DEFECTIVE CALCIUM SIGNALING AS A TOOL IN AUTISM SPECTRUM DISORDERS

Abstract

The present invention features methods that allow for diagnosing a risk for a patient or subject developing an Autism Spectrum Disorder, for identifying potentially therapeutic anti-ASD agents, and methods for treatment monitoring as specified in the independent claims.

Claims

1. A method for diagnosing a risk for developing Autism Spectrum Disorder (ASD) in a subject, the method comprising: a. obtaining a biological sample containing cells from the subject being evaluated for ASD; b. using a reference tissue type-matched cell from a control healthy neurotypically developing individual without known ASD risk factors and without ASD; c. using a positive reference tissue type-matched cell from ASD diagnosed individuals; d. independently culturing the cells from (a), (b), and (c); e. measuring the level of inositol triphosphate receptor (IP.sub.3R) calcium (Ca.sup.2+) signaling activity in all sets of the cultured cells from (d) in response to an agonist of IP.sub.3R Ca.sup.2+ signaling using a Ca.sup.2+ fluorescent probe and measuring the amount of fluorescence emitted by the probe; f. comparing the: a) peak signal height; b) area under the signal curve; and c) signal rate of rise of IP.sub.3R Ca.sup.2+ signaling activity obtained from (e); and g. identifying zones of susceptibility to determine a susceptibility to developing ASD based on the levels of IP.sub.3R Ca.sup.2+ signaling activity in (f), wherein the zones of susceptibility comprise: 1) signal dead zone, wherein the subjects have undetectable calcium signaling, seen only in subjects diagnosed with ASD, and therefore the subjects are designated as a first group susceptible to developing ASD; 2) neurotypical zone, wherein subjects have at least 40% of control cell IP.sub.3R Ca.sup.2+ signaling activity, a level rarely seen in ASD subjects, and therefore the subjects are designated as a second group who are less susceptible than the first group to developing ASD, and 3) indeterminant zone between 0-40% control signal, wherein the subjects are designated as a third group who have indiscriminate susceptibility to developing ASD and requiring further evaluation for susceptibility of developing ASD.

2. (canceled)

3. A method for screening a test agent to treat a subject with Autism Spectrum Disorder (ASD), the method comprising: a. using a reference tissue type-matched cell from control neurotypical individuals without known ASD risk factors; b. using a positive reference tissue type-matched cell from subjects diagnosed with ASD; c. independently culturing the cells from (a) and (b); d. Leaving one sample of the (a) population unexposed, exposing one sample of the (a) population and one of the (b) population of isolated cells to a range of doses of the test agent; e. contacting each of the cultured cells from (d) with an agonist of IP.sub.3R Ca.sup.2+ signaling and fluorescent Ca.sup.2+ indicator; f. measuring fluorescence emitted by the fluorescent Ca.sup.2+ indicator in the ASD (b) population of isolated cells with different doses of test agent to determine a test agent dose dependent IP.sub.3R Ca.sup.2+ signaling activity; g. measuring an amount of fluorescence emitted by the Ca.sup.2+ fluorescent probe in the (a) population of isolated cells unexposed to the test agent to determine the neurotypical control IP.sub.3R Ca.sup.2+ signaling activity; and h. detecting a dose-dependent increase in IP.sub.3R Ca.sup.2+ signaling activity in the test agent-exposed ASD (b) population of cells and comparing the neurotypical (a) control population IP.sub.3R Ca.sup.2+ signaling activities in (g) over the range of doses of the test agent, wherein the IP.sub.3R Ca.sup.2+ signaling activity in the ASD (b) population of isolated cells increases potentially to become comparable to that of the (a) population of isolated, untreated control cells, indicating that the test agent is a potentially therapeutic anti-ASD agent. An ideal agent would not have a significant effect on the (a) neurotypical cells.

4. (canceled)

5. A method of treating Autism Spectrum Disorder (ASD) in a subject, comprising: a. providing behavioral therapy; and/or b. providing a composition comprising one or more activators of dysfunctional inositol triphosphate (IP3) mediated calcium (Ca2+) signaling; and c. administering a therapeutically effective dosage of the composition in (b) to the subject.

6. (canceled)

7. The method of claim 1, wherein the subject and the individual are human, of similar sex, gender, ethnicity, and age.

8. The method of claim 1, wherein the biological samples comprise skin, foreskins, amniotic fluid, blood, and/or, cheek-swabbed epithelial cells.

9. The method of claim 1, wherein the cell type comprises a fibroblast obtained from skin, a fibroblast obtained from foreskin, a fibroblast obtained from amniocentesis, an iPSC (induced-pluripotent stem cell)-derived cell, a blood cell, and/or an epithelial cell from a cheek-swab.

10. The method of claim 1, wherein the matched tissue type consists essentially of skin fibroblast cells or amniocyte obtained prenatally by amniocentesis.

11. The method of claim 1, wherein measuring IP.sub.3R Ca.sup.2+ signaling activity level comprises: a. obtaining equivalent amounts of separately cultured skin fibroblast cells from the subject and from the control individual, wherein the cultured skin fibroblast cells from each of the subject and the control individual have been: i. loaded with a Ca.sup.2+ fluorescent probe, and ii. contacted with an agonist of IP.sub.3R Ca.sup.2+ signaling; and b. separately measuring, in each of the cultured skin fibroblast cells from the subject and the individual obtained in (a), an amount of fluorescence emitted by the Ca.sup.2+ fluorescent probe; and c. comparing the amounts of emitted fluorescence measured in (b).

