are not limited to, Bb, Masp1 / 2 and / or C3 and its activation products (C3b, ic3b, C3d), C4 (C4A, C4B, C4b, C4d)), SLC44A1, VCAN, ANXA1, CADM4 and NCAM2. In some aspects, C3, C4A, C4G,ATTORNEY DOCKET NO.37759.0498P1 C5-C9 or their activation fragments can be elevated in Alzheimer’s, Multiple Sclerosis, vascular dementia, and other neurodegenerative conditions with white matter damage (WMD) and myelin loss that can be detected by MRI.

[0084] In some aspects, one or more of the pathology-related or pathology-protective pathway molecules can be used to assess vascular dementia, AD, MS, and several other conditions with white matter loss. In some aspects, alphaSynuclein can be used to detect multi-system atrophy (MSA). iii. Microglia

[0085] In some aspects, targets (e.g. pathology-related or pathology-protective pathway molecules) on microglia for a second ligand to bind, which reflect disease altered processes include, but are not limited to, CXCR1, CD11b, TREM2 and its partner DAP12 / TyroBP, ITM2B, Thy1, C4b and CD59. In some aspects, these molecules are elevated in AD. In some aspects, CD11b and C4b are more generally upregulated with microglial activation, neuroinflammation and neurotoxic activity in many CNS diseases while the two homeostatic microglial (HM) specific surface markers, TMEM119 and P2RY12, are generally reduced in AD and can be reduced or unchanged in common CNS diseases. In some aspects, for AD, CD11c, TREM2 and TyroBP are upregulated on phagocytic disease-associated microglia (DAMs) involved in the response to amyloid plaques and amyloid clearing anti-abeta immunotherapy.

[0086] In some aspects, the disclosed methods of combining one or more HM microglial markers with antibodies to TMEM119 or P2RY12 with a pathology-related or pathology- protective pathway molecules and then normalized to a signal representing a total microglial particle population. In some aspects, options for the total population include combining a TMEM119 / P2RY12 antibody pair or antibody to either microglial marker combined with a less microglial-specific extracellular innate immune extracellular particle ligand (for example, anti- CD11b or ligand Isolectin IB4) or alternatively combined with a more general extracellular particle surface marker (for example antibody to flotillin-1 or anti-annexin or the HSP ligand Vp96). In some aspects, normalization can provide a signal common to the majority of microglial sourced particles including microglial exosomes and larger extracellular particles and corrects for variation in peripheral clearance rates. In some aspects, in the more specialized use of TMEM119 / P2RY12, because TMEM119 and P2RY12 are homeostatic markers downregulated in more activated microglia normalizing to the pair provides a ratio indexing microglial polarization and functional status. In some aspects, assessing functional status can be critical because microglia play both neurotoxic and neuroprotective roles in disease, acting as a so-called “double edged sword” with negative and positive roles.ATTORNEY DOCKET NO.37759.0498P1 iv. Neurons and neuronal compartments

[0087] In some aspects, targets (e.g. pathology-related or pathology-protective pathway molecules) on neurons or their compartments (e.g. synapses, axons and dendrites) for a second ligand to bind, which reflect disease altered processes can include, but is not limited to, complement activation markers (C1q, Bb, Masp1 / 2 and / or C3 and its activation products (C3b, ic3b, C3d), C4 (C4A, C4B, C4b, C4d)) that detect complement mediated clearance and the complement inhibitors CD59 (on Gabanergic synaptic sourced EV) and NPTX2 (on glutamatergic sourced EV). In some aspects, targets and sources can include excitatory synapse restricted markers (notably GluR2, GluR4, neuroligin-1 and neurexin 2) which are altered by excitotoxic and more disease-specific synaptotoxic mechanisms and often preferentially lost reflecting synapse loss in many CNS conditions and diseases with cognitive dysfunction. In some aspects, pathology-related target molecules can be more specific for a disease, for example for AD, where they include amyloid beta protein (total abeta, abeta 42, abeta 40, pyroglutamate modified abeta and phosphotau 181, 217, and 231). In some aspects, PrPc and synaptogyrin-3, both receptors for pathological abeta and ptau, which can be elevated in L1CAM positive extracellular vesicles in blood samples after brain injury, can be pathology-related or pathology- protective pathway molecules.

[0088] In some aspects, combining a neuronal cell source specific marker (e.g. NCAM1, L1CAM, EAAT1 / GLAST, EAAT2 / GLT-1, Kir4.1, AQP4 and GFAP) with pathology marker antibodies to total alpha synuclein (or more pathology-specific phospho-ser129 or aggregated alpha-synuclein) can detect alpha synuclein pathology and signals can be normalized for example for neuron-derived particles (NCAM1 / L1CAM) together or either alone or normalized by combining cell source reagent with a more general extracellular particle marker including but not limited to flotillin-1, ligands for HSPs. In some aspects, this ratio provides a ratio of total or pathological alpha-Synuclein per neuron-derived extracellular particle, a measurement which is elevated in Parkinson’s and other neuronal syncleinopathies. In some aspects, total alpha- synuclein in plasma is not elevated in Parkinson’s presumably as it includes a-synuclein found in other non-neuron sourced pools including RBC-associated fractions. 3. Donor Bead

[0089] The disclosed methods include combining a donor bead to the sample, wherein the donor bead binds to the first ligand or second ligand. In some aspects, the donor bead is a light- stimulated donor bead. In some aspects, a donor bead is a bead that, upon triggering or activating, provides a stimulus to the acceptor bead. In some aspects, the stimulus is a proximity limited chemical stimulus. In some aspects, the acceptor bead is linked to extracellular particlesATTORNEY DOCKET NO.37759.0498P1 via a second ligand to emit light at levels reflecting the second ligand levels. In some aspects, the donor bead can bind to the first ligand. In some aspects, the donor bead can bind to the second ligand. As long as the donor bead binds to one ligand and the acceptor bead binds to the other ligand it does not matter which one binds to the first ligand and which one binds to the second ligand.

[0090] In some aspects, the donor bead comprises a binding partner for the first ligand, wherein the binding partner binds to the first ligand. For example, in some aspects, the first ligand comprises a label. In some aspects, the label can be biotin. In some aspects, the binding partner for the first ligand is streptavidin, wherein the streptavidin binds to the biotin on the first ligand. In some aspects, the binding partner can be any molecule, protein, or nucleic acid that can bind to the first ligand or a label on the first ligand. In some aspects, a label on the first ligand can be any molecule, protein or nucleic acid that can be bound by the binding partner on the donor bead. As mentioned previously, the donor bead can bind the first ligand or the second ligand. Therefore, if the donor bead is binding the second ligand then as described above for the first ligand, the second ligand could comprise a label and the donor bead can comprise a binding partner for the second ligand (e.g. the label). In some aspects, beyond antibody-epitope interactions, receptor-ligand interactions can be assessed with examples including recombinant CD21 or its active fragments binding to activated complement C3, biotinylated cholera toxin binding to GM1 ganglioside (elevated in Alzheimer’s Disease) and PET TSPO probe PK11195 for activated microglia

[0091] In some aspects, donor beads can be polymeric matrix (typically polystyrene or polyacrylamide) nanoparticles doped with photoactivated molecules and covalently derivatized for coupling (for example, carboxyl or amino groups or otherwise coated with a derivatized material suitable for coupling chemistry). In some aspects, donor beads can be from the commercial Perkin Elmer AlphaLisa platform with proprietary 250-350 nm polystyrene hydrogel coated AlphaLisa donor beads containing phthalocyanine as a photosensitizer activated to generate singlet oxygen excited from oxygen in solution after excitation with 680 nm light. In some aspects, AlphaLisa donor beads are inactivated by ambient laboratory light requiring room darkening and potential cross-site variation. In some aspects, before excitation decays, singlet oxygen can travel in an aqueous solution for a distance up to about 200 nm to meet and excite a neighboring or “proximal” acceptor bead to emit the signal used for measurements. In some aspects, alternative photosensitizers include naphthalocyanine which excites at 775 nm to generate singlet oxygen with robust quantum yields; naptholocyanine is more photostable with less site-dependent decay from cross-site ambient laboratory light. Other photosensitizerATTORNEY DOCKET NO.37759.0498P1 alternatives include, but are not limited to, porphyrins and ruthenium II polypyridyl complexes (excitation 400-500 nm). 4. Acceptor Bead

[0092] The disclosed methods include combining an acceptor bead to the sample, wherein the acceptor bead binds to the first ligand or second ligand, wherein the donor bead and acceptor bead do not bind to the same ligand. Thus, the acceptor bead is present on whichever of the first ligand or second ligand that does not comprise the donor bead. In some aspects, the acceptor bead can bind to the first ligand. In some aspects, the acceptor bead can bind to the second ligand. As long as the donor bead binds to one ligand and the acceptor bead binds to the other ligand it does not matter which one binds to the first ligand and which one binds to the second ligand.

[0093] In some aspects, the acceptor bead directly or indirectly binds to the first ligand or second ligand. In some aspects, an example of direct binding can be when the acceptor bead is conjugated directly to an antibody that specifically binds the pathology-related or pathology- protective pathway molecule. In some aspects, an example of indirect binding can be when the acceptor bead is conjugated to an anti-IgG antibody or fragment thereof. Thus, in some aspects, the anti-IgG antibody or fragment thereof, which is conjugated to the acceptor bead, can bind to a pathology-related or pathology-protective pathway molecule-specific antibody. In some aspects, either the acceptor or donor bead (but not both) can be bound to the second ligand via a biotinylated ligand binding molecule to the streptavidin coated beads.

[0094] In some aspects, the acceptor bead is activated by the donor bead to produce or emit a signal. In some aspects, the acceptor bead is a polymeric matrix nanoparticle, typically polystyrene, that contains a fluorophore activated by the donor bead to produce or emit a signal. In some aspects, for the acceptor bead to be activated by the donor bead, the beads must be co- localized on an extracellular vesicle or particle of 10 to 1000 nm diameter. In some aspects, when using singlet oxygen emitting donor beads, when an acceptor bead is within that proximity, energy is transferred from the donor singlet oxygen to acceptor bead fluorophores, for example, a thioxene +anthracene cascade to rubrene (AlphaScreen) or a redshifted europian chelate emitting at 615 nm in AlphaLISA beads that provide the final signal emission flurophore. In some aspects, acceptor beads can use thioxene and anthracene derivatives within the acceptor bead and emit at 520-620 nm (some beads use rubrene). In some aspects, Alphaplex acceptor beads doped with final emitter chelates of terbium (545nm emission) or Samarium (645 nm emission) can be used and provide for multiplex simultaneous double labeling pairing with two different ligand directed acceptor beads with a donor bead directed by a third ligand. Thus,ATTORNEY DOCKET NO.37759.0498P1 in some aspects, three different ligands can report from one extracellular vesicle. For example, a donor and dual acceptor bead system can combine a cell source marker coupling with a donor bead and one pathology-related and one protective pathway specific marker or two pathology or protective markers or have a third marker used for normalization to total particles from the cell type or used to obtain a ratio of postranslationally modified to total marker levels.

[0095] In some aspects, 250-350 nm AlphaLisa donor and acceptor bead sizes and steric hindrance on extracellular targets creates a sphere packing theoretical limitation on the number of beads coating extracellular vesicles and therefore, one limitation on the dynamic range per vesicle. Therefore, in some aspects, smaller (25-200 nm) donor and / or acceptor beads with ligands for the pathway rather than cell source analyte (or both) can be employed to increase the dynamic range of the pathway marker detection in any specific application. In some aspects, the cell source ligand can be diluted to limit the donor beads per target extracellular particle and thus maximize the range of acceptor beads docking per extracellular particle, the dynamic range for the pathway marker. In some aspects, the dynamic range in multiplex extracellular particle assays can be limited by the large commercial acceptor bead (250-350 nm) size in relationship to target EV and other particles. In some aspects, alternative singlet oxygen sensing doped fluorophores include anthracene-derivatives Singlet Oxygen Sensing Green (emission 525 nm), anthracenylmethy-modified fluorphores of rhodamine-6g and fluorescein, Si -9,10- dimethylanthracene (DMA) (emission 640 nm) and paired singlet oxygen sensor doped and FRET responder quantum dots ( 605 nm FRET emission). 5. Signal

[0096] In some aspects, the disclosed methods include triggering the donor bead to activate the acceptor bead to produce a primary signal (S1). In some aspects, the amount, presence, or absence of signal (S1) indicates the presence of a pathology-related or pathology-protective pathway in the subject. In some aspects, the magnitude of a signal (S1) indicates the presence and / or extent of a pathology-related or pathology-protective pathway in a specific cell-type of a subject.

[0097] In some aspects, triggering the donor can comprise exposing (or exciting) the donor bead to a specific wavelength (e.g.680nm or 775nm (naptholocyanine), wherein the signal produced by the acceptor bead can be read using a specific wavelength different from what was used for the donor (e.g.615nm, 545nm, or 645nm). In some aspects, alternative methods for triggering a proximity-sensitive signal can be used. In some aspects, proximity ligation and FRET between two target ligands tagged with FRET interactive probes can detect proximity of two targets with close angstrom to low nm proximity, for example, when two EV targets areATTORNEY DOCKET NO.37759.0498P1 together in a receptor or signaling complex on the fluid particle, but singlet oxygen triggering has a ~200 nm range for acceptors and therefore, detect ligands on the same EV or particle surface but not in the same complex. Thus, while there are good alternatives for donor bead photoactivated singlet oxygen generation and sensing fluorophores or quantum dots in acceptor beads, the 200 nm range for singlet oxygen triggering can be particularly suitable for distances on the surface of extracellular vesicles and particles, including microparticles.

