ADDL Binding to Hippocampal Neurons

Abstract

Disclosed herein are methods for the quantification of ADDL binding to neuronal cells, including, but not limited to, primary cultures of hippocampal neurons. The method identifies and selects neurons based on any means capable of distinguishing neuronal cells, including, but not limited to, MAP2 immunoreactivity, which ensures that glial cells are excluded from an ADDL binding analysis; antibodies selective for neuronal cell surface receptors and/or other surface markers; reagents specific for neuronal signalling markers present intracellularly; and the like. Furthermore, ADDL binding occurs in a sub-population of 16 DIV neurons and is heterogeneous in intensity among individual cells. Also, ADDL binding can be further specified and quantified by using additional markers. Additionally, the presence or absence of ADDL binding is used to identify, characterize, analyze, assess, and/or evaluate agents (e.g., including, but not limited to, small molecules, antibodies, chemical compounds, dietary components, environmental conditions, etc.) that modulate ADDL binding. Such modulation can be positive or negative, including, but not limited to, ADDL binding inhibition, either total inhibition or partial inhibition, and the like.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the selection of ADDL positive neurons. Total number of objects in each field was identified using DAPI nuclear stain (left panel). Neurons were visualized using the dendritic marker MAP2 (middle panel). Blue circles represent selected objects positive for the neuronal marker MAP2. Orange circles represent MAP2 negative cells excluded in downstream image processing and quantification. ADDLs were visualized using the anti-oligomer specific antibody 2B4 (right panel). ADDL binding intensities in individual neurons was measured in soma (red circles) and proximal dendrites (green).

FIGS. 2A & 2B depict single cell binding intensities in ADDL treated cells. Histograms of average dendritic intensities measured by the ArrayScan® HCS Reader in cultured hippocampal neurons. The graphs show the distribution of intensities in single cells taken from 3 wells each (approximately 800 cells/well). The histograms were fitted by two Gaussian distributions. Dashed lines indicate the individual Gaussian, and the sum is shown by a solid line. Note the ADDL concentration-dependent shift to the left of the average Ringspot binding intensities. The images on the right show representative pictures of ADDL binding at the indicated concentrations (left panels) and areas of the image measured for binding intensity quantification are outlined in green (right panels). Note that incubation with the primary ADDL-specific antibody yields a single distribution, representative of non-specific binding activity.

FIG. 3 depicts selective ADDL binding to hippocampal neurons. Image shows superimposed staining of nuclei (blue), neurons (red) and ADDLs (green). Arrows indicate binding of ADDLs to a subset of MAP2 positive neurons (2) versus (3). Note that glia, represented by nuclear stain (1) do not exhibit ADDL staining.

FIG. 4 depicts FIG. 4: Co-localization of ADDL binding to neuronal cells. Double-labeling of neuronal cultures with MAP 2 antibody (A) and anti ADDL antibody (B). Co-localization (C) shows overlap of ADDL binding cells (green) and MAP 2 positive cells (red). Nuclei identified with Hoechst in blue. Identification of glial cells with GFAP (D) and double-labeling with ADDLs (E). Overlay of ADDL positive and GFAP positive cell types (F) excludes ADDL binding to GFAP positive cells.

DETAILED DESCRIPTION

Aβ (Abeta)-derived diffusible ligands (ADDLs) comprise the neurotoxic subset of Abeta 1-42 oligomers now implicated in synaptic malfunction and early stage memory loss en route to Alzheimer's disease (AD). Disruption in neuronal signaling and synaptic plasticity is caused by soluble Abeta (Ab) assemblies, rather than the fibrillar Ab deposited in plaques, suggesting a likely mode of action involving specific receptor-ligand interactions, rather than non-specific cellular damage.

Methods for the analysis of amyloid are known in the art (see e.g., U.S. Pat. No. 5,164,295; U.S. Pat. No. 5,223,482; U.S. Pat. No. 5,348,963; U.S. Pat. No. 5,547,841; U.S. Pat. No. 5,576,209; U.S. Pat. No. 5,652,092; U.S. Pat. No. 5,656,477; U.S. Pat. No. 5,693,478; U.S. Pat. No. 5,703,209; U.S. Pat. No. 5,721,106; U.S. Pat. No. 5,843,695; U.S. Pat. No. 6,001,331; U.S. Pat. No. 6,004,936; U.S. Pat. No. 6,194,163; U.S. Pat. No. 6,284,221; U.S. Pat. No. 6,294,340; U.S. Pat. No. 6,441,049; U.S. Pat. No. 6,518,011; U.S. Pat. No. 6,589,747; U.S. Pat. No. 6,600,947; U.S. Pat. No. 6,639,058; U.S. Pat. No. 6,677,299; U.S. Pat. Nos. 6,825,164; 6,949,575; and the like).