12. The method of claim 11, wherein the Ca.sup.2+ fluorescent probe is an intracellular-loaded fluorescent calcium indicator dye and comprises at least one member selected from the group consisting of a Fluo-8 AM, a Fluo-3, a Fluo-4, a Rhod-2; a Cal 520; a Calcium Green, a Calcium Orange; an Oregon Green BAPTA; a Fura Red; and a GCaMP.

13. The method of claim 11, wherein the agonist of IP.sub.3R Ca.sup.2+ signaling is at least one member selected from the group consisting of an adenosine triphosphate (ATP), photo-released-intracellular caged inositol triphosphate (IP.sub.3); an adenophostin A; a nucleotide; a glutamate, or a GPCR agonist.

14. The method of claim 12, wherein the ATP is extracellularly applied at 100 M.

15. The method of claim 11, wherein the emitted fluorescence is measured using a fluorometer, fluorescent imaging plate reader (FLIPR).

16. The method of claim 3, wherein the test agent is selected from the group consisting of a chemical compound, an antibody, an antibody fragment, an siRNA molecule, antisense RNA molecules, or an aptomer.

17. (canceled)

18. The method of claim 11, wherein the IP.sub.3 mediated Ca2+ signaling is neuronal.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0030] FIG. 1 shows a schematic overview of the present invention.

[0031] FIG. 2 shows a cartoon illustration of a proposed spatial organization of IP.sub.3R in endoplasmic reticulum (ER) membrane. FIG. 2 illustrates different types of Ca.sup.2+ signals produced by one or more IP.sub.3R(s) in response to low, intermediate, and high IP.sub.3 concentrations, which are, respectively, single-channel events, blips; elementary events, puffs; and global events, waves. FIG. 2, right traces, are fluorescence traces of blip, puff, and wave Ca.sup.2+ signaling events mediated by IP.sub.3R(s).

[0032] FIG. 3A bottom, shows plots of mean peak Ca.sup.2+ release exhibited by control and FXS human skin fibroblast cells loaded with calcium indicator Fluo-8 AM in response to ionomycin, a potent and specific calcium ionophore. FXS and control cell lines demonstrate no difference between cells in maximal calcium signal (the calcium pool size). FIG. 3A top, shows plots of mean peak Ca.sup.2+ release exhibited by control and FXS human skin fibroblast cells loaded with calcium indicator Fluo-8 AM in response to ATP, the agonist that tests for the ASD phenotype. FIG. 3B shows plots of mean peak Ca.sup.2+ release exhibited by 5 independent individual control and 5 independent individual FXS skin fibroblast cells induced with ATP normalized to corresponding maximal response to ionomycin. All FS lines are significantly different (p<0.05) from controls. FIG. 3C shows a mean response of TSC1 and TSC2 and control cell lines to ATP as in FIG. 3B. FIG. 3D shows a scatter plot showing IP.sub.3R expression levels in TSC and FXS cell lines as % of matched controls vs. the mean ATP-evoked Ca.sup.2+ signals in these cells relative to matched controls. Different symbols represent different cell lines. Here, and in other figures, error bars show 1 SEM.

[0033] FIG. 4A shows a plot of mean peak Ca.sup.2+ release exhibited by control and TS human skin fibroblast cells loaded with calcium indicator Fluo-8 AM in response to ionomycin, a potent and specific calcium ionophore. Three TS and three corresponding control cell lines were combined and averaged, and demonstrate no difference between cells in maximal calcium signal (the calcium pool size). Fluorescence signals are expressed as a ratio (F/F0) of changes in fluorescence (F) relative to the mean resting fluorescence of the same well before stimulation (F0). FIG. 4B shows a plot of mean peak Ca.sup.2+ release exhibited by individual control and TS skin fibroblast cells induced with ATP normalized to corresponding maximal response to ionomycin. Similar to the effects observed in FIG. 3B for the FSX cell lines, all TS lines are significantly different (p<0.05) from controls. FIG. 4C shows a mean response of TS and control cell lines to various concentrations of ATP.

[0034] FIG. 5A shows a plot of calcium indicator Fluo-8 AM fluorescent traces of Ca.sup.2+ release in individual control and FXS human skin fibroblast cells following photolysis of caged inositol triphosphate. FIG. 5B shows a plot of mean peak Ca.sup.2+ release exhibited by control and FXS skin fibroblast cells following photolysis of caged inositol triphosphate. All FS lines are significantly different (p<0.05) from controls in the percent of cells responding with any calcium wave, the slope of the wave, the latency of the calcium wave and the peak height of the wave. FIG. 5C shows a plot of mean peak Ca.sup.2+ release exhibited by control and TS human skin fibroblast cells following photolysis of caged inositol triphosphate. All TS lines are significantly different (p<0.05) from controls in the percent of cells responding with any calcium wave, the slope of the wave, the latency of the calcium wave and the peak height of the wave. Fluorescence signals are expressed as a ratio (F/F0) of changes in fluorescence (F) relative to the mean resting fluorescence of the same well before stimulation (F0).