[0098] In some aspects, primary signals for cell-source pathological or protective pathways can be normalized to produce ratios to other signals and plate to plate results normalized by including internal standards derived from pooled EV. Examples of normalization within assays include normalization to total cell-type specific extracellular particles measured in assays that take the same cell source marker used to get the primary signal and combine it with either a general exosome surface marker (CD81, CD63, CD9, Alix) or the same identical primary marker or additional different markers also constitutively expressed on the same cell source EV (examples for neurons include L1CAM and NCAM1). For example, if a primary cell source biotinylated mouse monoclonal antibody to epitope X was paired with a pathology or pathway marker to obtain a primary signal, one could use the same biotinylated monoclonal antibody to epitope X combined with the same monoclonal antibody with an alternative tag (e.g. other than biotin) and acceptor beads that recognize that tag or combine with a rabbit antibody to the same epitope and use anti-rabbit acceptor beads or combine with a ligand for another surface marker generally present on EV from the same cell source. These ratios are valuable because numbers of fluid markers per cell-type can change due to altered release, differences in escape into the blood or other fluid compartments or altered clearance, for example, reduced clearance from the blood due to altered kidney function. Thus, normalized ratios may often be better in describing the status of cell source specific pathological process marker levels (“Path”) in the tissue and originating cells. And normalized ratios also provide unitless numbers and control for site specific variance with reagents, lab temperatues or plate readers.

[0099] In some aspects, an alternative to normalization for total number of cell source-derived particles, a ratio of primary signal for post-translationally modified pathology or pathway marker (for example, cleaved by a pathology related protease or phosphorylated by a kinase) to a signal from the same cell-type specific source combined with a ligand for the total marker levels. For example, the “activated” complement pathway cleaved C3 product detected by its ligand CD21 can be normalized by the convertase enzyme substrate, total C3, on astrocyte EV. In other aspects, one can normalize with a ligand for a second pathological process marker that remains unchanged or moves in the opposite direction, which can also be used to produce a unitATTORNEY DOCKET NO.37759.0498P1 free ratio as an index of cell-type specific pathology in the source tissue. Additional markers for choosing pathology or pathway markers can be selected from “omics” data from cell source- derived material (proteomics, transcriptomics or lipidomics). In some aspects, selected candidate criteria include fold change, statistical significance, predicted or measured presence in EV with a surface accessible epitope or other surface accessible ligand. Significance to health or disease and surrogate biomarker potential for interventions is an additional criterion.

[0100] In some aspects, triggering is an excitation of the donor bead with a laser resulting in the release of a singlet oxygen, wherein the singlet oxygen excites a fluorescent signal from the acceptor bead. 6. Treating

[0101] In some aspects, the methods can further comprise treating the subject after determining the presence of a pathology-related or pathology-protective pathway in the subject. In some aspects, the treating comprises administering to the subject a known treatment for the disease or condition associated with the pathology-related or pathology-protective pathway identified in the subject.

[0102] In some aspects, the dose and efficacy of a treatment can be more rapidly and repeatedly assessed and monitored with the disclosed methods. Thus, in some aspects, treatment can be monitored and changed / altered in real time so that a subject does not stay on a treatment that does not work. C. Kits

[0103] The compositions and materials described above as well as other materials can be packaged together in any suitable combination as a kit useful for performing, or aiding in the performance of, the disclosed method. It is useful if the kit components in a given kit are designed and adapted for use together in the disclosed method. For example disclosed are kits comprising one or more of a first ligand, a second ligand, a donor bead, and an acceptor bead as disclosed herein. The kits also can contain equipment for detecting the signal produced by the acceptor bead. Examples A. Example 1 Alphalisa assay for biomarker related to cell specific derived extracellular vesicles

[0104] A method is needed to detect EV biomarkers from neuron or Astrocyte in peripheral system for neurodegenerative disease. Conventional methods: first use immunoprecipitation toATTORNEY DOCKET NO.37759.0498P1 purify neuron or Astrocyte EVs then run ELISA, western-blot or Flow cytometry to measure the target protein biomarkers. The limitations of these methods were obvious: including low and variable yields, highly variable platelet fragment impurities, extensive technician time, difficulty to scale and do high- throughout, and the use of Biotinylated antibodies in purifying cell source specific EV, but followed by subsequent ELISA assays for the biomarkers that also use biotinylated antibodies to detect, which may cause some artificial results in detecting signal from residual biotinylated antibody for the cell source, not the analyte.

[0105] The EXOLISA approach was tested on some human total EV samples and plasma samples. Signal was obtained from them, but most signals were low and only a few cases were high enough to detect. Overall, the utility of the assay was unsatisfactory. The main reason was that the assays used a cell source antibody combined with tetraspannin antibodies (CD9 or CD63 of CD81) and gave variable signals limited to and reflecting the number of exosomes from a cell source but not the disease parameters that clinicians and researchers need to measure.

[0106] Cell sourced alphaLisa assays were explored for blood EV markers without requiring an exosome marker. The plasma samples from SHR Rat models were tested – brains were taken and disease maker levels confirmed. After optimizing the protocol, it was found that a sample could provide dose-dependent signals from the assays. The assays worked to give signals that were sample and disease-specific in mouse models and human samples.

[0107] Therefore, assays using this approach were explored for multiple animal model and human samples and its high sensitivity and general application potential were analyzed.

[0108] FIG.1 shows an example of one of the disclosed methods, wherein the system utilizes streptavidin-coated donor beads to capture an analyte-specific biotinylated antibody, and acceptor beads conjugated to a second antibody that recognizes an epitope of the analyte. The donor beads are excited with a laser at 680 nm, resulting in the release of singlet oxygen, which excites an amplified fluorescent signal in the acceptor beads. As a result, the acceptor beads emit light at 615 nm, but only if they are within 200nm of the analyte captured by both antibodies.

[0109] An example of the disclosed methods is as follows: Platelet depleted plasma (5 ^l - 10 ^l) is incubated with biotinylated antibodies to the surface marker for a specific cell source (10 ^l) and a surface biomarker antibody (10 ^l) at 4 ^C overnight. Then the mixture is transferred into a AlphaplateTM384-plate and incubated at RT for 60min.10 ^l AlphaLISA Acceptor beads conjugated with anti-relative antibodies IgG (final Conc.10 ^g / ml) is added and incubated at RT for 60min. Then 10 ^l AlphaScreen streptavidin-coated donor beads (final Conc.40 ^g / ml) is added and incubated at RT for 30-60min. The assay is no wash. The plate is read on an EnSpire Alpha 2300 Multilabel Plate reader using an excitation wavelength of 680nm and emissionATTORNEY DOCKET NO.37759.0498P1 detection set at 615 nm. Background signals obtained from PBS were subtracted from the measured signals and produced using PBS substituted for sample controls with added reaction mixture as the negative control.

[0110] The plasma volumes, antibody dilutions, their incubation times and donor and acceptor bead ratios and numbers can be adjusted to optimize signal and group differences in different assays as they depend on the target extracellular particle abundance and analyte levels per particle. For example, overnight antibody incubations are required to detect rarer particles and, if necessary, larger sample volumes can be incubated with the antibodies and the beads targeting the cell source (usually donor beads) followed by pelleting to capture rare extracellular particles or other particles followed by resuspension in assay buffer.

[0111] The disclosed methods focus on detecting disease or process- specific surface biomarkers on extracellular vesicles (EV) or particles released from a specific cell type, instead of requiring an exosome-specific marker. In general, known assays capture or purify exosomes and measure exosome contents. Exoscreen is built to detect exosome numbers from specific cell sources with the idea of identifying tumor cell specific exosomes in liquid biopsy. In contrast, the disclosed approach is built to measure specific disease surface markers on cell sourced particles that may include ectosomes, exosomes, microparticles, blebs, debris, small apoptotic bodies can provide very useful information reflecting many critical disease processes. Of course, there is also the option to normalize to account for the variance in the numbers of cell sourced particles as well as peripheral particle clearance in a sample using two cell source markers or a cell source marker and a generic exosome marker for normalization to give a per particle level or ratio. With an exosome cancer-related marker, this is analogous to using an Exoscreen assay for normalization of the assay data.

[0112] Many studies show that EVs carry surface proteins which can be representative of their cells of origin, and EVs carry cargo that contain some pathological information. One antibody against the surface marker of specific cells type and another antibody or recombinant protein that binds a surface pathological marker were used. Candidate target analytes in EV proteomics papers have been screened using samples prepared from plasma or specific cell types or tissues.

[0113] The amount of candidate analytes detected are rare and below the limits of detection compared to the levels of the exosome markers like CD9 detected in earlier work. Sensitivity was likely a main obstacle preventing success in adapting that protocol to detect disease-related surface markers rather than exosome markers.

[0114] There are a few key points to the disclosed methods. In some aspects, it can beATTORNEY DOCKET NO.37759.0498P1 important to choose the surface exposed analyte epitopes and pair the antibodies or detector proteins with appropriate dilutions that make the assays depend on the levels of analyte rather than simply total cell type-specific particle numbers. Analytes can be preselected for proteomic or other evidence for their presence in extracellular vesicle enriched factions and selected for the extracellular domain epitopes on the surface of cells. The abundance of the analytes can be an additional concern or criterion.

[0115] Overnight incubation is used to allow time to let low levels of biotinylated antibody and analyte antibody to capture their target. Since a long incubation time can be used to detect low abundance biomarkers, the standard AlphaLisa approach with antibodies directly conjugated to AlphaLisa acceptor beads may not always be able to be used. In some aspects, this can cause higher background with the longer incubation times.

[0116] In pursuing biomarkers related to Alzheimer’s disease it was found that the disease risk gene ApoE4 reduced the release of exosomes. It was then realized that many disease processes can increase or reduce exosome release from the cells or organ or impact exosome or EV clearance. Therefore, 2 assay sets are best deployed and data analyzed using both the individual marker data and their ratio as candidate meaningful biomarkers.

[0117] These disclosed methods can have several applications. In general, the ability to require multiple identifiers per EV or particle can increase specificity for cell source. For example, both L1CAM and NCAM1 have been used to affinity-purify “neuron-derived” extracellular vesicles, but neither are entirely neuron specific. NCAM1 is also expressed in NK cells, rare subsets of T and B lymphocytes, dendritic cells, and mesenchymal stem cells. L1CAM is also expressed in some oligodendrocytes, Schwann cells; melanocytes, T cells, B cells, monocytes, certain epithelial and endothelial cells and upregulated in various cancer cells, notably colorectal cancers. Therefore, using either of these candidate general neural EV markers with a second neural cell source marker, for example, synapse-enriched markers including GluR1-4, SNAP25, CamKIIa, synaptophysin or a syntaxin, synaptotagmin, neuroligin or neurexin family member can provide more specific markers related to loss or alterations in synaptic sources.

[0118] They can also provide biomarkers for diseases, especially degenerative disease processes occurring in real time- the slope of the curve for the disease process. This is distinct from measuring slowly cumulative changes with imaging or other methods. The methods can also be used for monitoring the progress of intervention or treatment effects in real time. The assay approach can also work on cell culture medium in systems used to screen for drug efficacy, then it can benefit the screen for effective therapeutic compounds by addingATTORNEY DOCKET NO.37759.0498P1 biomarkers to detect cell source target engagement and monitor dosing and efficacy. B. Example 2: Complement C3 & active C3 (CD21) from astrocyte derived extracellular vesicles 1. Tau related complement C3 activity:

[0119] Plasma samples from rT1 human tau transgenic mice were run with Tau positive (n=8) vs Tau negative (knockout) (n=6) background. The data (see FIG.2) showed Tau dependent astrocyte derived C3 (p=0.063) and CD21 detection of cleaved active C3 elevation (p=0.050) consistent with the literature showing tauopathy induced complement activation is a major cause of neurodegeneration in mouse models for AD, frontal temporal dementia (FTD-17), progressive supranuclear palsy and CTE. 2. Age-related complement C3

[0120] Complement C3 expression in astrocytes is also known to rise with age. In a study using mouse strain-PS19, combined Tg+ and Tg- group, a trend for age-related complement C3 elevation was seen. BioGlast1 / C3 level from 9 months old mice (n=4) was increased compared to 4 months old (n=4), p=0.07 (FIG.3). Even with small numbers this trend was observed by ICC for C3 in brain. FIGS.32-33 also illustrate aging effects in normal WKY rats. 3. Excitatory amino acid transportor (EAAT2) from astrocyte derived extracellular vesicles:

[0121] Studies show increased excitotoxicity that is believed to be due to decreased astrocytic glutamate transporter EAAT2 expression in aging and sporadic AD patients and AD model mice. EAAT2 removes excess glutamate. Plasma samples from WKY / FAD (APP / PS1) positive (n=7) vs negative rats (n=7) were tested. WKY is the background strain for SHR rats and WKY-FAD represent an amyloid rat AD model. The data showed BioGlast1EAAT2 were reduced in WKY / FAD positive, p=0.056 (FIG.4). If normalized with BioGlast1CD81, BioGlastaEAAT2 was significantly reduced, p=0.009. Excitotoxicity is relevant to epilepsy and AD. For example, there is a clinical trial for AD using an anti-epilepsy drug that was reported to raise levels of EAAT2 in mouse brains. Other groups have developed drugs to elevate EAAT2 to treat AD and many other CNS conditions with excitotoxicity. As these rat models have seizure activity, this assay can be used with the disclosed methods on these models. 4. Excitatory synapse loss

[0122] Glutamate receptors (GluR) including GluR1-4, are enriched on excitatory synapses that are preferentially lost in many chronic diseases with different pathologies contributing to memory deficits and where synapse loss is the proximal cause of cognitive decline but lacks blood biomarkers. For example, excitatory synaptic marker loss is found in vulnerable brainATTORNEY DOCKET NO.37759.0498P1 regions in Alzheimer disease (AD) and AD animal models with amyloid or tau pathology or both. Consistent with this loss, while not synapse enriched, antibody to L1CAM has been used to affinity purify plasma EV preparations and detect losses in excitatory synaptic markers by commercial ELISA in samples from patients with AD.

