A method for the diagnosis of fragile x syndrome using a sigma-1 receptor-targeted positron emission tomography probe

Abstract

The present invention discloses a positron emission tomography (PET) probe (+)-[ 18 F]YL1 method and its use in fragile X syndrome (FXS), comprising the following: 1. (+)-[ 18 F]YL1 synthesis and characterization; 2. Cognitive impairment and stereotypic compulsive behavior testing in FXS mouse models; 3. (+)-[ 18 F]YL1 positron emission tomography (PET); 4. Correlation analysis between the quantitative results of PET in each brain region and the severity of cognitive impairment and stereotypy. 18 By quantitatively analyzing F]YL1 PET and analyzing its correlation with different dimensions of cognitive impairment and the severity of stereotypy, we can determine the changes in Sig-1R expression in various brain regions in FXS, thereby deepening our understanding of the potential relationship between Sig-1R-related brain functional networks and external behavioral manifestations. This invention provides new insights into the field and is expected to provide new target insights and effective imaging methods for the diagnosis and treatment evaluation of FXS.

Description

Technical Field

[0001] The present invention belongs to the field of medical imaging technology, and particularly relates to a method for diagnosing fragile X syndrome using a positron emission tomography probe targeting a Sigma-1 receptor. Background Art

[0002] FXS is the leading genetic cause of intellectual disability and autism spectrum disorders (ASD). Its primary pathogenesis is due to silencing of the Fmr1 gene on the X chromosome, resulting in the loss of FMRP, a protein crucial for brain development. Although numerous hypotheses have been proposed for the diagnosis or treatment of FXS, these remain far from exhaustive and satisfactory. Therefore, further efforts are needed to decipher the complex pathophysiology of FXS and advance the development of therapeutic drugs. Summary of the Invention

[0003] FMRP deficiency is associated with endoplasmic reticulum stress or dysfunction of many ion channels, particularly abnormal calcium ion flux, leading to impaired synaptic function and structure. In animal models of FXS, altered synaptic function is thought to underlie this neurodevelopmental disorder. Geng et al. discovered that FMRP interacts with VDAC to regulate the formation and function of endoplasmic reticulum-mitochondrial contact sites (ERMCS), termed MAMs, which are crucial for mitochondrial calcium (mito-Ca2+) homeostasis. FMRP-deficient cells exhibit excessive ERMCS formation and endoplasmic reticulum-mitochondrial Ca2+ transfer, promoting mitochondrial ROS accumulation and dysfunction. Therefore, regulating MAM-related signaling pathways may help rescue FMRP function. The multiple regulatory mechanisms of Sig-1R, which is primarily enriched at MAMs, are closely related to the pathophysiology of FXS and are considered one of the potential diagnostic and therapeutic targets for FXS.

[0004] PET is an imaging technique that can visualize biomolecule metabolism and receptor binding in vivo. PET imaging targeting Sig-1R can track changes in Sig-1R expression in vivo during disease states. This study developed a PET molecular probe (+)-[18F]YL1 that specifically targets Sig-1R. PET / CT-MRI imaging was performed on FXS model mice to investigate changes in Sig-1R expression in the brains of these mice. Correlation analysis between PET quantitative results and behavioral phenotypes was performed to explore the potential application of this probe in FXS. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the defect of the existing technology that there is a lack of effective imaging means for diagnosing fragile X syndrome and provide a method for diagnosing fragile X syndrome by using a positron emission tomography probe targeting the Sigma-1 receptor.

[0007] The present invention primarily solves the above-mentioned technical problems through the following technical solutions. One of the technical solutions of the present invention is: an imaging method for assisting the diagnosis of fragile X syndrome, comprising: 1. Synthesis and characterization of (+)-[18F]YL1; 2. Testing for cognitive impairment and stereotypic compulsive behaviors in the FXS mouse model; 3. Positron emission tomography (PET) of (+)-[18F]YL1 targeting Sig-1R; and 4. Correlation analysis between PET quantitative results in various brain regions and the severity of cognitive impairment and stereotypic compulsive behaviors.

[0008] When the mice show the following changes, it indicates that the (+)-[18F]YL1 can evaluate the disease:

[0009] (1) (+)-[18F]YL1 PET imaging showed increased uptake in the brain of FXS mice.

[0010] (2) Correlation analysis between the quantitative results of (+)-[18F]YL1 PET and various brain regions related to spatial cognition, social cognition and stereotyped repetition showed a strong correlation.