[0035] FIG. 6A shows a plot of the number of local Ca.sup.2+ events in control and FXS human skin fibroblast cells loaded with EGTA and Fluo-8 AM and following photolysis of caged inositol triphosphate. FIG. 6B shows a distribution of local Ca.sup.2+ events of various amplitude in control and FXS cells following photolysis of caged inositol triphosphate. It demonstrates FXS cells to have a preponderance of small local events and a deficit of large events compared to control.

[0036] FIG. 7 is a plot of mean peak Ca.sup.2+ release exhibited by FXS and matching control human skin fibroblast cell lines treated with 0 M cyclic adenosine monophosphate (cAMP) or 25 M cAMP following photolysis of caged inositol triphosphate. It demonstrates a normalization of the calcium signal by cAMP in FXS cells, comparable to control, with little effect on control cells.

[0037] FIGS. 8A-8E show IP3-mediated Ca2+ signaling in FXS and TSC fibroblasts is impaired at the level of local events. Data are from 17 FXS cells, 17 TSC cells, and 16 control cells (Ctr) matched to both experimental groups. FIG. 8A shows representative traces of individual events to illustrate their kinetics. FIG. 8B shows a single Ca2+ event shown on an expanded scale to illustrate measurements of peak amplitude and event duration at half-maximal amplitude. FIG. 8C shows the mean total numbers of Ca2+ release sites detected within cells during 40 s imaging records following uniform photo-release of i-IP3 within a cell.

[0038] FIG. 8D shows the mean amplitude of all events following the photolysis within a cell. FIG. 8e shows the distributions of event durations (at half maximal amplitude) derived from all events identified in FXS (open diamonds), TSC (stars) and control cells (black squares) (o*). The data are fit by single-exponential distributions with time constants (t.sub.0) of 15 ms (both FXS and TSC) and 32 ms (control). *=p-value<0.05; **=p<0.01, n/snon-significant.

[0039] FIGS. 9A-9E show reduced constitutive Ca2+ signals in FXS and elevated autophagy markers in FXS, TSC, and ASD. FIG. 9A shows locations of spontaneous Ca2+ signals in WT fibroblasts. FIG. 9B shows Ca2+ events from selected sites in FIG. 9A. FIG. 9C shows numbers of sites in WT and FXS cells. FIG. 9D shows GFP-LC3 expression in WT cells showing ring-shaped structure characteristic of autophagosomes FIG. 9E shows background-subtracted fluorescence of GFP-LC3 for WT, FXS, TSC2 and sporadic ASD fibroblasts. N=10 for all experiments.

[0040] FIG. 10A shows the ATP response in fibroblasts from a highly heterogenous cohort of subjects sporadic ASD as well as from controls and those with syndromic ASD and as percent of a reference cell line. Average Ca2+ response in skin fibroblasts from unaffected neurotypical controls (Ctr), Prader-Willi syndrome (PWS), fragile X syndrome (FXS), tuberous sclerosis syndrome 1 and 2 (TSC), Rett syndrome (Rett) and from subjects with sporadic ASD (ASD). N below each cell line represents number of individuals tested. (GM03440) run on the same FLIPR plate. Bar graphs show mean+/SEM for each group. Data points represent responses from an individual. FIG. 10B shows an ROC Curve showing 73% sensitivity and 92% specificity of the high throughput assay in discriminating ASD samples from control samples.

[0041] FIG. 11A shows traces of ATP-induced Ca.sup.2+ events in zero Ca.sup.2+ solution. FIG. 11B shows traces of ionomycin-induced (IM) Ca.sup.2+ events in zero Ca.sup.2+ solution. FIG. 11C shows percent change of Ca.sup.2+ release relative to basal measurement in ATP induced Ca.sup.2+ signaling. FIG. 11D shows maximum Ca.sup.2+ release relative to basal signal in IM induced Ca.sup.2+ signaling. FIG. 11E shows normalized values of ATP responses to IM responses.

[0042] FIG. 12A shows that differentiation of human iPSC to GABA interneurons involves 4 stages, including embryonic body (EB) formation, induction of neuroepithelial cells (NE), patterning of MGE progenitors and differentiating to GABA neurons. FIG. 12B shows that under a defined system, hiPSCs were differentiated into neurons.

[0043] FIGS. 13A-13C show IP.sub.3-mediated Ca.sup.2+ signaling is decreased in neuronal progenitors from an FXS patient, similar to fibroblasts. FIGS. 13A-13B show superimposed traces of single-cell Ca.sup.2+ response to uncaging of ci-iP.sub.3 in control (FIG. 13A) and FXS (FIG. 13B) progenitors. Arrow indicates time of the UV flash. FIG. 13C shows mean amplitudes and latencies to peak of Ca.sup.2+ fluorescence signals in FXS progenitors (red) and matched controls (black).

[0044] FIGS. 14A-14E show optical single channel recording using optical patch clamp technique. FIG. 14A shows TIRF imaging of the local Ca.sup.2+ microdomain around an open IP.sub.3R located in close proximity to the plasma membrane. FIG. 14B shows a comparison of puffs recorded by conventional wide-field fluorescence (grey) and by TIRF imaging with EGTA loaded (black). FIG. 14C shows an example of sites that show exclusively single-channel activity. FIG. 14D shows fluorescence trace showing multiple puffs evoked at a single site following photo-release of IP.sub.3. FIG. 14E inset shows an individual puff recorded using the optical patch clamp on an expanded time scale illustrating step-wise changes in fluorescence arising from closings and openings of individual IP.sub.3R channels. Histogram shows the distribution of step levels as multiples of the single-IP.sub.3R channel (blip) fluorescence.