[0123] Here, the plasma from hypertensive SHRSP rats with and without familial AD genes leading to amyloid and tau pathology, SHRSP (n=7, 13-17 months old) vs age-matched SHRSP FAD+ (n=7) were compared pairing biotinylated GluR1+2 and a rabbit antibody to L1CAM using an ExPath assay with streptavidin coated donor and anti-rabbit coated AlphaLisa acceptor beads. Consistent with significant synapse loss, plasma from SHR FAD+ rats (with AD pathology) displayed a significant reduction, p<0.01 (FIG.5 A). Since Complement C1q is known to tag neurons and excitatory synapses lost in analogous mutant FAD transgenic AD model mice, an ExPath assay was used on the same samples using bio-NCAM1 antibody paired with anti-rabbit C1q using the same AlphaLisa platform and reagents and found a similar trend (p=0.09) for deficits in rats with AD pathology (FIG.5B). Using a larger rat plasma sample set (n=55, aged 12-20 mo age, including n=11 WKY, WKYFAD, SHRSP and SHRSP-FAD+), plasma results were compared from two additional ExPath assays designed to reflect loss of CNS excitatory synapses, one paired biotinylated GluR1,2 and recombinant CD21 binding nickel chelate coated acceptor beads and the other ExPath assay used bio-NCAM1 moAb and rabbit C1q with detection by anti-rabbit coated AlphaLisa acceptor beads. Results with these two very different assays across all samples showed a very high 0.925 correlation that cross- validates the methods (FIG.6). Similar plasma EV assays for excitory synapse loss are shown using other variations of the method in Fig.34A-C, illustrating a robust result consistent with the well-known reduction of the putative CNS excitatory synapse source in AD and AD mouse models. 5. Aquaporin 4 (AQP4) from astrocyte derived extracellular vesicles:

[0124] AQP4 is a water channel expressed on astrocyte endfeet lining the blood brain barrier (BBB). Loss, damage and retraction of astrocyte endfeet occurs in AD, CTE and aging and contributes to BBB damage. In WKY FAD+ (n=7) vs WKY FAD- (n=8) Rats, it was found that FAD+ rats show BioGlast1AQP4 trends for reduction, p=0.126 (FIG.7).

[0125] In many sample sets, there were good correlations between the astrocyte biomarker levels. Overall, group differences or trends consistent with data from direct measurements in brain were found. The table below shows the assays that have been tested and some applications of each.ATTORNEY DOCKET NO.37759.0498P1 6. Vascular Cell Adhesion Molecule 1 (VCAM1) on endothelial derived extracellular vesicles

[0126] VCAM1 is one of several markers of endothelial “activation” caused by localized damage and inflammation and serves as an adhesion site for invading circulating leukocytes on their luminal surface. VCAM1 is known to be chronically elevated in SHRSP and parent SHR rat strains with chronic hypertension compared with t normotensive (same background strain) WKY rats. Therefore, an ExPath assay was used pairing biotin-CD144 (endothelial marker) with rabbit anti-VCAM1 using plasma from age-matched WKY and hypertensive SHRSP rats and found significantly elevated VCAM1 in the SHRSP (FIG.8) with an outstanding ROC analysis curve (FIG.9).

[0127] Vascular inflammation and downstream endothelial activation are further elevated by the presence of AD amyloid and tau pathology in brains of SHRSP-FAD+ mice, a model for mixed neurovascular and AD pathology that contributes to cognitive deficits in millions of patients with more difficult to diagnose “mixed dementia”. When the same ExPath assay used in FIG.8 and FIG.9 to compare plasma samples from age-matched SHRSP and SHSP-FAD+ rats and then normalized the results for total ExPath biotin-CD144 and CD81 to obtain a ratio reflecting VCAM1 elevation per particle, results revealed a significant (p<0.01) elevation in VCAM1 in the AD pathology bearing SHRSP-FAD+ group (FIG.10). The elevated ratio shows that the VCAM1 elevations in endothelial derived EV do not simply reflect reduced clearance or increased secretion but VCAM1 levels per EV that are elevated in response to brain pathology.

[0128] Overall, these results illustrate that ExPath assays in animal models reflect differences or trends consistent with data from direct measurements in brain. 7. Human plasma from age-matched dementia versus cognitively normal controls.

[0129] PET imaging with highly radioactive probes for amyloid or tau fibrils are the current gold standard for detecting AD pathology in patients. However, a sizeable (~25-30%) fraction of aged, cognitively normal controls are amyloid positive and similar numbers or cognitively impaired, suspected AD patients in clinical trials have been amyloid PET negative, particularly in patients without the major genetic risk factor for amyloid, ApoE4. Thus, analysis of 24 subjects where amyloid PET status (SUVR) in the dementia (Dem) and normal cognition (NC) cohorts revealed both amyloid positive and negative patients, with ROC analysis AUC=0.72 for discriminating dementia (FIG.11A). Comparing plasma samples from the same 24 patients with the ExPath assay for active complement C3 (CD21) on astrocyte-derived EV (biotin-Glast) normalized to total C3 with bio-Glast, the Dem patients show the expected trend for an increase and Roc analysis produces an AUC=0.807. This discrimination is similar to, or better than,ATTORNEY DOCKET NO.37759.0498P1 amyloid PET, but non-invasively reports on a specific, druggable neuroinflammatory process implicated in both the neurodegeneration and synapse loss that drive progressive cognitive deficits that are only marginally slowed in patients with successful anti-amyloid treatment. These results are also consistent with reports of elevations of multiple complement factors that converge on and include active complement C3 assayed by sandwich ELISA in affinity purified astrocyte-derived EV preparations from AD and other CNS conditions. This data shows that no wash ExPath assays developed using animal model samples can be applied to detect cell source specific pathologic processes in human fluid samples, illustrated here with plasma from well- characterized normal controls and dementia patients and complement activation in astrocyte- derived EV.

[0130] The table below shows the assays that have been tested and some applications of each. Table 1: complement related assays Assay Analyte Extracellular Human Mouse Rat Applications , ,ATTORNEY DOCKET NO.37759.0498P1 Table 2: Non-complement related assays al alC. Examples of Using the Disclosed Methods to Detect Diseases / Conditions 1. Detecting Neurodegenerative Diseases of Aging

[0131] Because excessive synaptic elimination in neurodegenerative diseases of aging is typically mediated by complement activation, active excitatory synapse loss in dementias can be assessed by combining antibody to GluR2 / 3 with an antibody or other ligand for a pathological marker representative of an active complement component, for example CD21 (a receptor for active C3 where the classical and alternative complement pathways converge). Signal for total; excitatory synaptic extracellular particle obtained by combining GluR2 / 3 with another excitatory synaptic marker or a tetraspannin exosome marker (CD81, CD9, CD63) or flotillin can be used to assess total excitatory extracellular particle signal which can be stage-dependently elevated by seizure activity, TBI, stroke and other acute injury, but ultimately declines with severity of disease and loss of excitatory neuronal sources. The disclosed methods for synapse-derived extracellular particles permit monitoring of synaptic pathology and synapse elimination in real time. 2. Detecting acetylcholinesterase loss related to cognitive decline

[0132] A significant loss of acetylcholinesterase (AchE) occurs at synaptic sites in AD and contributes to cognitive impairment as shown by improvements from FDA-approved acetylcholinesterase inhibitors. AchE loss can be assessed by combining a ligand for a general orATTORNEY DOCKET NO.37759.0498P1 cholinergic selective neuronal or synaptic marker with a ligand for AchE. A loss of AchE in neural exosomes occurs at least 5 years prior to cognitive decline. 3. Detecting Prion-Related Diseases

[0133] The prion protein PrPC serves as receptor not only for prions, but also other soluble pathological protein aggregates including abeta and tau oligomers and it is upregulated in CNS in AD and other CNS diseases. However, PrPC is also found in glial cells and multiple peripheral cell types so measuring PrPc in blood without cell source information is insufficient to detect a pathological process. CNS neuronal levels of PrPC can be assessed by combining a neuronal or synaptic cell source-selective extracellular particle marker with a ligand for pathology-relevant (Path) marker PrPC. Similarly, endoethelial or glial specific extracellular particle marker pairing with a PrPc ligand reports on PrPc in endothelial cells, astrocytes or microglial cells. 4. Detecting Vascular Damage

[0134] Ligands for cell source markers include (most selective: CD144 and CD31 as well as CD34 and Lyve1, enriched in EC-derived nanoparticles (EV or exosomes or debris and even larger microparticles. Ligands to pathological processes in EC include responses to local inflammation and tissue damage by upregulating cellular adhesion molecules (CAMs) including Path markers (VCAM1, ICAM1, E-selectin, P-selectin). Additional path markers include the complement activation markers (C1q, Bb, Masp1 / 2 and / or C3 and its activation products (C3b, ic3b, C3d), C4 (C4A, C4B, C4b, C4d)). For example, vascular inflammation reflected in increased plasma endothelial EV complement C3 and Bb activation are markedly elevated with white matter damage and vascular cognitive impairment. Additional path markers include Glut1, LAT-1 / SLC7A5 and P-GP / MDR1 which are elevated in subjects with white matter hyperintensities reflecting damage to axon tracts associated with vascular damage with aging. 5. Detecting Cerebral amyloid angiopathy (CAA)

[0135] Detecting Cerebral amyloid angiopathy (CAA) in amyloid positive AD patients who are candidates for new FDA approved immunotherapies is now a major concern because the patients with CAA can have dose-limiting and severe, even deadly side-effects due to increased cerebrascular inflammation detected as ARIA. Plasma biomarkers relevant to CAA with ExPath assays include CD144 or CD31 for endothelial source and Abeta, VCAM1 or TLR4 (elevated) or LRP1 (reduced). These may be combined with evidence of amyloid positivity and vascular fibroblast Expath EV measures using PDGFRalpha for cell source coupled tenascin C and Col6A2 as CAA pathology markers.ATTORNEY DOCKET NO.37759.0498P1 6. Detecting Long-Covid.

[0136] Complement activation plays a central role in both acute and Long Covid detected in many reports and uncovered in unbiased plasma proteomic assays that highlight persistent endothelial complement activation and CNS complement activation in pathology or CSf reports. The plasma ExPath assays show that plasma can be used to detect endothelial and CNS complement activation in Long Covid patients with neural symptoms as well as discriminate Long Covid from healthy controls with high sensitivity and accuracy. The results are consistent with a report using a multi-plex panel with multiple endothelial activation / inflammation markers to distinguish Long Covid from acute Covid and healthy controls. They reported elevations in plasma CD144 (VE-Cadherin) but also in other surface endothelial proteins including P-Selectin, PECAM1, E-Selectin and ICAM-1 consistent with vascular inflammation. P-Selectin alone was most elevated and worked better to diagnose Long Covid in combination with a second factor, soluble secreted Ang-1. As discussed by Patel, P-Selectin is highly expressed on both endothelial cells and platelets so values reflect both sources as illustrated by data showing that plasma P-selectin is markedly reduced after platelets have been removed from patient samples. Thus, using the ExPath approach and measuring Bio-CD144 / P-Selectin reflecting endothelial P-selectin and its ratio to Bio-CD144 / CD81 or CD144 / CD31 as an index of total EC derived particles can be predicted to be a better biomarker for monitoring Covid and Long-Covid than a conventional immunoassay of P-selectin where a lot of signal is platelet- derived. Similarly, circulating E-selectin has been reported to be a useful blood biomarker for ICU admission predicting severe Covid and could also be measured with the ExPath approach, for example, as Bio-CD144 / E-selectin for Covid. While E-selectin (CD62E) is more endothelial specific and not on platelets like P-selectin (CD62P), soluble E-selectin appears much less useful as a biomarker for Long Covid. Thus, the ExPath assays for active complement in astrocyte and endothelial EV can be augmented by the proposed ExPath assays. 7. Detecting Cancer

[0137] A liquid biopsy can be used for tumor epitopes. Multiple supersensitive approaches to detect tumor cells, DNA, proteins and other markers are in trials or have even been approved for monitoring patients. Nevertheless, most cancers are now classified not only by traditional pathogenomic tissue source (breast, colon, prostate, lung etc.) and cell type (carcinomas, sarcomas, leukemias or lymphomas) but also by their individual tumor specific oncogene and tumor suppressor pathway mutations that inform on more selective therapeutic options.

[0138] ExPath assays, as described herein, for adenocarcinoma EVs with epithelial cell origin can pair an epithelial cell subclass antigen, for example, (Lgr5 / GPR49 on intestinal stem orATTORNEY DOCKET NO.37759.0498P1 crypt cells for colon cancer), (ITGA6 or NGFR for lung basal cell carcinoma), (MUC5AC or TSPAN8 for lung goblet cell) , (CDHR3 for ciliated lung epithelial cells), CEACAM5 or 6 for secretory epithelia) with a more general marker, for example EpCAM that is commonly elevated across most adenocarcinomas (e.g. colon, stomach, pancreas, breast, and prostate) and their EV or with a surface marker more specific for an individual cancer as listed above.

[0139] Cancer stem cell markers can be paired with cell source markers or each other. Alternatively, the epithelial cell subclass antigen can be paired with cancer stem cell markers (e.g. prominin1 / CD133 or CD44) found on many stem cell derived cancers (including colorectal, melanoma, ovarian, prostate and many others) where elevated levels predict more aggressive tumors and the pairing and detection of CD133 and CD44 on the same EV predicts more malignant tumors.

[0140] Cancer cell type or stem cell marker can be paired with an MMP marker predicting invasiveness. Similarly, a ligand for the main metalloproteinase involved in invasive spread, MMP-14 (MT1-MMP) which is elevated on invasive tumors and their secreted EV can be paired with a cell type or stem cell marker to create assays predicting more aggressive tumors and the need for more aggressive treatment.

[0141] Pairing a ligand for a cancer cell type or stem cell marker with a ligand for checkpoint inhibitor target marker can be used to guide check point inhibitor therapy. Check point inhibitor targets are often elevated on and used by tumor cells (most commonly PD-L1 but alternatively GPNMB, CTLA-4, CD20 and CD52) to help escape immune surveillance. Thus, identifying elevated checkpoint inhibitor targets on tumors can be used to predict successful responses to checkpoint inhibitor therapies. 8. Detecting Heart Function and Failure

[0142] Heart function and failure. Cardiomyocyte sourced EV can be detected in fluids as positive for ligands for connexin 43, Troponin T and / or Cav3 which can be paired together to index the numbers of cardiomyocyte -derived EV which increase with heart hypertrophy at early stages of heart disease. Glucose demand is associated with compensatory increased Glut1 expression at early stages of hypertrophy but followed by decreased Glut1 expression with failure. Combining one of the cardiomyocyte markers with Glut1 reports on heart specific glucose use, heart health and progressive disease. Similarly, combining a cardiomyocyte marker with a ligand for active complement will provide a measure of inflammation in heat disease. D. Example 4. Endothelial cell-derived extracellular vesicle VCAM1 as a biomarker for blood vessel inflammation

[0143] Among the deleterious effects of high blood pressure described in theATTORNEY DOCKET NO.37759.0498P1 literature, endothelial dysfunction is an essential cause of the blood vessel hyperplasia that leads to coronary artery disease and ultimately heart failure or stroke. During endothelial dysfunction, pro-inflammatory cytokines including TNFalpha and other factors that recruit white blood cell (leukocyte) infiltration through the vessel walls, a process mediated by increased focal expression of Vascular cell adhesion molecule 1 (VCAM-1) driven by activation of the transcription factor NFkB.