Additional methods are disclosed in U.S. Pat. No. 5,137,873; U.S. Pat. No. 5,200,339; U.S. Pat. No. 5,262,332; U.S. Pat. No. 5,434,050; U.S. Pat. No. 5,506,097; U.S. Pat. No. 5,514,653; U.S. Pat. No. 5,523,295; U.S. Pat. No. 5,538,845; U.S. Pat. No. 5,567,724; U.S. Pat. No. 5,622,981; U.S. Pat. No. 5,876,948; U.S. Pat. No. 5,958,964; U.S. Pat. No. 6,011,019; U.S. Pat. No. 6,107,050; U.S. Pat. No. 6,140,309; U.S. Pat. No. 6,210,655; U.S. Pat. No. 6,268,479; U.S. Pat. No. 6,274,119; U.S. Pat. No. 6,331,408; U.S. Pat. No. 6,413,512; U.S. Pat. No. 6,428,950; U.S. Pat. No. 6,555,651; U.S. Pat. No. 6,579,689; U.S. Pat. No. 6,649,346; U.S. Pat. No. 6,660,530; U.S. Pat. No. 6,737,038; U.S. Pat. No. 6,815,175; U.S. Pat. No. 6,878,363; and the like.

Disclosed herein is an image-based method for quantification of ADDL binding to primary hippocampal neurons in culture using the Cellomics ArrayScan imaging platform. Dissociated neurons from E18 rat hippocampus were cultured in 96-well microtiter plates for 16 days and exposed to increasing concentrations of ADDLs for 15 minutes, followed by fixation. ADDLs were visualized via immunohistochemistry utilizing an ADDL-specific monoclonal antibody, and neurons were identified using MAP2 immunostaining. Images were acquired at 10× magnification, and average ADDL binding intensity in MAP2 positive cells was measured in the proximal dendritic compartment.

ADDLs show selective binding to a sub-population of hippocampal neurons. Binding to individual neurons is heterogeneous and most prominent at higher concentrations suggesting a differential expression of ADDL binding sites or selective binding of ADDL species to particular neurons. Detection of ADDL binding is limited at low magnification, thus reducing the number of selected neurons at lower ADDL concentrations. This image analysis method is a valuable and quantitative tool for characterizing ADDL binding to synaptic receptors and evaluating molecules that block specific ADDL binding.

EXAMPLE 1

Primary Hippocampal Neuron Cultures.

Primary hippocampal cultures were prepared from embryonic day 18 (E18) rat brains. Cells were plated on 96-well microtiter plates, coated with poly-D-Lysine (50 mg/ml) at a density of 10,000/well. Hippocampal cultures were grown in Neurobasal medium supplemented with B27, 0.5 mM glutamine, 12.5 mM glutamate and penicillin/streptomycin. At 4DIV neurons were treated with AraC (2 mM) to inhibit glia proliferation.

ADDL Labeling and Immunocytochemistry of Hippocampal Neurons.

ADDL were assembled according to Chromy et al., 2003. Live neurons (16 DIV) were incubated with increasing ADDL concentrations at 37° C. for 15 min. After washing with phosphate-buffered saline (PBS), the neurons were fixed for 15 min with 4% paraformaldehyde/4% sucrose in phosphate-buffered saline (PBS). After fixation cells were washed two times for 30 min at room temperature and incubated with primary antibodies 2B4 and MAP2 (Upstate) in buffer (2% BSA, 0.1% Triton X-100, 30 mM phosphate buffer pH 7.4) overnight at 4° C. Neurons were then washed three times in PBS for 30 min at room temperature and incubated with secondary antibody conjugated to Alexa 488 and Cy5 in buffer (2% BSA, 30 mM phosphate buffer pH7.4, 300 nM DAPI) for 2 hr at room temperature and washed three times in PBS for 30 min.

Image Analysis and Quantification.

Images of labeled neurons were acquired and analyzed on a Cellomics ArrayScan® HCS Reader. Acquisition settings included imaging 10 fields per well at a 10× magnification. A proprietary modification of a Cellomics BioApplication was used for image analysis. Nuclei were identified using DAPI (Channel 1) and neurons were identified and selected for analysis by their staining by the MAP2 antibody (Channel2, CY5). The neuronal subpopulation was analyzed for ADDL binding in Channel 3 (FITC). BioApplication automatically reports the percentage of MAP2-labeled cells in each sample (well average of the 10 field) as well as level of ADDL binding in each individual cell. Images and numeric data were automatically transferred to Cellomics Store®, where well- and cell-level data were viewed for analysis. Cell level data were exported to Origin for Histogram analysis.