DETAILED DESCRIPTION OF THE INVENTION

[0045] Referring now to FIGS. 1-14, the present invention features methods for diagnosing a risk for a patient developing an ASD and for identifying potentially therapeutic anti-ASD agents and methods for treatment monitoring. As summarized in FIG. 1, the present invention for diagnosing susceptibility to ASD in a subject comprises: obtaining a skin sample from the subject to be diagnosed; assaying the sample utilizing high throughput screening to determine IP.sub.3R Ca.sup.2+ signaling activity levels (e.g., using FLIPR); and comparing signal activity level to a reference value from a healthy control subject; wherein a low activity level beneath the threshold for the reference control samples is indicative of susceptibility to ASD.

[0046] Embodiments of the invention provide methods of diagnosing a risk for a patient developing ASD. Such methods involve a step of identifying a reduced IP3R Ca2+ signaling activity level in cells from the patient comparable to matched cells from a known ASD (positive control) and substantially reduced compared to a known neurotypical (negative control) individual; and diagnosing a risk of the patient developing ASD when the IP3R activity level is reduced comparable to that of the known ASD positive control individual. Typically, in such methods, the patient and positive and negative control individuals are both human beings; and the cells from the patient and the cells from the control individuals are matched in tissue type.

[0047] In some embodiments, the patient and the positive and negative control individuals are of similar sex, gender, ethnicity, and age.

[0048] In some embodiments, the matched human tissue type consists essentially of skin fibroblast cells, peripheral blood cells, keratinocytes, umbilical cord or amniocentesis-derived cells. The biological samples comprise skin, foreskins, amniotic fluid, blood, umbilical cord and/or, cheek-swabbed epithelial cells. The cell type comprises a fibroblast obtained from skin, a fibroblast obtained from foreskin, a fibroblast obtained from umbilical cord or amniocentesis, an iPSC (induced-pluripotent stem cell)-derived cell, a blood cell, and/or an epithelial cell from a cheek-swab.

[0049] In some embodiments, the identification of the reduced IP3R Ca2+ signaling activity level in the patient further involves obtaining equivalent amounts of separately cultured and matching cells from the patient and from the control individual that have been loaded with a Ca2+fluorescent indicator and contacted with an agonist of IP3R Ca2+ signaling. Then measuring, in the so loaded and contacted cells, an amount of fluorescence emitted by the fluorescent Ca2+ indicator; and comparing the measured amounts of emitted fluorescence.

[0050] In some embodiments, the fluorescent indicator of IP3R mediated Ca2+ signaling is a Fluo-8 AM, Fluo-3, Fluo-4, Rhod-2 and related derivatives; Cal 520 and its analogues; Calcium Green, Calcium Orange and related derivatives; Oregon Green BAPTA and related derivatives; Fura Red, GCaMPs or other genetically encoded calcium indicators.

[0051] In some embodiments, the agonist of IP3R Ca2+ signaling is at least one of an adenosine triphosphate and a caged inositol triphosphate or its analogues. Other agonists include, but are not limited to: Adenophostin A; nucleotides; glutamate or other GPCR agonists.

[0052] Certain embodiments of the present invention provide a method of identifying potentially therapeutic anti-ASD agents. Such methods include using two populations of cells comprising 1) a reference tissue type-matched cell from control neurotypical individuals without known ASD risk factors and 2) a positive reference tissue type-matched cell from subjects diagnosed with ASD. The steps of the method comprise: 1) independently culturing these two populations of isolated cells; 2) leaving one sample of the neurotypical population unexposed and exposing one sample of the neurotypical population and one of the ASD population of isolated cells to a range of doses of the test agent; 3) contacting each of the cultured cells from (2) with an agonist of IP.sub.3R Ca.sup.2+ signaling and fluorescent Ca.sup.2+ indicator; 4) measuring fluorescence emitted by the fluorescent Ca2+ indicator in the ASD population of isolated cells with different doses of the test agent to determine a test agent dose dependent IP3R Ca2+ signaling activity; 5) measuring an amount of fluorescence emitted by the Ca.sup.2+ fluorescent probe in the neurotypical population of isolated cells unexposed to the test agent to determine the neurotypical control IP.sub.3R Ca.sup.2+ signaling activity; and 6) detecting a difference (e.g., increase) in IP.sub.3R Ca.sup.2+ signaling activity in the test agent-exposed ASD population of cells across the range of doses of the test agent. For the control IP3R Ca2+ signaling activities, the same comparison is made with the neurotypical (a) control population for IP.sub.3R Ca.sup.2+ signaling activities over the range of doses of the test agent. In such methods, an increased IP3R Ca2+ signaling activity in the test agent-exposed ASD population of isolated cells to potential levels observed in the untreated (e.g., not exposed to the test agent) control population of isolated cells identifies the test agent as a potentially therapeutic anti-ASD agent. An ideal agent would not have a significant effect on the (a) neurotypical cells. Also, in such methods, each of the first and the second populations of cells: were isolated from the same type of tissue of an ASD patient; exhibit a reduced level of IP3R Ca2+ signaling activity as compared to matched cells isolated from an individual that does not have ASD; and comprise substantially the same number of cells.

[0053] In some embodiments, anti-ASD therapeutic agents of the invention are chemical compounds, antibodies, antibody fragments, siRNA molecules, antisense RNA molecules, aptomers, or the like.