[0144] VCAM1 plays an important role in amplifying local inflammation because it facilitates the initial vascular wall interaction with circulating and invading immune cells. VCAM1 is upregulated on the luminal endothelium in response to local inflammation where it facilitates leukocyte tethering to the endothelial cells via the integrin receptor α4β1 (also known as VLA-4) which triggers additional pro-inflammatory signaling that ultimately weakens the tight junction and permits transmigration of the circulating immune cells into injured and inflamed tissues.

[0145] In order to maintain long term control of this inflammation triggering, the extracellular domain of the transmembrane protein VCAM1 is constitutively shed through proteolytic cleavage by the membrane-bound metalloproteinase ADAM17, resulting in normal relatively high and detectable levels of baseline plasma sVCAM1. One limitation in the use of sVCAM1 is the high background of this baseline shedding and variable control of its clearance and factors that modulate the shedding metalloproteinase activity, notably the metalloproteinase inhibitor TIMP3.

[0146] The potential relevance of sVCAM1 as a biomarker for vascular injury associated with hypertension was previously made and showed increased sVCAM-1 levels in older adults with uncomplicated essential hypertension. This was subsequently confirmed by others in subjects with or without the coexistence of other cardiovascular diseases but challenged by other studies who found no elevation of sVCAM-1 raising doubts that it can ever be reliably used as a single specific biomarker for hypertension because VCAM1 is also increased by other causes of vascular injury including atrial fibrillation, obesity, diabetes, air pollution and atherosclerosis. Because elevated VCAM1 is an excellent global marker for ongoing vascular injury contributing to chronic disease risk stemming from chronic, low grade inflammation, this study sought to develop a simple and rapid test to monitor elevated endothelial VCAM1 rather than its more complex and constitutively released product soluble VCAM1.

[0147] One of the most widely used animal models of chronic hypertension and vascular injury is the Stroke-Prone Spontaneously Hypertensive (SHRSP) rat. Aging SHRSP rats have progressive vessel wall damage, thrombotic occlusions and reductions of cerebral blood flow linked to cerebrovascular disease (CBVD), white matter damage and cognitive impairment. TheATTORNEY DOCKET NO.37759.0498P1 data on this established model for aging induced cerebral small vessel disease showed systolic and diastolic pressure were increased by 50% and 42% respectively in the aging SHRSP and normotensive control WKY rats with and without Alzheimer-related amyloid plaque pathology used in this study.

[0148] Here, surface VCAM1 was measured on extracellular vesicles derived from endothelial cells in the plasma of SHRSP and WKY rats and it was found that the direct measure of intact VCAM1 can separate SHRSP and WKY very well. The effect of mutant familial Alzheimer disease (AD) presenilin and Amyloid precursor protein (APP) transgenes that result in AD amyloid plaque and tau pathology in SHRSP were also examined and it was shown that FAD transgenes and or resultant CNS amyloid deposition significantly increases the expression of VCAM1 normalized per endothelial-derived EV particle. The results show that extracellular vesicle endothelial VCAM1 can be a useful biomarker not only for cardiovascular disease, but also in AD, CBVD and the mixed AD and CBVD present in SHRSP-FAD rats. 1. Methods i. Rat

[0149] Rats used were: (1) WKY rat: non-hypertensive rats, 14-22 months old, n=7, include female and male and (2) SHRSP rat: Stroke-Prone Spontaneously Hypertensive rats, 14-20 months old, n=9, include female and male. Additional rat groups of varying ages with and without human ApoE3 or ApoE4 targeted replacement of rat ApoE are described in figure legends and numbers used in each assay are shown in scatter gram plots. ii. Other supplies

[0150] Additional supplies used were: Biotinylated anti-CD144 antibody: anti-CD144 were mouse monoclonal antibody; Anti-VCAM1 antibody: rabbit monoclonal antibody; AlphaLISA Acceptor beads conjugated with anti-rabbit IgG; AlphaScreen streptavidin-coated donor beads; 600 ^l microtube; AlphaplateTM384-plate; EnSpire Alpha 2300 Multilabel Plate reader; Pipettes and Pipette tips; and Plate Sealers. iii. Protocol

[0151] Blood was collected from the heart at euthanasia under terminal general anesthesia in tubes with 250 mM EDTA and immediately transferred to ice. Blood was centrifuged at 2200×g for 20 min at 4°C to remove debris. One hundred microliter (100 ^l) of plasma was aliquoted to avoid multiple freeze-thaw changes and stored at -80 ^C.

[0152] Prior to the assay, thawed plasma was centrifuged at 1500g for 15min to deplete platelets.10 ^l platelet-depleted plasma was incubated with 10 ^l biotinylated anti-CD144 antibody plus 10 ^l anti-VCAM1 antibody in a microtube by gentle mixing at 4 ^C overnight.ATTORNEY DOCKET NO.37759.0498P1 The mixture was transferred into wells of AlphaplateTM384-plates and incubated at RT for 60min.10 ^l AlphaLISA Acceptor beads conjugated with anti-rabbit IgG (final Conc.10 ^g / ml) was added and incubated at RT for 60min.10 ^l AlphaScreen streptavidin-coated donor beads (final Conc.40 ^g / ml) was added and incubated at RT for 30-60min. Plate was read on the EnSpire Alpha 2300 Multilabel Plate reader using an excitation wavelength of 680nm and emission detection set at 615 nm. No sample added reaction mixtures served as the sample negative controls. Background signals obtained from negative control “blanks” were subtracted from the measured signals for all sample measures. Measured fluorescence emission from samples were used for further data analysis. 2. Results

[0153] A plasma biomarker for CD144 positive endothelial cell sourced upregulation of VCAM1 ( BioCD144 / VCAM1) can distinguish the hypertensive SHRsP from WKY rat blood plasma samples with a significant elevation in the hypertensive rats (FIG.8, FIG.9),

[0154] This is a measure of endothelial “activation” in response to hypertension or other causes of endothelial stress or inflammation without the complication of factors modulating proteolytic cleavage to release soluble sVCAM1 or the wash steps in a conventional ELISA or the many preparation steps if endothelial exosomes are purified, extracted and assayed for VCAM1. Because VCAM1 is also expressed on subsets of B cells, splenic macrophages and dendritic cells, sVCAM1 is not entirely endothelial cell sourced. CD144 are both relatively but not entirely specific for endothelial cells. Similarly, CD144 is expressed by endothelial cells but also not entirely endothelial specific as it is also expressed by adipocytes. For this reason, studies double immunopurify endothelial exosomes sequentially using CD144 and CD31 before extracting assaying markers for inflammation and activation. But because VCAM1 is also endothelial enriched, using the combination of CD144 and VCAM1 provides a relatively specific measure of endothelial activation, typically in response to cytokines and reactive oxygen species (ROS) in the context of inflammation, for example in heart disease or small vessel disease.

[0155] This assay had very high sensitivity and specificity for SHRSP rats compared with normotensive WKY with ROC Area =1 (FIG.9). This perfect separation is consistent with SHRSP’s known endothelial activation and elevated VCAM1 at the level of mRNA which is also confirmed at the protein level with VCAM1 / actin roughly 2-fold elevated in SHRSP vs WKY.

[0156] Next the levels of BioCD144-VCAM1 in aging WKY and SHRSP rats that also expressed human mutant APP / PS1 genes were evaluated, a model for mixed small vesselATTORNEY DOCKET NO.37759.0498P1 disease (SVD) and Alzheimer pathology or “mixed dementia”. Mixed vascular and AD dementia is highly prevalent in human populations. No effect of FAD transgenes and pathology on WKY rats was found but a selective reduction in BioCD144 VCAM1 in the SHRSP-FAD+ rats with a mixed vascular SVD and AD pathology was seen, but reductions in signal were even greater in BioCD144-CD81 assays for endothelial cell EV bearing a classic tetraspannin marker enriched in EV and exosomes. Then the BioCD144-VCAM1 signal was normalized to BioCD144-CD81 to examine VCAM1 per endothelial cell particle and found a significant elevation in SHRSP rats with FAD transgenes and AD pathology compared with SHRSP rats (FIG.10). 3. Discussion

[0157] Collectively, these results show that the disclosed methods can be used to detect elevated endothelial VCAM1 as a function of chronic hypertension in vascular disease and the loss of endothelial CD144-VCAM1 or CD81 EV particles in SHRSP with FAD, a model for mixed dementia; but also an elevation of endothelial VCAM1 normalized to CD81 in this new mixed dementia model.

[0158] These assays illustrate the ability to use this invention to quickly and simply monitor endothelial cell inflammation / activation that often occurs with hypertension, vascular and heart disease, Covid, and aging. The same assay shows highly variable sample-dependent levels in plasma samples from aging humans in a clinical trial for MCI or subjective cognitive impairment but there is insufficient additional patient data to evaluate their endothelial cell activation status. Combined with other biomarkers and clinical data, this approach may also be useful in diagnosing mixed vascular and AD dementia, and evaluating interventions.

[0159] For example, inflammation is known to be a significant factor driving atherosclerosis and cardiovascular events. The traditional biomarker for these studies is C-reactive protein (CRP) and the recent Novartis Cantos trial treating patients at risk for CVD events with antibody to the pro-inflammatory cytokine IL-1beta reported that in patients with baseline CRP above 2mg / ml, antibody treatments significantly and dose-dependently reduced CRP and CVD events.

[0160] CRP is an acute phase protein primarily produced by the liver and not an ideal biomarker of chronic vascular / endothelial inflammation which is a critical factor in traditional vascular disease but also in Long Covid. For example, a multiplex panel with multiple endothelial activation / inflammation markers was recently used to distinguish Long Covid from acute Covid and healthy controls and they reported elevations in plasma CD144 (VE-Cadherin) but also in other surface endothelial proteins including P-Selectin, PECAM1, E-Selectin and ICAM-1. P-Selectin alone was markedly elevated and had an AUC of 1 but worked better toATTORNEY DOCKET NO.37759.0498P1 diagnose Long Covid in combination with a soluble secreted Ang-1. P-Selectin had been previously reported elevated with acute Covid but it is highly expressed on both endothelial cells and platelets so values reflect both sources. But plasma P-selectin was markedly reduced when platelets were removed illustrating more significant endothelial sourced signal even though levels were still elevated in Covid patients. Thus, using the approach disclosed herein and measuring Bio-CD144 / P-Selectin and its ratio to Bio-CD144 / CD81 is predicted as a better biomarker for monitoring Covid and Long-Covid than a conventional immunoassay of P- selectin. Similarly, circulating E-selectin has been reported to be a useful blood biomarker for ICU admission predicting severe Covid and could also be measured with the disclosed approach, for example, as Bio-CD144 / E-selectin for Covid. While E-selectin (CD62E) is more endothelial specific and not on platelets like P-selectin (CD62P), soluble E-selectin was reported much less useful as a biomarker for Long Covid. Moreover, endothelial complement activation has been confirmed in proteomic assays of Long Covid vs control samples and CNS complement activation is found in Long Covid patients with neural symptoms. The ExPath assays for endothelial and CNS complement activation illustrate their utility in rat models for Long Covid and in Long Covid patient samples.

[0161] In conclusion, we have illustrated a simple assay for CD144 / VCAM1 as an example of a suitable blood biomarker for endothelial inflammation in connection with chronic hypertension and vascular disease and additional endothelial activation by Alzheimer pathology in a mixed dementia model. Deploying the same rapid no wash method for other endothelial cell sourced markers of inflammation including P-Selectin (for Long Covid), E-selectin (predicting severe Covid) or Complement activation markers C3b, C4b and BB for conditions with significant complement activation will provide a new tool kit for non-invasive assessment of endothelial cell health. E. Example 5: Monitoring persistent CNS Complement activation after Traumatic Brain Injury (TBI) 1. Introduction

[0162] Traumatic brain injury (TBI) is a common injury in auto and other accidents, sports and with military exposure in Veterans. The U.S. CDC reported 223, 136 hospitalizations and 64,362 deaths from TBI in 2019 and estimated 280,000 hospitalizations and about 50,000 deaths in 2021. The numbers of mild TBI cases are harder to estimate and in the range of 200 to 700 per 100,000 person in global annual cases. With ~ 8,000,000,000 people this means 200 to 700 x 80,000 = 16 to 56 million global cases per year. While the majority recover in the short run, millions have persistent damaging processes and the long-term consequences for many can beATTORNEY DOCKET NO.37759.0498P1 quite serious including risk for post-traumatic epilepsy, Parkinson or Alzheimer Disease (PD or AD) or chronic traumatic encephalopathy (CTE).

[0163] Three of these diseases involve accumulations of insoluble, fiber forming protein aggregates or proteinopathies. PD has accumulations of aggregated alpha synuclein while both AD and CTE have accumulations of aggregated tau protein or tauopathy as a hall-mark debilitating neuropathology found in postmortem brain, but it is currently impossible to diagnose and treat this underlying process in patients in the early stages of developing chronic, costly debilitating, incurable conditions. AD also features accumulation of extracellular beta amyloid protein and recent progress has been made clearing amyloid accumulation in AD with Aduhelm and Lecanemab antibodies to amyloid, but even successful abeta treatment has left behind ongoing established and self-sustaining tauopathy and persistent cognitive decline. Mouse models for amyloid accumulation with rare autosomal dominant familial mutations in amyloid precursor protein (APP) were created, but they failed to show the robust tauopathy, neurodegeneration and associated neuroinflammation, including complement activation that was found in AD.