TABLE 1
Number of selected nuclei (DAPI)
	ADDL Concentration
	1 μM	0.5 μM	0.25 μM	0.125 μM	No ADDL
Well A	597	728	607	707	665
Well B	679	706	695	736	744
Well C	694	713	895	708	692
Total	1970	2147	2197	2151	2101

TABLE 2
Number of selected neurons (MAP2)
	ADDL Concentration
	1 μM	0.5 μM	0.25 μM	0.125 μM	No ADDL
Well A	564	705	587	671	625
Well B	643	663	663	692	713
Well C	652	674	823	667	662
Total	94 ± 0.3%	94 ± 1.5%	92 ± 2.5%	94 ± 0.4%	96 ± 1%

Summary of Tables 1 & 2 (refer to FIG. 1):

Table 1: Number of selected nuclei identified in each well (n=3 for each concentration of ADDLs).

Table 2: Number of selected MAP2 positive cells in each well. The total neuronal population measured for each ADDL concentration is expressed as a percentage of nuclei identified. Non-neuronal cells are not included in the analysis.

TABLE 3
Summary table of ADDL positive neurons
	ADDL Concentration
	1 μM	0.5 μM	0.25 μM	0.125 μM	No ADDL
No. of Cells	1,859	2,042	2,073	2,030	2,000
ADDL	74%	26.6%	24%	2.8%	0%
positive
ADDL	26%	73.4%	76%	97.2%	100%
negative

Table 3: Quantification of ADDL binding to hippocampal neurons in culture expressed as percentage of MAP2 positive cells at increasing ADDL concentrations. Note that the number of identified ADDL positive cells at low magnification is reduced with decreasing ADDL concentrations. (refer to FIGS. 2A, 2B, & 3)

This application is related to U.S. patent application Ser. No. 08/796,089, filed Feb. 5, 1997, now U.S. Pat. No. 6,218,506, issued Apr. 17, 2001; International Patent App. No. PCT/US98/02426, filed Feb. 5, 1998; U.S. patent application Ser. No. 09/369,236, filed Aug. 4, 1999, now U.S. patent application Ser. No. 11/100,212, filed Apr. 6, 2005; International Patent App. No. PCT/US00/21458, filed Aug. 4, 2000; U.S. patent application Ser. No. 09/745,057, filed Dec. 20, 2000, now U.S. patent application Ser. No. 11/130,566, filed May 16, 2005; U.S. patent application Ser. No. 10/166,856, filed Jun. 11, 2002; International Patent App. No. PCT/US03/19640, filed Jun. 11, 2003; U.S. patent application Ser. No. 10/676,871, filed Oct. 1, 2003, now U.S. patent application Ser. No. 10/924,372, filed Aug. 23, 2004, now U.S. patent application Ser. No. 11/142,869, filed Jun. 1, 2005; International Patent 15 App. No. PCT/US03/30930, filed Oct. 1, 2003; International Patent App. No. PCT/US05/17176, filed May 16, 2005; International Patent App. No. PCT/US05/23958, filed Jul. 5, 2005; and the like. All of the foregoing patents and patent applications are incorporated herein in their entirety by reference.

Unless otherwise noted, all patents, patent applications, as well as any other scientific and technical writings mentioned herein are incorporated by reference to the extent that they are not contradictory.

The preceding description of preferred embodiments is presented for purposes of illustration and description, and is not necessarily exhaustive nor intended to limit the claimed invention to the precise form(s) disclosed. The description was selected to best explain the principles of the invention and practical application of these principles to enable others skilled in the art to best utilize the claimed invention in various embodiments and with various modifications as are suited to the particular use contemplated. The scope of the claimed invention is not to be limited by the specification, but defined by the claims herein.

Claims

1) A method for quantification of ADDL binding to neuronal cells to which ADDLs bind.

2) The method of claim 1, wherein the method identifies and selects neurons based on MAP2 immunoreactivity, thereby ensuring that glial cells are excluded from the ADDL binding analysis.

3) The method of claims 1 or 2, wherein ADDL binding occurs in a sub-population of 16 DIV neurons and is heterogeneous in intensity among individual cells.

4) The method of claims 1, 2, or 3, wherein the presence or absence of ADDL binding is used to evaluate molecules and antibodies that block ADDL binding.

5) A method of screening for ADDL binding inhibitors.

6) A method of screening for ADDL signalling.

7) A method of screening for cell types and receptors involved in ADDL signalling.

8) The method of claim 1 wherein the primary cultures of neurons are hippocampal cells.