[0054] In some embodiments, the IP3R Ca2+ signaling is neuronal.

Examples

[0055] The following are non-limiting examples of the present invention. It is to be understood that said examples are not intended to limit the present invention in any way. Equivalents or substitutes are within the scope of the present invention.

Cell System Models for ASD

[0056] This invention utilizes fibroblasts, which are readily obtained from skin biopsies and are already in routine clinical use for the diagnosis and development of therapeutic strategies of mitochondrial, peroxisomal and lysosomal organellar-based neurological diseases. The physiology of IP3 signaling in fibroblasts is well studied, providing a validated and convenient model that complements advanced imaging technologies to resolve IP3R functioning in intact cells at the single-molecule level. Although fibroblasts and neurons express differing proportions of the three subtypes of the IP3R, it has been recently demonstrated that the single-channel gating and conductance properties of the three types of IP3R are essentially the same. Finally, fibroblasts are readily obtainable from both disease and matched control subject populations. Thus, the present invention utilizes fibroblasts, which serve as a valid model to investigate the fundamental properties of neuronal IP3 signaling and an amenable model system for IP3/Ca2+ signaling as a biomarker and potential diagnostic tool for ASD. The present invention features a method to investigate the molecular mechanisms underlying this shared defect and its downstream signaling consequences.

Cell Culture

[0057] Human skin fibroblasts were purchased from Coriell Cell Repository. Cells were cultured in Dulbecco's Modified Eagle's Media (ATCC 30-2002) supplemented with 10% (v/v) fetal bovine serum and 1 antibiotic mix (penicillin/streptomycin) at 37 C. in a humidified incubator gassed with 95% air and 5% CO2, and used for up to 20 passages. Cells were harvested in Ca2+, Mg2+-free 0.25% trypsin-EGTA (Life Technologies) and sub-cultured on 96-well plates at a seeding density of 1.510.sup.4 cells/well for 2 days before use.

High-Throughput Ca2+ Imaging

[0058] Skin fibroblasts were seeded in 96-well plates (e.g., clear-bottom black 96-well plates; Greiner Bio One catalogue #T-3026-16) at 310.sup.4 cells per well and grown to confluency. On the day of the experiment, cells were loaded with membrane-permeant Ca2+ indicator Fluo-8 AM 4 M in standard buffer solution (130 mM NaCl, 2 mM CaCl2), 5 mM KCl, 10 mM glucose, 0.45 mM KH2PO4, 0.4 mM Na2HPO4, 8 mM MgSO4, 4.2 mM NaHCO.sub.3, 20 mM HEPES and 10 M probenecid) with 0.1% fetal bovine serum for 1 h at 37 C., then rinsed with standard buffer solution. 100 l of Ca2+-free solution was added to each well, and cells were allowed to equilibrate for 5 minutes prior to the experiment. The assay was then performed with a FLIPR instrument (Fluorescent Image Plate Reader, Molecular Devices, Sunnyvale, Calif.). Relative Fluorescent Units were measured during 120 s to determine kinetics reflecting the change in intracellular Ca2+ levels according to ATP addition. A basal read of plate fluorescence (470-495 nm excitation and 515-575 nm emission) was read for 2 seconds on the FLIPR. Next, 100 l of 2ATP (1 M, 10 M, 100 M final concentration) in Ca2+-free Hank's Balanced Salt Solution (HBSS), or HBSS alone, were added to the appropriate wells. A real-time fluorescence measurement was immediately performed for 180 seconds of the assay, followed by addition of 100 l of 3 ionomycin (to 10 M final concentration), and the recording continued for another 30 sec. Fluorescence signals are expressed as a ratio (F/F0) of changes in fluorescence (F) relative to the mean resting fluorescence of the same well before stimulation (F0). Individual data were normalized to the maximum ionomycin response for each well obtained at the end of the experiment. Bars represent standard error mean. For experiments studying local Ca2+ signals, cells were loaded with Ca2+ indicator Ca1520, c-iIP3 and additionally incubated with 10 M EGTA-AM for an hour. [Ca2+]i signals were imaged using an Apo TIRF 100 (NA=1.49) oil objective.

Single-Cell Ca.SUP.2+ Imaging

[0059] Cells seeded in glass-bottomed dishes were loaded with 4 M Fluo-8 AM and 1 M caged i-IP.sub.3 (ci-IP.sub.3) for 45 mins. [Ca.sup.2+]; changes were imaged with a 40 oil objective at 30 frames sec.sup.1. A single flash of UV light was used to uncage i-IP.sub.3. For local Ca.sup.2+ signals, cells were loaded with Ca.sup.2+ indicator Ca1520, c-iIP.sub.3 and 10 M EGTA-AM for an hour. [Ca.sup.2+]; signals were imaged using an Apo TIRF 100(NA=1.49) oil objective at 129 frames sec.sup.1.

Example 1 IP3-Mediated Ca2+ Signaling is Depressed in FXS and TSC Fibroblasts

[0060] To examine for defects in IP3-mediated signaling associated with ASD, a fluorometric imaging plate reader (FLIPR) was used to monitor cytosolic Ca2+ signals in skin fibroblasts from FXS, TS, and matched control subjects. Adenosine triphosphate (ATP) was applied to activate G-protein coupled receptors (GPCR)-linked purinergic P2Y receptors in Ca2+-free extracellular solution to exclude Ca2+ influx through plasmalemmal channels.