[0164] This has led to an intensive investigation of tauopathy and a search for biomarkers that can identify individuals with ongoing neurodegenerative processes and be used to monitor process activation and responses to treatments as surrogate biomarkers for tauopathies. Because rare mutations in tau, for example at amino acid proline 301 (p301L, p301S) are sufficient to cause pure tauopathy in patients with frontal temporal dementia, multiple lines of transgenic mice with these or similar rare tau mutations were created as models of tauopathy and succeed in displaying synapse and neuron loss and related cognitive deficits as well as an associated neuroinflammatory response in microglia and astrocytes.

[0165] For example, PS19 (p301S) tau transgenic mice have been used to show that this model features complement activation and, more importantly, that reducing complement activation by blocking initiating C1q or downsteam complement C3 can dramatically slow synapse and neuron loss and cognitive deficits. This is consistent with reduced neurodegeneration and therapeutic benefits from eliminating microglia or blocking complement receptor pathways at C3aR or C5aR in tauopathy model mice, including PS19. Collectively, these data imply that complement pathways contribute to synapse and neuron loss and are activated by tau mutation induced tauopathy.

[0166] The majority of tauopathy patients including almost all burdened with AD or CTE, don’t have tau causal tau mutations and the tauopathy is initiated by other factors an forms with wild-type (WT) tau aggregates initiated or accelerated by other risk factors. With CTE and someATTORNEY DOCKET NO.37759.0498P1 cases of AD, traumatic brain injury (TBI) has been identified as a critical initiating factor and acute TBI-induced complement activation has been identified as a persistent critical factor in the maintenance chronic neuroinflammation for up to 2 years after TBI in mice. Therefore, in this study, the rT1 tau transgenic model was used, which overexpresses a CamKII doxycycline- regulated wild type human tau transgene to test the impact of repeat mild TBI on acute (1 to 7 days post TBI) initiation of tau aggregate formation and persistent (3 months post-TBI neuroinflammation, neurodegeneration, tauopathy, complement activation, cognitive deficits and blood biomarkers reflecting tauopathy. F. Example 6 1. Specific Aims

[0167] Aim 1. To determine whether Spike 1 (S1) accelerates Alzheimer's disease (AD) progression and whether AD modulates S1 effects on AD and cerebrovascular disease (CBVD) pathology. Rationale: Acute SARS-CoV2 (SC) infection can lead to Long Covid (LC) 3 months post infection, often associated with chronic neurological, conditions, from mild symptoms to 'brain fog' independent of age, particularly in the elderly. LC appears to accelerate progression of early AD or conversion to AD in patients with mild cognitive impairment. Clinical evidence and current models of LC have led to great strides in understanding LC, but there are many knowledge gaps including the interaction with comorbidities and genetic risk factors, which limits development of treatments and preventive strategies. Some SC strains infect rats, possibly because rat and human ACE2, the S1 receptor, share an S1 hydrogen bonding residue 24 of the ACE2 binding region. Data shows that subcutaneous injection of S1 spike (a candidate pathology mediator in LC) into AD Tg rats exacerbated complement activation pathways implicated in LC and AD as well as amyloid and tau pathology. Rats treated with recombinant S1 peptide or the rat transmissible B.1.351 can show increased ptau and vascular pathology and leukocyte infiltration interacting with ApoE, vascular amyloid, SPP1, complement-driven neuro- inflammation. Design: At 11 mos. age, transgenic rats positive (FAD) or negative for AD with targeted replacement of human variant of Apolipoprotein E (ApoE3) are treated with recombinant S1 (of B.1.351 variant) or vehicle and after behavioral testing, euthanized at 60 days post injection for tissue analysis. Two experiments were performed. One used 40 females with n=10 were 40 [n=10 x 2 treatments (vehicle or S1 spike) x 4 models (WKY rats positive or negative for FAD and SHRSP rats positive or negative for FAD). The other used n=5 rats for each of the same groups with and without human ApoE3 or ApoE4 knocking genes replacing rat ApoE and comparing veh or S1 spike injection. Primary OutcomesL Executive function (set shift) deficits, leukocyte infiltration, vascular and WM damage, BBB leakage,ATTORNEY DOCKET NO.37759.0498P1 microhemorrhages, demyelination, amyloid and tau pathology and synapse loss and CSF, CNS and plasma extracellular vesicle surrogate markers (of CNS complement activation) confirmed in plasma of LC patients with brain fog.

[0168] Aim 2: To determine if Apolipoprotein E4 (ApoE4), a major genetic risk factor for LC, exacerbates the CNS response to S1 in normotensive FAD rats during AD progression. Rationale: Increasing evidence implicates E4 as a risk fact for Neuro LC. Transgenic AD models have been developed with targeted replacement of ApoE4. Compared to Aim 1 ApoE3, ApoE4 in rats can increase cerebrovascular pathology including CAA, BBB damage, neuroinflammation, demyelination and tau pathology in S1 treated E4 rats. H2. S1, E4 and FAD can interact to increase vascular pathology. Design: Human E4 rats (with and without FAD) can be treated with S1 or a vehicle and compared to E3 rats in Aim 1. A total of 80 rats are needed (n=10 x 2 sexes x 2 transgene (E4FADWKY and E4WKY). The paradigm and outcomes are as in Aim 1. S1 can amplify E4 amyloid and vascular pathology.

[0169] Aim 3: To examine if hypertension, a major comorbidity for LC, exacerbates the S1 response in AD rats. Rationale: Hypertension is one of the largest risk factors for severe LC. However, which pathways are influenced and how it affects neuropathology are unknown. S1 and hypertension increase leukocyte infiltration, cerebrovascular disease and the severity of tauopathy Design: E4FADSHRSP and E4FADWKY rats are treated with and without S1). n=10 x 2 sex x 2 treatment (S1 or Vehicle) x 2 models (hyper-or normo-tensive) = 80 rats. Paradigm and outcomes parallel Aim 2.

[0170] Aim 4: To evaluate the efficacy of small molecules that antagonize either C5a receptor C5AR1 or upstream complement pathways. Rationale: C5aR1 targeting is safe and works in AD models. Multiple small trials for LC show efficacy of curcumin, a small molecule in turmeric, as an adjunct therapy reduces severity in acute SARS-CoV2. It reduces amyloid, C1q an early component in the pathway, NFKB and oxidative triggers of complement. Design: E4FADSHRSP +S1 with and without drugs). Outcomes are as listed in Aim 1. n=10 x 2 x 3 treatment (curcumin, C5aR1 inhibitor or vehicle) x 1 model (hypertension with FAD and huApoE4) = 60 rats. The paradigm and outcomes are as in Aims 1 and 2.

[0171] Aim 5: To determine how FAD or hypertension modulates the neuropathological response to live SARS-CoV2 in E4 vs E3 rats. Rationale: Injection of ACE2 binding S1 alone can miss many immune responses to live virus, notably more severe infection and interferon pathways. Hypothesis: Neuropathological and neuroinflammatory responses to the transmissible virus will overlap with S1 but be more pronounced particularly in hypertensive E4 rats. Design: Rats are infected intranasally with rat transmissible B.1.351 variant.76 rats are used [(16 forATTORNEY DOCKET NO.37759.0498P1 pilot studies) + (n=10 x 1 sex x 2 transgenes (E4FAD or E4 or E3FAD) x 2 blood pressure (normotensive or hypertensive)]. As in Aims 2-4, rats are euthanized 45 DPI and pathology and biomarkers assessed. 2. Background, Significance and Rationale

[0172] Long Covid (LC), Alzheimer (AD) and Complement Hyperactivation. Comorbidities like ApoE isoform, hypertension and Alzheimer's affect Long Covid (LC) risk and severity and share common mechanisms involving complement activation. Modern genetics of AD risk implicates ApoE4 (E4) and innate immune genes (TREM2, CD33, CR1, Clu, CFH, C1s, etc.) that control the neuroinflammatory response to Abeta and tau, including complement pathways that are implicated in synapse loss, neurodegeneration, neurovascular damage, leukocyte invasion and BBB leakage or bleeds. C3 antagonism protects from synapse loss and cognitive deficits. E4 is a risk factor for Long Covid (LC), which increases risk for all cause dementia including Alzheimer Disease (AD), accelerating demyelination and pre-existing AD. Neuropathology of LC includes microglial activation, vascular and BBB damage with fibrin leakage and macrophage invasion. Widespread microvascular and associated white matter (WM) damage and demyelination is common in LC with symptoms at 7 months follow-up after hospitalization consistent with earlier autopsy series of severe COVID cases. Persistent post- COVID neuropsychiatric symptoms in a study of 1.28 million patients included an increased risk of cognitive deficits, dementia, psychotic disorders, and epilepsy or seizures lasting up to 2 years after diagnosis. However, risks for mood and anxiety disorders subsided early and showed no excess over the 2-year follow-up. Microglial activation is the most consistent post-Covid neuropathological finding arguing for biomarkers that reflect neurotoxic inflammation and treatments to limit harmful aspects of neuroinflammation. Covid-induced inflammation and complement can interact with AD and ApoE4 at multiple levels with evidence for increased Abeta, ptau, inflammation, vascular injury, BBB leakage and demyelination. This study clarifies the interaction of risk factors and develops a model that more accurately reflects key mechanisms in LC.

[0173] While Abeta directly binds C1q to activate complement pathways, AD pathology more generally activates all three classical, alternative and lectin pathways that converge at the C3 level to promote synapse loss. With more vascular involvement from Covid or CAA, the endothelial complement attack directs BBB damage and leukocyte invasion. ApoE4 is known to increase vascular involvement and directly modulate the complement pathway increasing inflammation. Acutely, S1 Covid spike protein targets endothelia and activates C3 via the alternative pathway, reportedly by interfering with both inhibitor CFH and the lectin pathways.ATTORNEY DOCKET NO.37759.0498P1 Continuous endothelial involvement can be an underlying mechanism in the LC syndrome. AD and Covid converge on persistent complement effector pathways. Acute Covid infection results in very complex direct and indirect immune responses in many organs that mostly recover. In cases of “Long Covid” (LC), with symptoms from any system, serum proteomics of 6,500 proteins and machine learning has implicated proteins reflecting persistent complement activation pathways down to the C5 level as the best biomarker signature with endothelial cells as the common tissue target. Covid targets endothelial cells (EC), initially most prominent in lung where it causes vascular inflammation triggering EC activation increasing expression of EC adhesion molecules (VCAM1, ICAM1, SELP) and the complement C3 activation pathway that strongly overlaps increased platelet activation, adhesion and stroke pathogenesis including hypertensive arterial damage that facilitates platelet adhesion. Persistently elevated plasma C3 and a downstream C5b-9 complement pathway attack on endothelial cells is central to Long Covid including CNS (Neuro-Covid). It is implicated in complement activation as downstream from monocyte and platelet adhesion and “thromboinflammation”, as well as upstream (thrombo-inflammation is mediated by complement C5b-9 attack). Complement activation in endothelial-derived extracellular vesicles appeared diagnostic for VCID diagnosed using MRI for white matter. Relevance to AD pathogenesis has already been confirmed by unbiased comparative Covid and AD transcriptomic analysis using large data sets where algorithms identified the target complement C5a receptor C5aR1 as a key driver. Thus, the associations of Complement, Covid and AD are confirmed. Additional genetic analysis of not only AD, but Covid severity risk has also identified multiple immune genes that support a causal role for complement pathway activation. Finally, because unresolved complement activation is a required key driver in neuroinflammation that persists for years after traumatic brain injury (TBI) and TBI significantly increases risk of AD or rarer CTE, it is also plausible with Covid. Thus, complement is a very strong candidate driver of Covid’s persistent neurovascular and neuroinflammatory pathways relevant to synapse loss, cognitive decline and AD, but tools for monitoring and targeting these pathways in CNS remain under-developed.

[0174] The known and unknown from human LC and mouse LC models. Mouse-adapted Covid infection in old C57 mice elevates an immune gene hub implicated in AD, with complement C4 and C5aR1. Interactions of AD and Covid have been reviewed. There is evidence for Covid-induced increases in about 30% of LC cases with neurological manifestations (fatigue, headache, loss of smell and taste, delirium, insomnia, brain inflammation, stroke, brain hemorrhage, brain fog and other cognitive impairment). Morbidity and mortality rates for COVID-19 were increased in subjects with AD, and interactions involvedATTORNEY DOCKET NO.37759.0498P1 AD pathology, increased ACE-2, elevated proinflammatory molecules, advanced age, BBB disruption and the presence of APOE4. Acute viral infection of brain is present in some severe Covid cases, human organoid cultures and some mouse models, but little or no CNS virus is found at later LC stagesApoE4 is known to increase risk of neurovascular amyloid and BBB damage but also associated with increased Covid severity, risk of micro-hemorrhage and Long Covid neuro symptoms. ApoE4 association with Covid severity was experimentally confirmed. Increased mortality and more severe lung disease and neutrophil expansion in ApoE4 vs ApoE3 mice infected with mouse-adapted Covid was seen, but the brain was not examined. Thus, ApoE4 clearly interacts with Covid-19 in both mice and humans, but the relationships between Covid, ApoE4 and AD pathogenesis remain unexamined. Most cases of Long Covid with neurological symptoms exhibit neuroinflammation, early leukocyte invasion and BBB breakdown, but do not show any remaining significant viral infection of brain. The acute leukocyte invasion normally subsides, but persistent activated microglial nodules and perivascular macrophages remain, even in the absence of AD pathology. This is consistent with data showing S1 Spike leads to persistent increased microglial activation and active CNS complement C3 even in the absence of AD pathology.