[0061] Skin fibroblast cell lines from each of five FXS patients and five ethnicity-, sex-, and age-matched unaffected donor-derived control fibroblast cell lines were obtained from the Coriell Cell Repository. Skin fibroblast cell lines from each of three TS, two TSC1 patients and one TSC2 patient, and three corresponding sex-, age- and ethnicity matched control fibroblast cell lines were also obtained from the Coriell Cell repository.

[0062] Responses were significantly depressed in FXS cells (FIG. 3A, top; FIG. 3B). This was not due to deficits in ER Ca2+ stores in FXS cells, as application of ionomycin in Ca2+-free media to completely liberate intracellular Ca2+ stores evoked similar signals in FXS and control cells (FIG. 3A, bottom). Cell lines from tuberous sclerosis (TSC1 and TSC2) patients further demonstrated deficits in ATP-evoked Ca2+ signals (FIG. 3C), again without any appreciable difference in Ca2+ store content. Further, the diminished Ca2+ signals in FXS and TS cells cannot be substantially attributed to diminished expression of IP3R proteins because IP3R expression showed little correlation with Ca2+ signaling depression (FIG. 3D).

[0063] To then discriminate whether the observed deficits in ATP-induced signals in FXS and TSC cells arose through defects in GPCR-mediated generation of IP3, or at the level of IP3-mediated Ca2+ liberation, the GPCR pathway was circumvented by loading cells with membrane permeant, biologically inert caged IP3 (ci-IP3). Concordant with defects in ATP-induced Ca2+ signals, global cytosolic Ca2+ responses evoked by photo-released i-IP3 in FXS cells were depressed and displayed slower kinetics. Corresponding measurements from TSC cells revealed even greater deficits in Ca2+ signal amplitudes.

[0064] Single-cell assays. Cells were loaded for imaging using membrane-permeant esters of Fluo-8 and c-IP3. Cells were incubated at room temperature in HEPES-buffered saline (in mM: NaCl 135, KCl 5, MgCl2 1.2, CaCl2) 2.5, HEPES 5, and glucose 10) containing 1 M ci-IP3/PM for 45 mins, after which 4 M Fluo-8 AM was added to the loading solution for a further 45 minutes before washing three times with saline solution. [Ca2+]i changes were imaged using a super-resolution N-STORM Nikon Eclipse microscope system with a 40(NA=1.30) objective. Fluo-8 fluorescence was excited by 488 nm laser, and emitted fluorescence (>510 nm) was imaged at 30 frames sec-1 using an electron-multiplied CCD Camera iXon DU897 (Andor).

[0065] Photolysis of c-IP3 was evoked by a millisecond standardized single flash of UV (ultraviolet) light (350 to 400 nm) from an arc lamp focused to uniformly illuminate a region slightly larger than the imaging frame to uncage biologically active IP3 from c-IP3, a metabolically stable and biologically inert isopropylidene analog of IP3. The amount of IP3 released is standardized by selecting a flash duration, but is ultimately a function of several factors, including length of the flash, power of the Arc lamp, and neutral density filters inserted on the light path. Image data were acquired as stack .nd2 files using Nikon Elements for offline analysis using Nikon Elements. Calcium-evoked fluorescence signals from the whole cell are expressed as a ratio (F/F0) of changes in fluorescence (F) relative to the mean resting fluorescence at the same region before stimulation (F0). Bars represent standard error mean.

[0066] UV flash photolysis of cells loaded with biologically inert c-IP3 to photorelease active IP3 bypasses the GPCR signaling pathway and produces IP3 mediated IP3R activation. By controlling UV flash length and intensity, equivalent quantities of active IP3 were delivered to both control and FXS and TS cells, stimulating Ca2+ release. Consistent with the observations of defects in ATP-induced Ca2+ signaling in FX and TS cells, defects in global Ca2+ signaling were also observed in FXS and TS cells following UV flash photolysis of c-IP3 (FIGS. 5A-5C).

[0067] The results of these experiments indicate that the peak ATP-induced release of Ca2+ in 0 Ca2+ solution is significantly (p<0.05) depressed in the FXS and TS patient fibroblast lines, as compared to matched control cell lines (FIGS. 3B and 4B). This depression is not simply due to deficits in ER Ca2+ stores as application of the Ca2+ ionophore ionomycin in Ca2+-free media, which completely liberates all intracellular Ca2+ stores, demonstrated similar total Ca2+ content in FXS and control fibroblast cells (FIG. 3A) as well as TS and control fibroblast cells (FIG. 4A).

[0068] These results suggest that the defect in Ca2+ signaling in these three independent ASD models is not due to altered signaling to the IP3R via the GPCR or IP3 pathway, but instead implicates altered IP3R function.