[0175] Advantages and limitations of rat versus mouse models. Mouse models have clear advantages in that they are cheaper, better studied, produced in many facilities and offer a wider variety of tools as well as transgenes and knockouts to cross in and explore mechanisms. Current mouse models for AD and Long Covid (LC) show induction of neuroinflammation, but so far, LC models generally lack prevalent and important human pre-existing conditions and resultant comorbidities known to impact CNS diseases and rate of progression. Mice are naturally resistant to human-adapted Covid because the murine sequence of ACE2, the Covid S1 spike protein receptor used by virus to internalize and infect, has low affinity binding to spike binding in mice. Therefore, researchers developed K19-ACE2 mice expressing high levels of human ACE2 to cause neuroinflammation and BBB damage, but artificially high ACE2 expression in brain resulted in robust CNS viral infection and often lethal inflammation. This fails to model LC which features negligible or no CNS viral load or LC in AD as amyloid induces endogenous ACE2 upregulation. With a newer approach using knocked in human ACE2, human strains infect and the pathogenic delta results in milder Covid, vascular damage and synaptic deficits, a more useful model for acute NeuroCovid. Several groups have similar results but human ACE2 is no longer essential as new engineered mouse ACE2 adapted strains can now infect and cause neuroinflammation in mice as models for LC / Neuro-Covid. That said, while AD and LC mouse models have neuroinflammation overlapping human AD and LC, lab mice have lowATTORNEY DOCKET NO.37759.0498P1 complement levels and mouse Abeta models display only limited complement activation, synapse and neuron loss compared with human AD. This may reflect significant differences in immune response hubs identified in mouse vs human CNS “omics” studies at the single cell level warranting human iPSC-derived astrocyte and microglia and human brain in a dish studies. Or it may reflect a failure of mouse Abeta models to induce tangles without human tau, typically human mutant tau with mutations unrelated to AD.

[0176] Surprisingly, common mouse models have very low complement levels in blood. Further, an attempt to model neuromyelitis in mice, a classic complement-mediated disease found “mouse serum is a strong inhibitor of AQP4-IgG-dependent CDC produced by complement from multiple species, including human” and “mouse serum contains inhibitor(s) of the classical complement pathway”, possibly elevated C1-inhibitor. In contrast to rats, “mouse models require direct injection of human complement together with AQP4-IgG into brain”. This review cites other differences and concludes that mice have limited utility as models involving the classic complement pathway originating with C1q. These concerns are most relevant in the context of BBB leakage or hemorrhage and complement factor leakage into the brain, which is evident with ApoE4, Covid, CAA and / or chronic hypertension and SVD. Consider the case of C5 which shows very low mRNA expression in brain compared to liver secreted C5 (~1,000x higher C5 levels protein in blood than CSF). With hemorrhage and C5 influx, CSF C5a levels rise ~1,400 fold. More modest C5a CSF increases are found with meningitis accompanied by C5a>C5aR1 mediated leukocyte (including neutrophil) invasion which can be targeted by C5a receptor antagonist. Neutrophil invasion is a common feature in acute Covid and AD that is directly implicated in ApoE4 females.Therefore, CSF C5a was evaluated and it was predicted more focal C5a>C5aR1 pathway activation that compounds vascular damage due to ApoE4, CAA, hypertension and Covid interactions. Vascular adhesion and invasion of activated neutrophils cause myeloperoxidase -induced oxidative damage to endothelial cells amplifying vascular damage. Unlike mice, rats and humans have more comparable serum complement levels and share similar serum classical complement activation and related neutrophil-mediated toxicity in vitro. Persistent microglial activation is an LC hallmark, but unlike mice, rats share similar microglial complement C2 and C3 expression. This matters because complement activation converging on C3, including the classical C1q initiated pathway is clearly implicated in AD, Vascular dementia / VCID and tauopathies.

[0177] Development of a Rat Model with genetic and environmental risk factors for LC (FIG. 12). The Mixed Dementia (MxD) rat model was developed using severely spontaneously hypertensive rats (SHRSP) strains with and without the Familial Alzheimer’s Disease [(FAD)ATTORNEY DOCKET NO.37759.0498P1 that express presenilin (PS1) and amyloid precursor protein (APP) mutations]. Normotensive rats (WKY-the strain upon which SHRSP was developed) were also created. Coexistence of hypertension and FAD, each risk factors for both Long Covid (LC) and acute COVID severity, exacerbates cerebrovascular disease (CBVD) pathology, neuroinflammation, amyloid angiopathy (APP) and tau hyperphosphorylation. Given that ApoE4 (an AD risk factor) is linked to Long COVID risk or acute COVID severity, E4- (and E3-targeted replacement rats for controls) was bred, yielding the following strains (E3FADWKY, E4FADWKY, E3FADSHRSP and E4FADSHRSP). Heterozygous FAD are backcrossed to Tg- rats yielding control littermates, while retaining 100% of respective strain background. Compared to E3, E4 rats display significantly more tau pathology (particularly in the amygdala).

[0178] Up to 80% of AD cases have SVD or other contributing vascular pathology that is believed to be significant in ~33% of cases while ~50% of AD cases have ApoE4 risk. So, the models can reflect AD with both genetic and vascular disease risks expected to interact with Covid induced endothelial pathology and chronic inflammation in LC. While complement activation is a clear feature of AD and related neurodegenerative diseases as well as Long Covid with Neuro-Covid, the CNS levels are difficult to monitor requiring complicated purification of CNS derived plasma EV or CSF, which limits CNS complement pathways as a therapeutic or preventive target.

[0179] Validation of clinical relevance of animal models. This proposal interweaves human plasma samples from LC and MarkVCID cohorts to understand aspects of the model that overlap with the clinical disease. Covid-19 activates endothelial cell (EC) complement and with 12-month follow-up proteomics, complement C3 and P-selectin (SELP) remain significantly elevated with AUC~1 discriminating LC / PASC) where 36% had lingering neurological symptoms. LC-increased SELP is produced from both activated platelets and endothelial cells, but origins cannot be distinguished with current approaches so it is proposed to use endothelial EV SELP as an exploratory assay. Proteomics revealed significant biomarker candidates; but lacked sensitivity to identify organ-specific biomarkers linked to tissue specific pathology and symptoms with high sensitivity and selectivity. Aim 2 will obtain data from Ontario Cohort plasma samples with 40 nucleocapsid antibody confirmed LC patients, 20 with neuro symptoms including brain fog and 20 without neuro symptoms. A larger blinded confirmation cohort has been drawn from a 1000+ LC patient cohort with extensive multiomic workup completed. LC data show elevated plasma ptau, including ptau217 in their larger LC cohort. Data supporting the plasma EV assays in LC and AD is presented. LC has a major vascular component. Well- characterized AD, MCI, VCID and NC plasma samples are provided.ATTORNEY DOCKET NO.37759.0498P1

[0180] Rationale to use both peripheral spike protein injection (FIGS.13-16) and. viral infection (FIG.17). As reviewed below, spike protein directly and indirectly activates complement in vitro and in vivo. Direct injection into the brain activates microglia via TLR4, but direct injection limits the many proven vascular and regional effects including brain uptake via ACE2 after peripheral i.v. or s.c. injection. Using recombinant S1 spike subunits permits work in BLS2 labs and to access behavior and eventually imaging facilities. Therefore, in this proposal, both S1 spike injection (Aims 1-4) and viral infection (Aim 5) are used to determine how comorbidities and genetic factors modulate LC-like symptoms and pathology. Specifically, these studies explore how hypertension or ApoE isoform can synergize with Covid Spike protein (or live virus) to exacerbate BBB damage, dysregulation of the complement cascade and tauopathy (evaluated by immunohistochemistry, biochemistry, CSF and plasma markers for CNS complement activation, Abeta and tauopathy. How these factors impact cognitive deficits, Alzheimer's neuropathology, synapse loss, demyelination and cerebrovascular CAA and SVD pathology relevant to bleeds and Vascular Cognitive Impairment are evaluated. As discussed above, this is done in rats because unlike mice, rats and humans share key similarities in Abeta induced WT tauopathy and neuron loss, complement expression profiles and natural vulnerability to Covid-19 while their large size facilitates candidate surrogate biomarker tests. Persistent sequelae of spike protein exposure is modeled on the prodromal stages of Alzheimer's and VCID that include increased Abeta, ptau, chronic neuroinflammation, cerebrovascular dysfunction and leukocyte invasion.

[0181] In summary, rat models are used because, compared with mice, rats have high levels of microglial-driven complement activation like humans consistent with more neuron loss and real non-FTD mutant tauopathy with the human mutant APP / PS1 transgenes expressed in rats but not mice. This is confirmed using imaging with Abeta and tau aggregate PET to show tauopathy following amyloid as in humans. The Abeta-driven wild type tauopathy pathway matters as the data shows peripheral S1 Covid spike injection increases Abeta and downstream ptau217 in FAD transgene carriers. This can model Covid- induced reductions in plasma Abeta 42 and 42 / 40 ratio and increases in Abeta-responsive ptau181 shown in Chinese AD patients, extending a larger NYC study that found increased serum ptau181 post-Covid. CSF profiling of Covid patients with neurologic problems also demonstrated lower CSF Abeta 42 / 40, an index of Abeta deposition along with altered processing of the beta Amyloid Precursor protein (APP) supporting a possible increase in Abeta generation by it rate limiting enzyme, BACE1. Rats have much larger brain, CSF and plasma volumes for biomarker assessment in vivo as well as higher level executive function permitting biomarker studies of progressive decline that parallelATTORNEY DOCKET NO.37759.0498P1 clinical studies. Rats also have better homology with human ACE2 than mice which explains why sewer rats were recently shown to carry Covid; rats infect in the lab, notably with the delta variant S1 spike with 2x affinity for ACE2 vs. the Wuhan Hu.1 strain, the two spike variants used in early studies. As discussed below, the B.1.351 variant human strain shares high affinity binding with human and rat ACE2 permitting infection and transmission in rats, but not mice.

[0182] Increased Abeta production or reduced clearance could cause the accelerated amyloid deposition. The analysis of LC plasma proteomics pointed to a pattern of chronic vascular inflammation, hypoxia and HIF1alpha activation, a transcription factor (TF) known to upregulate BACE1, a rate limiting enzyme in Abeta production, along with inflammation. Transcriptomic analysis of Covid and AD identified upregulated STAT3, an additional TF BACE1 inducer that is elevated with both AD and Covid-19. Further, increased chronic complement C3a signaling through C3aR1 in AD inhibits microglial Abeta clearance and antagonizing C3aR1 effectively reduced Abeta accumulation by enhancing microglial phagocytic clearance in vitro and in vivo. Chronic C3a >C3aR1 signaling is also implicated in increasing STAT3 in AD and in tauopathy model mice, where targeting C3aR1 reduced the level of STAT3 and was highly protective at every level. Thus, Covid induction of chronically increased CNS C3 activation, inhibition of Abeta clearance and TFs for BACE1, can form a feedback loop interacting by amplifying pre-existing Abeta and ptau both upstream and downstream of C3 that is modified by chronic hypertension and ApoE4. PCR panels can evaluate upregulation of HIF1a, STAT3 and BACE1 expression.

[0183] For this purpose, new rat AD / VCID models have been developed with human ApoE3 or ApoE4 knocked in along with heterogenous (+ / -) co-integrated mutant APP / PS1 transgenes. The transgenes are from the Cohen F344 AD rat model with plaques, tangles and neuron loss that progress from amyloid PET positive to tau PET positive with significant neuroinflammation. The rats are bred with and without a spontaneously hypertensive WKY background providing one of the best models of small vessel disease / VCID with immune systems closer to humans. An initial phenotype of the rat lines describes their SVD in the context of Abeta and tau as mixed dementia pathology.

[0184] Unlike mice, rats have been naturally infected with human Covid virus and demonstrated to both infect and transmit human Covid virus in the lab. But this is not widely appreciated, and this will be the first use of a rat model used for Long Covid. The utility of the innovative model is already demonstrated by peripheral S1 spike protein injection resulting in dramatic increases in AD pathology. The consequences of Covid virus injection may be even more robust.ATTORNEY DOCKET NO.37759.0498P1

[0185] This is the first animal model for Covid interactions with hypertension. The first animal model for Covid interactions with human ApoE isoforms and AD pathology.

[0186] The development of simple high throughput assays for CNS cell source-dependent complement activation validated in an animal model. These assays are pathway- and cell- specific plasma extracellular vesicle (EV) assays to track dysregulation of CNS complement non-invasively. The simplicity of the new assays which do not require EV purification, makes them scalable. De-identified human MCI, AD, VCID and LC (with and without brain fog) and sex and age-matched healthy control plasma samples are used to validate clinical relevance of new EV biomarker plasma assays for vascular inflammation and CNS complement pathways to LC Neuro-Covid symptoms. They include the innovative use of the natural CR2 receptor for active C3.

[0187] Curcumin had multiple trials mitigating acute Covid and it reduced complement activation in the curcumin MCI trial. This is the first test in a Long Covid model and validate translatable new surrogate biomarkers.

[0188] This study describes the first rodent model use of CSF biomarkers for complement activation in response to S1 spike or Covid.

[0189] This study provides the validation of endpoints responsive to peripheral spike protein injection in models that can be exported to widely available BSL2 facilities.

[0190] These studies describe the first model for LC / AD using a natural human Covid strain which also addresses rat infection with human adapted virus as potential Covid host, ApoE4 risk for viral infection and severity and public health threat.

[0191] These studies address Covid elevated SPP1, a putative driver of border macrophage / microglial mediated synaptic loss in AD amyloid models that is known to interact with aging, ApoE4 and gender effects. 3. Results

[0192] The data show that peripheral Covid spike protein injection increases amyloid, ptau and CNS and vascular complement activation that can be monitored in blood using new assays. This brief rationale guides the overall perspective: Covid induction of Abeta, ptau and persistent CNS complement activation can interact with and amplify pre-existing pathology and injury responsible for cerebrovascular damage, neutrophil, lymphocyte and monocyte / macrophage invasion, BBB leakage, CAA-associated bleeds and Abeta- or tau-initiated synapse loss, all known to be influenced by ApoE4 and hypertension risk. This proposal seeks to model human risk for Covid interactions with ApoE4, AD and related cerebrovascular pathology. It can enable the development of simple high throughput plasma EV assays for overlapping AD and COVID-ATTORNEY DOCKET NO.37759.0498P1 relevant CNS complement and related inflammatory pathways with surrogate biomarker potential demonstrated by response to treatments already available for clinical trials for both treatment and prevention.