Example 2 IP3-Signaling is Affected at the Level of Local Events

[0069] Without being bound by any particular theory, experimental data support a model in which IP3-mediated Ca2+ signaling exists as a hierarchy of Ca2+ events of differing magnitudes. In this model, a coordinated recruitment of clusters of IP3Rs located on the ER is responsible for generating global Ca2+ waves. It is possible that deficits in global Ca2+ waves observed in FXS and TS human skin fibroblasts result from alterations in local Ca2+ signals. Control and FXS skin fibroblasts were then loaded with the Ca2+ buffer EGTA to restrict the diffusion of Ca2+ between puff sites and prevent CICR between clusters of IP3Rs. In this way global Ca2+ waves can be devolved into multiple discrete puff sites whereupon the kinetics of Ca2+ release from IP3Rs can be observed. Cells were stimulated by photo-release of c-IP3 as described above, and individual puffs were resolved, and the results graphed in FIG. 6. FIG. 6A shows that number of local events is lower in FXS compare to control cells. Puff amplitude distribution in FXS cells is shifted toward smaller events, whereas control cells have more events with larger amplitude (FIG. 6B), corresponding to a bigger local Ca2+ release. In a physiological setting without the EGTA present, larger elementary Ca2+ events should be more successful in activating neighboring clusters, leading to further IP3R activation. The net result should be a higher probability of successful production of calcium waves that arise with a shorter latency, steeper slope and larger maximum, as is observed in FIGS. 3-5. These results suggest that IP3-mediated Ca2+ signaling in FXS cells is altered at the level of both local and global IP3R signals.

[0070] IP3-mediated cellular Ca2+ signaling is organized as a hierarchy, wherein global, cell-wide signals arise by recruitment of local, elementary events involving individual IP3R or small numbers of IP3Rs (FIG. 2). These elementary events were then imaged to elucidate how deficits in the global Ca2+ signals in FXS and TSC cells may arise at the level of local IP3R clusters and individual channels. Ca2+ release evoked by spatially uniform photolysis of ci-IP3 across the imaging field was apparent as localized fluorescent transients of varying amplitudes, arising at numerous discrete sites widely distributed across the cell soma (FIGS. 8A-8B). To quantify differences in elementary Ca2+ events between the cell lines, a custom-written, automated algorithm was utilized to detect events and measure their durations, numbers and amplitudes. Local events were appreciably briefer in FXS and TSC cells (FIG. 8C), suggesting a shortening in mean open time of IP3R channels. A second key difference lay in the numbers of detected sites, which were strikingly different between control and ASD lines (FIG. 8D), although mean event amplitudes were similar (FIG. 8E).

Example 3 cAMP Partially Restores Ca2+ Signaling in FXS Human Fibroblasts

[0071] Several kinases modulate IP3R Ca2+ signaling, including protein kinase A (PKA). PKA is a cAMP-dependent kinase, and reduced levels of cAMP have been shown to exist in drosophila and mouse FXS models, as well as in peripheral blood of human FXS subjects. To determine whether altered PKA activity leads to decreased IP3 Ca2+ signaling in FXS skin fibroblasts, the inventors conducted assays with the cell membrane permeant cAMP analog, 8-bromo-cAMP.

[0072] Fibroblast skin cells were loaded for imaging using membrane-permeant esters of Fluo-8 AM and c-IP3 and imaged as described above. Global Ca2+ responses were obtained before and after 20 minute incubation with 25 uM 8-bromo-cAMP (Tocris, cat. #1140). Image data were acquired as stack .nd2 files using Nikon Elements for offline analysis using Nikon Elements. Fluorescence signals are expressed as a ratio (F/F0) of changes in fluorescence (F) relative to the mean resting fluorescence at the same region before stimulation (F0). Bars represent standard error mean.

[0073] Incubation of skin fibroblasts with 8-bromo-cAMP partially rescued the dampened global Ca2+ response to photo-release of IP3 observed in human FXS skin fibroblast cells (FIG. 7, left). Strikingly, cAMP had minimal effect on control cells, actually tending to lower the peak amplitude (FIG. 7, right).

Example 4 Mitochondrial Energetics; a Putative Link Between Disrupted Ca.SUP.2+ and ASD

[0074] Low-level constitutive IP3R-mediated transfer of Ca2+ from the ER to mitochondria maintains basal levels of oxidative phosphorylation and ATP production. In its absence, ATP levels fall, inducing AMPK-dependent, mTOR-independent autophagy. Because of the mitochondrial energy deficient endophenotypes of autism, this study investigated whether constitutive Ca2+ signaling is impaired in ASD fibroblasts, leading to autophagy. Fibroblasts from FXS subjects displayed fewer sites of local constitutive Ca2+ release than control cells (54 vs. 186 per cell), and while single channel amplitudes were similar, with channel open time reduced, total calcium flux was decreased in FXS. (FIGS. 8C and 9C) To then investigate whether autophagy is upregulated in ASD, GFP-LC3 (a marker for autophagosomes) was expressed in fibroblasts from WT, FXS, TSC2 and a sporadic ASD subject recently enrolled in CART. GFP-LC3 fluorescence was significantly elevated in all ASD cases versus control (FIGS. 9D-9E). Significant elevations of lysotracker red fluorescence marking acidic lysosomes that bind autophagosomes were observed.

Example 5 Discriminating Between Syndromic or Sporadic ASD Samples and Controls Using Receiver Operator Characteristic (ROC) Curves

[0075] Currently, ASD is diagnosed using clinical, behavioral assessments that may be subject to human error. Without wishing to limit the present invention to any theory or mechanism, the invention uses intracellular calcium signaling as an ASD biomarker that can be detected using in vitro high throughput assay measurements. An ROC curve evaluates parameters to separate affected from unaffected individuals for diagnostic purposes. The area under the curve (AUC) in FIG. 10B shows that the assay of the present invention is quite robust (84% accuracy) in discriminating between syndromic or sporadic ASD samples and controls. Using the reference shown in FIG. 10A, 73% sensitivity and 92% specificity of the high throughput assay is observed in discriminating ASD samples from control samples. FIG. 10A shows that IP.sub.3-mediated Ca.sup.2+ response is significantly depressed across monogenic and sporadic forms of ASD.

Example 6 High-Throughput FLIPR Screen to Monitor IP.SUB.3.-Mediated Ca.SUP.2+ Signaling Changes in Response to Purinergic Activation

[0076] A high-throughput screen using FLIPR was developed to monitor IP.sub.3-mediated Ca.sup.2+ signaling in the monogenic ASD and typical, sporadic ASD samples. FIGS. 11A-11E show representative IP.sub.3-mediated Ca.sup.2+ signaling changes in response to purinergic activation and demonstrate that Ca2+ signals in response to ATP activation are lower in ASD and FSX samples.

[0077] IP3 signaling in the FLIPR assay is activated by bath application of an agonist (e.g., ATP) to activate metabotropic purinergic receptors. This introduces complications and potential variability in the pathway leading to IP3 production. To circumvent that, the present invention features a method for delivering IP3 directly to the ER of permeabilized fibroblasts. This will be based on established protocols utilizing a low-affinity fluorescent Ca2+ indicator (furaptra) trapped in the lumen of the ER and agents (e.g. saponin, streptolysin-O) to selectively permeabilize the cholesterol-rich plasma membrane, while sparing the cholesterol-poor ER. Moreover, this method will enable one to control and investigate variability that may arise from intracellular factors (such as ATP concentration, cytosolic Ca2+ buffers, phosphatases and kinases) known to modulate IP3R functioning.

Example 7 Human-Induced Pluripotent Stem Cells

[0078] Human induced pluripotent stem cells (hiPSCs) were generated from the fibroblasts using the Thermo-Fisher Sendai virus protocol. For the differentiation, hiPSCs form EBs in suspension culture for the first 7 days and then are plated and develop into colonies containing rosette, neuroepithelial cells. At day 16, neural progenitors can be observed in the edge and the rosette-containing colonies are detached and grown in suspension to form neuroepithelial sphere.

[0079] Differentiation of human iPSC to GABA interneurons involves 4 stages, including embryonic body (EB) formation, induction of neuroepithelial cells (NE), patterning of MGE progenitors and differentiating to GABA neurons (FIG. 12A). Under a defined system, hiPSCs were differentiated into neurons (FIG. 12B). FIG. 13 shows IP.sub.3-mediated Ca.sup.2+ signaling is decreased in neuronal progenitors from an FXS patient, similar to fibroblasts.

Example 8 Technical Innovation: Ca.SUP.2+ Fluorescence Signals from Individual IP.SUB.3.Rs; the Optical Patch Clamp

[0080] The optical patch-clamp technique allows the imaging of Ca.sup.2+ flux through single ion channels within intact cells with single channel resolution. Total internal reflection microscopy (TIRFM) (FIG. 14A) together with a slow Ca.sup.2+ buffer (FIG. 14B) is used to restrict excitation of a cytosolic fluorescent Ca.sup.2+ indicator to within 100 nm of the plasma membrane, thereby monitoring the local microdomain of elevated cytosolic [Ca.sup.2+] around the pore of Ca.sup.2+-permeable membrane channels. The resulting localized single-channel Ca.sup.2+ fluorescence transients (SCCaFTs) turn on and off rapidly, tracking channel openings and closings with a time resolution of a few milliseconds (FIG. 14C). Using this technique, the Ca.sup.2+ puffs arising from clusters of IP.sub.3Rs (FIG. 14D) can be dissected into the constituent openings and closings of individual receptor/channels (FIG. 14E).

[0081] As disclosed herein, reduced IP3-mediated Ca2+ signaling was shown in ASD in the context of fragile X (FXS) and tuberous sclerosis syndromes (TS). The inventors found that human fibroblasts from three genetically distinct monogenic models of ASD-fragile X and tuberous sclerosis TSC1 and TSC2uniformly display depressed Ca2+ release through IP3 receptors. They observed defects in whole-cell Ca2+ signals evoked by G-protein coupled cell surface receptors and by photo-released IP3, and at the level of local elementary Ca2+ events, suggesting fundamental defects in IP3R channel activity in ASD. Given its ubiquitous functions in the body, malfunctioning of IP3-mediated signaling can account for the heterogeneity of non-neuronal symptoms seen in ASD, such as gastrointestinal tract problems and immunological complications.

[0082] In summary, these results provide compelling evidence that IP3-mediated Ca2+ signaling is a common phenotype and a shared functional defect in three distinct monogenic models of ASD. The implications of this work are: GPCR-triggered intracellular Ca2+ release is decreased in three distinct monogenic modes of ASD; These pathological alterations are downstream of IP3 generation, as similar results are obtained using UV flash photolysis of membrane permeant caged IP3-AM; TIRFM imaging determined that a striking difference between control and ASD lines arose in the numbers of detected sites and the durations of the local events; IP3-mediated Ca2+ signaling is a common biomarker and a possible therapeutic target for ASD; and alterations in Ca2+ homeostasis can be a common pathogenic mechanism in ASD and explain the heterogeneity of its symptoms.

[0083] Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase comprising includes embodiments that could be described as consisting essentially of or consisting of, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase consisting essentially of or consisting of is met.