[0193] The data support the feasibility and strength of the proposed LC model. As described herein, six weeks following a single subcutaneous injection of S1 spike protein Abeta and tau accumulation dramatically increase including in the models with human ApoE. This includes ApoE4-dependent cerebrovascular amyloid (CAA) and downstream ptau217 by ICC and brain extract ELISA. Ptau217 has emerged as the single best plasma biomarker for CNS amyloid pathology in AD, so tauopathy evaluation can use a panel. S1 spike increased activated C3 on plasma astrocyte EV, CSF C3a, C3 activation in frontal cortex at the level of C3b’s stable product C3d by WB analysis which localized to peri-vascular C3d by ICC, consistent with increased leukocyte adhesion.

[0194] New high throughput cell-specific, pathway-specific plasma EV assays for CNS inflammation "ExPath" (FIGS.13-15, 17-19). The ability to characterize, measure, monitor and treat tissue-specific neuroinflammation mostly limited to GFAP. The use of “brain-derived” cell origin-specific plasma EVs was pioneered to inform on dysregulated CNS pathways. While the neuron-derived EV assays using L1CAM have been challenged, the Astrocyte-derived EV identified by biotinylated GLAST antibody have consistently identified increased CNS complement activation in MCI, AD, TBI and multiple cohorts with cognitive deficit and purified endothelial EV show elevated VCAM1 and vWF that is not diagnostic for white matter hyperintensities WMH. In contrast, elevated endothelial-derived EV complement pathway markers including C3b and C5b-9 were diagnostic for subjects with WMH and correlated with cognitive and volumetric deficits but ultimately their demanding methods evaluating endothelial EV C3b failed in multi-site consortium for VCID / vascular dementia VCID (MARKVCID) even though endothelial C3b appeared very accurate in distinguishing advanced CeSVD from clean controls at some sites. This has led to ExPath plasma EV assays using proven cell source tagged biomarkers to detect vascular (endothelial / CD144) and astrocyte (astrocyte / Glast1) extracellular vesicles and their levels of biomarkers for complement (C3, active complement C3b), C5b-9) or vascular (SELP, ITGB2, ICAM1 or VCAM1). ExPath plasma EV analysis is performed on the animal studies and validated in human samples using plasma from LC, Neuro LC, and controls and MCI, AD and VCID samples.

[0195] ExPath Methodology: A high-throughput measurement of plasma organ- and pathway- specific extracellular vesicle surface biomarkers was developed without the need to prepare exosomes. Because exosome preparation is labor intensive and difficult to reproduce, a highATTORNEY DOCKET NO.37759.0498P1 throughput method that does not require EV isolation, called ExPath, was developed. FIG.13 compares conventional and ExPath methods.

[0196] To test these ExPath plasma assays for CNS complement activation and their treatment responses in humans, FIG.14A confirms increased C3 activation in AD vs Ctrl NC plasma, while B and C demonstrate surrogate CNS active C3 assays responses with one proposed Covid / AD animal model treatment in humans with MCI

[0197] ExPath assays also demonstrate active C3 in astrocyte and endothelial EVs with LC (FIG.15). While FIG.15 demonstrates increased CNS and vascular complement activation in LC NeuroCovid patients at least 6 months post infection from the first cohort where most are too young to have significant pre-existing AD pathology. But samples from the 1,000+ LC cohort have a wider age range and evidence of lower 42 / 40 and increased ptau with LC- where validation of the plasma ExPath assays can be used in rat models.

[0198] Even though endothelial complement activation is not brain specific, the two assay results were significantly correlated suggesting more widespread persistent endothelial inflammation and CNS involvement extending to brain in Neuro-Covid patients from collaborator Fraser’s Ontario cohort. Active C3 was normalized to total C3 in these figures. Because of uneven variance, FIG.15A used square root, but significant NC increases are still obtained if outliers are removed. Mean age is 43 so these LC patients are unlikely to have AD pathology, but still show evidence of increased CNS and vascular activated C3. i. Hypertensive AD rat with knockin of human ApoE4 (or control human ApoE3 (also see Background and Significance).

[0199] The MxD rat was developed using the Cohen Tg AD Rat (PS1 / APP), with each FAD cross to pure spontaneously hypertensive -stroke prone (SHRSP) background (and normotensive background WKY from which the SHRSP were derived). The SHRSPFAD rats have extensive amyloid and increased vascular pathology including SVD and CAA as well as greater mitochondrial deficits, WM damage and tauopathy compared with WKYFAD controls. They can also have microinfarcts (FIG.16). It is widely agreed that SHRSP rats effectively model hypertension-associated human cerebral small vessel disease, a known cause of VCID, neuroinflammation and white matter damage that also accelerates progression as a co-morbidity with AD. Because LC also causes primary vascular damage and persistent microglial activation in NeuroCovid, interactions are predicted with hypertension and SVD in SHRSP rats.

[0200] This model was used to replace rat ApoE with ApoE3 and ApoE4. ApoE4, increases vascular amyloid, neuroinflammation and vascular / BBB damage in mouse models, and pro- inflammatory responses in human iPSC-derived glia in vitro and patient brain samples. ApoE4ATTORNEY DOCKET NO.37759.0498P1 also increases vWF in human iPSC-derived endothelial cells and ApoE4 / 4 AD patient brain EC and may interact with post-Covid risk and chronic BBB leakage in Long Neuro-Covid patients. Cre-regulated liver expression of ApoE3 vs ApoE4 in mice causes cerebrovascular pathology with higher endothelial VCAM1 while proteomic analysis of plasma identified ApoE4 increased complement proteins as a factor contributing to CNS endothelial pathology supporting increased inflammation, VCAM1 and SELP and leukocyte adhesion in primary endothelial cell cultures from ApoE4 knockin mice (Marottoli, et al., 2021). Therefore, the rat models with human ApoE3 vs ApoE4 KI permit investigation of ApoE4 effects on neurovascular inflammation and interactions with CVSD, AD amyloid and Covid spike or virus. ii. Spike Injection Protocol and Spike Effects on cell and pathway-specific plasma CNS and endothelial markers

[0201] Rat model endothelial ExPath EV biomarkers for endothelial-specific complement activation, Abeta or endothelial damage (FIG.17). Data from the ExPath assay for endothelial active C3 complement, VCAM1, Col6A and Abeta are shown in FIG.17. Using endothelial specific CD144 antigen paired with inflammation markers (active C3, or VCAM1) shows that plasma of SHRSPFAD rat exhibit high endothelial activation indexed by VCAM1 in EV (FIG. 17A) which is reduced by complement C5a receptor (C5AR1) inhibitor, PMX205 (FIG.17B), a drug that went into severe Covid trials as an adjunct and is proposed as a treatment in the rat LC / AD models. Covid S1 spike increased endothelial active C3 (FIG.17C) and endothelial COL6a1, a fibrosis marker (FIG.17D) in the EFAD+ rats. Consistent with apparent increases in total and especially vascular amyloid (CAA), S1 spike also increased endothelial EV Abeta (FIG.17E). If validated to reflect CAA, this has high impact potential as E4 CAA influences new FDA approved treatments and there is no plasma CAA biomarker. iii. Rat Model Astrocyte-specific activation of Complement using ExPath EV biomarkers.

[0202] FIG.18 shows complement activation in plasma astrocyte EVs. FIG.18A shows increased active C3 in plasma astrocyte EV in the SHRSPFAD rats relatives to other strains. FIG.18B, shows that a C5aR1 antagonist PMX205 reduces complement activation in astrocyte EVs in FADSHRSP rats, with elevated active C3 in plasma astrocyte EV without lowering normal levels in WT rats. FIG.18C shows that 6 weeks post single s.c. injection of S1 spike, active C3 in plasma astrocyte EVs was elevated in EFADSHRSP (human ApoE) rats.

[0203] Next, S1 spike effects were investigated in female FAD+ or FAD- (neg) rat models without human ApoE. As FIG.19 shows S1 spike again increased vascular and CNS active C3 in plasma assays and active complement (C5A) in CSF as well as ptau217 and C3d / intact totalATTORNEY DOCKET NO.37759.0498P1 C3 in TBS from cortex.

[0204] This in contrast to rat models with human ApoE3 or ApoE4 (FIG.20) where S1 spike increased Abeta, ptau217, C3d and C5b-9 by ICC across FAD+ treatment groups including WKYEFAD and SHRSPEFAD rats with either human ApoE3 or ApoE4 (FIG.20). Consistent with mice, all human ApoE FAD rats (EFAD) strains had less amyloid and ptau than strains with only rat ApoE (not shown), but peripheral spike injection markedly increased Abeta and ptau217 in all strains, illustrated in FIG.20. (ApoE4FAD injected with S1 spike apparently caused greater ptau217 pathology compared to E3.)

[0205] FIG.20 demonstrates examples of staining of spike-treated E4SHRFAD illustrating expected 6E10 Abeta-labeling of amyloid plaques, vascular amyloid with cellular elements and possible intraneuronal Abeta or Abeta containing APP fragments (FIG.20A). ptau 217 (FIG. 20B) labeled neurons and plaque neurites. Complement C3d showed prominent spike-related vascular and microvascular labeling in segments and cellular elements, likely endothelial cells and consistent with plasma EV data. Scattered weaker neuronal staining was also found (FIG. 20C). Prominent and highly variable C5b-9 neuronal and vascular labeling was present in all groups (FIG.20D). All of these were highly significantly increased with spike, quantified with image J (Fig.20I). 4. RESEARCH DESIGN AND METHODS

[0206] The overall approach for all aims is shown in the schematic diagram of FIG.21, using the human ApoE targeted replacement EFAD models with or without hypertension. In addition to measurement of blood pressure, further confirmation is provided by measuring mRNA levels of ATP11b, which are notably low in this polygenic hypertensive model. S1 injection or infection is performed at early AD stage, allowing assessment of long term effects during the rapid progression phase, for which preliminary data showed robust effects of a single S1 s.c. injection on many inflammatory parameters (see FIG.17 for spike injection protocol). After rats are tested for executive function rats, rats are euthanized, CSF, blood and brain collected. One hemibrain is immersion fixed in formalin, while the other is dissected into subcortical regions (thalamus and septum), hippocampus, frontal cortex. The "rest of cortex" is tissue banked as well as lungs, kidney and heart for future omics or sharing with other PI's. Rats are injected subcutaneously 250 ug / kg bw of recombinant pathogenic SARS-CoV-2 peptide (S1 Spike vs ctrl peptide) or intranasally inoculated with Sars-Cov2 at the USC BSL3 in Aim 5 which will be conducted across all years and in parallel with the spike injection studies.ATTORNEY DOCKET NO.37759.0498P1 i. Aim 1. To determine whether Spike 1 (S1) accelerates Alzheimer's disease (AD) progression and whether AD modulates S1 effects on AD and cerebrovascular disease (CBVD) pathology in normotensive rats.

[0207] Design: AD transgenic rats (FAD) with or without Alzheimer's pathology are treated with recombinant S1 (of B.1.351 variant) or vehicle at 13 months of age and evaluated 60 days later. Because Aim 1 addresses FAD effects independent of other comorbidities, the normotensive rats with targeted replacement of human variant of Apolipoprotein E (ApoE3) (which does not increase risk for AD) is used to isolate FAD effects,. As shown in the table a total of 80 rats are needed [n=10 x 2 sex x 2 treatment (vehicle or S1) x 2 models (E3WKY rats pos. or neg. for FAD)]. Primary endpoints for all aims are discussed in and in the Methods section. Analysis is by multi way ANOVA (Sex * spike * FAD) to identify independent effects of Spike and FAD and interactions of Spike effects with FAD.

[0208] Primary endpoints include Abeta, ptau, neurovascular and white matter damage, complement activation and synaptic marker loss. Spike injection has advantages in simpler mechanisms and BSL2 safety levels permitting routine use in the lab and export to other sites, more extensive behavior and future multi-model imaging studies.

[0209] Baseline bulk Frontal Ctx RNA seq GO analysis of SHRP-FAD rats without spike or E4 identified 46 viral response genes including 16 for Covid, which were upregulated relative to WKY controls (not shown). Thus these 16 genes can be measured to identify whether S1 exacerbates of SHRSP- or FAD- specific effects or interactions.

[0210] Secondary Endpoints C5aR1, C3aR1 and SPP1. As reviewed in the introduction, C5 and C5a receptor C5aR1 are up and candidate drivers in Covid and AD. C3aR1 is also upregulated by acute Sars-Cov infection and by Abeta, which upregulates hypoxia-induced factor 1 (HIF1alpha) to shift metabolism, promote lipid accumulation, reduce amyloid phagocytosis and upregulate SPP1, which is persistently elevated plasma SPP1 in severe Covid- 19 and AD. Thus components of these pathways can be assessed as secondary outcomes.

[0211] Potential Outcomes include confirmation of the data showing large Covid spike induced increases in AD pathology and CNS and vascular complement activation in new rat models and more in depth characterization of the model. Results for WB of APP, CTF, and BACE 1 and PCR of related transcription factors, and confocal colocalization of proteins in microglia will clarify mechanisms for S1 Spike effects on amyloid and inflammation. New plasma EV biomarkers can be generated for CNS and vascular complement endpoints at the level of cell sourced active C3 and possibly VCAM, C5, C3AR1, C5aR1 or SPP1. Because of the strong response in experiments, there was noo co inject wheat germ agglutinin (WGA)ATTORNEY DOCKET NO.37759.0498P1 following the method of (Erickson, et al., 2023) who reported ~10X greater brain uptake, but may be useful to improve robustness of the model. ii. Aim 2: To determine if Apolipoprotein E4 (ApoE4), a major genetic risk factor for LC, exacerbates the CNS response to S1 in normotensive FAD rats during AD progression.

[0212] Design: Human E4 rats (with and without FAD) will be treated with S1 or a vehicle and compared to E3 rats in Aim 1. A total of 80 rats are needed (n=10 x 2 sexes x 2 transgene (E4FADWKY and E4WKY). The paradigm and outcomes are listed in Aim 1. Statistical analysis can answer questions that are important to understand the interaction between FAD and ApoE genotype in response to S1 protein. This aim focuses on ApoE4, in dependent of hypertension. Added endpoints include 3 microglial genes sensitive to E4 (e.g. SPP1). Data from E3 groups in Aim 1 are included in the analysis of E4 effects and Spike *ApoE * FAD interactions.

[0213] Potential Outcomes: Specifically, the primary outcomes can help answer the following three questions: (a) compared to E3, does E4 differentially impact S1 responses in normotensive FAD negative rats, (b) or in normotensive FAD positive rats, and (c) the relative magnitude of response to spike protein comparing E4 vs E3 in Aim 1 outcomes. Outcomes can detail ApoE4’s interaction with NeuroCovid / AD. iii. Aim 3: To examine if hypertension, a major comorbidity for LC, exacerbates the S1 response in AD rats

[0214] Design: E4FADSHRSP and E4FADWKY are treated with and without S1). n=10 x 2 sex x 2 treatment (S1 or Vehicle) x 2 models (hyper-or normo-tensive) = 80 rats. Aim 3 comparisons include the normotensive groups from Aim 2. Multiway ANOVA (hypertension * FAD * spike) is used to assess outcomes (and MANOVA for similar variables) iv. Aim 4: To evaluate the efficacy of small molecules (such as those in FIG.22) that antagonize steps in the complement cascade.

[0215] Design: E4FADSHRSP +S1 with and without curcumin. Outcomes are as listed in Aim 1. n=10 x 2 x 3 treatment (curcumin, C5aR1 inhibitor or vehicle) x 1 model (hypertension with FAD and huApoE4) = 60 rats. The paradigm and outcomes are listed in Aim 1. Note Aim 4 multiway ANOVA analysis (drug * spike * sex) uses hypertensive no spike controls E4FAD SHRSHP from Aim 3 groups for control. Statistical analysis will answer questions that are important to understand drug efficacy in the most severe model. Specifically, the primary outcomes are to answers the following question: What are the relative efficacies of curcumin and C5aR1.ATTORNEY DOCKET NO.37759.0498P1

[0216] Potential Outcomes. Efficacy of upstream (curcumin) and specific downstream (C5aR1) targeting for control of multiple specific Covid and AD-relevant endpoints can give many possibilities. v. Aim 5: To determine how FAD or hypertension modulates the neuropathological response to live SARS-CoV2 in E4 vs E3 rats.

[0217] Design: Rats are infected intranasally with rat transmissible B.1.351 variant.76 rats are needed [(16 for pilot studies) + (n=10 x 1 sex x 2 transgenes (E4FAD or E4 or E3) x 2 blood pressure (normotensive or hypertensive)]. As in Aims 2-4, rats are euthanized 60 days later and neuropathology and biomarkers assessed.

[0218] Aim 5a SHR rat and Aim 5b EFADSHRSP rats are performed to optimize the infection paradigm that will be used in the main experiment (aim 5c). The design is as follows: First the virus is delivered intranasally using ~50-75 uL / nare. The viral dose is 1x105PFU, and dose will be adjusted to ensure infectivity at both of these time points. In both pilots there are two groups: one euthanized at Day 3 post infection and the other euthanized at Day 14 post infection. Outcomes for the pilots include ensure infectivity in lungs and brains as well as weight loss and behavioral symptoms of sickness (hunching, loss of appetite) can be assessed.

[0219] Aim 5c: Main Experiment: Please note that the non-infected controls are from Aims 1. Females are chosen because of known ApoE4 * Female interaction and limitations in budget and BSL3 space in the total number of rats that can be treated over the five-year period. The main goal is to determine the interaction between hypertension and FAD in hypertensive rat in terms of (a) relative effects among infected animals and (b) relative magnitude of each group normalized to non-infected animals within strain. (c) Finally, FAD * blood pressure interactions are evaluated in the E4 strains.

[0220] Potential Outcomes. A direct comparison of S1 injection versus infection in the same model is critical for clarifying similarities and differences. Viral infection may be higher and more severe with E4 modeling humans. PCR is used to define inflammatory responses including interferon expected with acute Covid virus based on mouse and hamster models and confirmed in human organoids with increased microglial C3. Although, IFN response genes are acutely increased in infected CNS, there is little evidence for their persistent elevation in the absence of ApoE4. Therefore, a more robust virus effect involving IFN with E4FAD in rats is expected and one E4FAD group was added for ApoE effects within AD Tg. 5. Other Methods

[0221] Model and Dosing. The COVID model and doses of S1 are described in FIG.17 and virus protocol in Aim 5.ATTORNEY DOCKET NO.37759.0498P1

[0222] Behavior. AD and VCID both impact executive function and flexibility (Trail Making Test B). To assess this in rodents, Attentional Set shifting Task (ASST) is commonly used. Rats are required to dig for rewards associated with 2 dimensions (compound discrimination (CD) of type of odor vs type of digging media) to determine which is associated with the reward. Tests include extradimensional (ED, when dimension associated with the reward is changed). Reversals (changing rule within dimension) are assessed requiring forgetting and relearning. These tests reveal executive function deficits associated with loss of WM integrity in SHR rats. The data show that AD-specific responses as well as Tg * spike protein interactions (Fig.22).

[0223] PCR-RNA from cortex can be assessed with PCR panel covering DEGs upregulated in human AD and Covid databases and bulk RNAseq from the mixed dementia rats: Type I IFN (IFITM3, IFI27), ApoE microglia (SPP1, GPNMB, Trem2, NLRP1, CD74), Complement (C1qa, C3, C3aR1 and downstream STAT3 and BACE1, T cell CD7, Neutrophil (MPO), PVM (CD163, S100A8 ), BBB (Cld9) Endothelial (Alas2, Pycard, ICAM1). Hypoxia- HIF1a. Required primers are designed using Rat Genome Database sequences and tools.

[0224] Antibodies, Western, ICC, Dyes, and ELISAs. Initial valuation is done using dyes: H&E for lacuna, cysts, petechial hemorrhages, Luxol blue for demyelination, Congo Red (CR for vessel damage as well as CR / Cresyl Violet for amyloid). AD pathology is assessed by plaque and vessel Abeta by 6E10 ICC and CR IHC. Biochemically P-tau (AT8, pT217tau and ptau 217 ELISA from TBS) and tau oligomer T22 (vessels, neurons, gift R Kayed) is used to assess tau changes in TBS and detergent-insoluble fractions. Abeta 40 and 42 these fractions are assayed by commercial ELISA. ICC and WB (as well as ExPath assays) are used to assess inflammatory pathways: complement (C3d / C3, C5b-9, C3aR1, C5aR1), Myelin loss- (MBP, MAG / PLP1), GFAP, ICC for microglia (Iba1, CD68, pilot activation state Trem2, SPP1, GPNMB and perivascular macrophages (PVM, CD204, CD163); WB is also used to assess spike effects on Abeta (APP681-695 Cterm, sAPPbeta, BACE1); ECM remodeling-ICC is assessed using Decorin, Col61, ColIV;CeSVD-Col4, VEGF-C; BBB leak ICC-rat IgM, fibrinogen and Col4 (for SVD). Details for the routine euthanasia, perfusion, fixation, dissection and methods for quantitative ICC, confocal double labeling, tissue preparation, PCR and ELISAs are found in the art. CSF (See FIG.19). C3a and C5a are measured in the CSF with commercial high sensitivity ELISA kits (MyBioSource / Invitrogen).

[0225] ExPath Method. ExPath pairs cell source antibodies (biotin-anti-cell source surface biomarkers (e.g GLAST1 for astrocytes, CD144 for endothelia etc.) to tag EV for source with reagents or antibodies for pathway / pathology biomarkers (e.g. antibody to C5b-9 attack complex, C3aR1, C5aR1, VCAM1, P-Selectin, C3, recombinant CD21, the CR2 receptor forATTORNEY DOCKET NO.37759.0498P1 active complement C3) and appropriate markers for normalization on most or all EV from the source (CD81 or a second cell source marker). Briefly, platelet depleted plasma (5μl -10μl) is incubated with biotinylated antibodies to a surface marker for a specific cell source (10μl) and a surface biomarker antibody or other ligand (viz.6-HIS recombinant CD21m(CR2) ligand for active C3 products) (10μl) at 4°C overnight. This mixture is already in or transferred to an Alphaplate TM 384-plate and incubated at RT for 60min.10μl AlphaLISA Acceptor beads conjugated with secondary antibody IgG (final Conc.10μg / ml) or alternatively, 6-His reactive Acceptor beads for 6-HIS tagged ligands like CD21) is added and incubated at RT for 60min. 10μl AlphaScreen streptavidin-coated donor beads (final Conc.40μg / ml) is added and incubated at RT for 30-60min. The assay is no wash. The plate is read on an EnSpire Alpha 2300 Multilabel Plate reader using an excitation wavelength of 680 nm and emission detection set at 615 nm. Assays are amenable to adaptation for high throughput scaling using Perkin Elmer’s automated Janus and HTS workstations. Background no sample or pathway antibody / ligand controls are typically low and subtracted. The final results are normalized to markers on all EV from the cell source or related pathway markers (C3d product / total C3 precursor) or both. This controls for variation in EV plasma clearance (kidney / spleen function), reagent or plate-reader performance. For further cross-validation with existing methods, for one experiment, one can Exoquick precipitate EV from 250 ul plasma, enrich EV for GLAST1 or CD144 EV, extract and run ELISAs for complement C3b and CD81 (Goetzl, et al., 2018, Goetzl, et al., 2020),

[0226] Statistics and Power Analysis. Mixed regression models can identify interactions between hypertension, ApoE, sex, AD and interventions with ExPath, ELISA, neuropath, symptoms / behavior data. MANOVA minimizes Type II errors when interactive variables are measured. Significant group differences have been reported in multiple sample sets with n=16- 40 using purified EV and Elisa or ExPath and the sample sets. Power calculations use 80% power to detect a difference as small as 1 S.D., determined by R Project for multiway ANOVA. The degree of relationship between ExPath, biochemical and pathology indices and any association with hypertension are determined by mixed regression models. Analytes includes IHC mean intensity, particles / area, percent staining, particle size (e.g. ptau217 / tangles, number and size of microinfarcts, 6E10 / plaques, collagen-IV or CAA vessels and associated astrocytes (GFAP) and microglia (Iba1). For Set shift ASST, a multiway (e.g. transgene * comorbidities * drug) and multivariate ANOVA is used (for each task, variables are trials to criterion, percent failure and total trials.

[0227] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the method andATTORNEY DOCKET NO.37759.0498P1 compositions described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

ATTORNEY DOCKET NO.37759.0498P1 CLAIMS We claim:

1. A method of detecting or quantifying a pathology-related or pathology-protective pathway in a subject comprising a) combining a first ligand with a sample obtained from the subject comprising an extracellular particle, wherein the first ligand binds to a cell-type specific marker on the extracellular particle; b) combining a second ligand with the sample, wherein the second ligand binds to a pathology-related or pathology-protective pathway molecule on the extracellular particle; c) combining a donor bead to the sample, wherein the donor bead binds to the first ligand or second ligand; d) combining an acceptor bead to the sample, wherein the acceptor bead binds to the first ligand or second ligand, wherein the donor bead and acceptor bead do not bind to the same ligand; e) triggering the donor bead to activate the acceptor bead to produce a signal; wherein the amount, presence, or absence of a signal indicates the amount, presence, or absence of a pathological condition in the subject.

2. The method of claim 1, wherein the extracellular particle is an ectosome, exosome, bleb, cell debris, or apoptotic body.

3. The method of any one of claims 1-2, wherein the first ligand is a protein, nucleic acid, compound, or fragment thereof.

4. The method of any one of claims 1-2, wherein the first ligand comprises a label.

5. The method of claim 4, wherein the label is biotin.

6. The method of any one of claims 3-5, wherein the first ligand is an antibody.

7. The method of claim 6, wherein the antibody is a biotinylated antibody.ATTORNEY DOCKET NO.37759.0498P1 8. The method of claim 7, wherein the biotinylated antibody is a biotinylated cell type- specific antibody.

9. The method of any one of claims 1-8, wherein the second ligand is a protein, nucleic acid, compound, or fragment thereof.

10. The method of any one of claims 1-9, wherein the second ligand is a pathology- related or pathology-protective pathway molecule-specific ligand.

11. The method of claim 10, wherein the pathology-related or pathology-protective pathway molecule-specific ligand is a pathology-related or pathology-protective pathway molecule-specific antibody.

12. The method of claim 10, wherein the pathology-related or pathology-protective pathway molecule-specific antibody is an antibody that specifically binds VCAM1, ICAM1, Pecam1, complement activation marker-specific (C1q, Bb, Masp1 / 2 and / or C3 and its activation products (C3b, ic3b, C3d), C4 (C4A, C4B, C4b, C4d)), CD59, cancer stem cell (CD133, CD44), cancer invasive (MMP-14),CEA, checkpoint specific (PD-L1,GPNMB, CTLA-4, CD20 and CD52), metabolic (Glut1), EAAT2.LAT-1, MDR1, proteinopathy specific (Abeta, p-tau (181, 231), tau oligomer, PrPsc, p-129SaSyn, aSyn aggregate), PrPc.

13. The method of any one of claims 1-12, wherein the donor bead comprises a binding partner for the first ligand, wherein the binding partner binds to the first ligand.

14. The method of claim 13, wherein the binding partner for the first ligand is streptavidin, wherein the first ligand comprises biotin, and wherein the streptavidin binds to the biotin on the first ligand.

15. The method of any one of claims 1-14, wherein the sample is plasma, serum, saliva, urine, spinal fluid or synovial fluid.

16. The method of claim 15, wherein the plasma is platelet depleted.

17. The method of any one of claims 1-16, wherein the acceptor bead directly or indirectly binds to the first ligand or second ligand.ATTORNEY DOCKET NO.37759.0498P1 18. The method of claim 17, wherein the acceptor bead is conjugated to an anti-IgG antibody or fragment thereof, wherein the anti-IgG antibody or fragment thereof binds to the pathology-related or pathology-protective pathway molecule-specific antibody.

19. The method of any one of claims 1-18, wherein triggering is an excitation of the donor bead with a laser resulting in the release of a singlet oxygen, wherein the singlet oxygen excites a fluorescent signal from the acceptor bead.

20. The method of any one of claims 1-18, wherein combining the first ligand and / or the second ligand with the sample is an incubation of the first ligand and / or the second ligand with the sample for 1-24hrs.

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