[0011] The positive progress effect of the present invention is:

[0012] The present invention has developed a probe (+)-[18F]YL1 that is metabolically stable in the brain and specifically targets Sig-1R, as well as a synthesis method thereof. The probe is used to non-invasively evaluate the Sig-1R expression level in the brains of Fmr1 gene knockout mice at the in vivo level, and simultaneously explore the correlation between Sig-1R expression levels and behavior in FXS. This further deepens the understanding of the relationship between Sig-1R-related brain functional networks and external behavioral manifestations, providing a basis for Sig-1R as a target for diagnosis and drug treatment of FXS. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The structure of a representative tracer targeting the Sigma-1 receptor is shown;

[0014] Figure 2 Design a flow chart for the experiment;

[0015] Figure 3 The radiosynthetic route and HPLC quality control results of (+)-[18F]YL1 are shown;

[0016] Figure 4 The metabolic stability results of (+)-[18F]YL1 in rat brain;

[0017] Figure 5 Schematic diagram of mouse behavioral testing and its test results;

[0018] Figure 6 (+)-[18F]YL1 PET-MRI images and quantification as well as in vitro autoradiography images and quantification results;

[0019] Figure 7 Staining of the cortex and hippocampus of WT and FXS mice;

[0020] Figure 8 This is a correlation analysis between the severity of different dimensions of cognitive impairment and stereotypy and the uptake of (+)-[18F]YL1 in different brain regions. DETAILED DESCRIPTION

[0021] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0022] 1. Experimental Design

[0023] First, the Fmr1 gene was knocked out using Crispr / Cas9 technology to simulate the gene methylation transcriptional silencing mechanism in humans. Then, the gene was identified and verified by PCR, and a mouse model that can be used for experiments was successfully constructed. Subsequently, the feasibility of (+)-[18F]YL1 for the diagnosis and treatment of FXS was evaluated through mouse behavioral tests, (+)-[18F]YL1 PET-MRI scanning targeting the Sigma-1 receptor, autoradiography, and immunofluorescence staining. Detailed experimental procedures are available at Figure 2 .

[0024] 2. Radiosynthesis and HPLC procedures of (+)-[18F]YL1

[0025] Experimental method: (+)-[18F]YL1 was synthesized by direct nucleophilic substitution reaction (see Figure 3 The product was then quality controlled and co-injected to verify its radiochemical purity by HPLC.

[0026] Experimental results: The radioactive peak of the final product and the UV absorption peak of the standard (+)-[ 19F]TZ3108 were 14.7 min and 13.9 min respectively, and the retention time was basically the same (see Figure 3 B and C in Figure 3). The average radiochemical purity of the final product was 98.9 ± 2.0%.

[0027] 3. Biodistribution experiment

[0028] Experimental Methods: Twenty Sprague-Dawley rats weighing approximately 200 g were randomly divided into five groups (5-minute, 30-minute, 30-minute, 60-minute, and 120-minute groups), with four rats per group. Five, 30, 60, and 120 minutes after injection of 2.2-3.7 MBq (+)-[18F]YL1 probe via the tail vein (for the 30-minute block group, 1 mg / kg SA4503 was injected via the tail vein 5 minutes before probe injection), the rats were sacrificed by cervical dislocation, and organs of interest (brain, blood, heart, liver, kidney, lung, spleen, pancreas, muscle, bone, and intestine) were rapidly dissected. Brain tissue was dissected and quickly placed on ice for brain sectioning, including cortex, striatum, hippocampus, thalamus, cerebellum, brainstem, and remaining brain tissue. After gently wiping off any blood from the surface of the organ with paper, the organ was placed in the bottom of a corresponding gamma counter flow tube and weighed. The weight of the gamma counter flow tube was calculated by subtracting the weight before and after sample insertion. The radioactivity of (+)-[18F]YL1 in each organ was counted using a gamma counter. All measurements were background subtracted and decay-corrected to the time of injection, and then averaged. Data are expressed as the percentage of injected dose per gram of tissue (%ID / g).

[0029] Results: As shown in Table 1, the biodistribution of the probe (+)-[18F]YL1 was assessed in adult male Sprague-Dawley rats at 5, 30, 60, and 120 minutes post-injection, and uptake was calculated as %ID / g. Specific uptake of the probe was assessed at 30 minutes after pretreatment with the blocker SA4503 (1 mg / kg) 5 minutes before tracer injection. SA4503, a well-established, highly potent and selective Sig-1R agonist, has been shown to exhibit neuroprotective effects.

[0030] (+)-[18F]YL1 rapidly penetrated the blood-brain barrier and accumulated in the rat brain. Brain uptake (%ID / g) of (+)-[18F]YL1 reached 2.257±0.184 at 5 minutes, remained at 2.082±0.197 at 60 minutes, and remained at 1.670±0.202 at 120 minutes, demonstrating good uptake and stability of the probe in the rat brain. Compared with the brain uptake of the racemic compound [18F]TZ3108 (1.586±0.505 and 1.321±0.082 at 5 and 60 minutes, respectively, with Ki for Sig-1R = 0.48 nM), (+)-[18F]YL1 was more potent (Ki for Sig-1R = 0.14 nM), which may contribute to the binding stability of the probe (+)-[18F]YL1 to Sig-1R and enhance the imaging effect; the higher brain uptake of (+)-[18F]YL1 may be due to the variability of the biodistribution experiment or the higher binding affinity of (+)-[18F]YL1 to Sig-1R. Furthermore, we found that at 5 minutes, (+)-[18F]YL1 uptake was relatively high in multiple brain regions (including the cerebellum, brainstem, cortex, thalamus, and hippocampus), with the highest uptake in the cerebellum (2.557±0.251) and the lowest uptake in the striatum (1.905±0.204). Although (+)-[18F]YL1 gradually washed out of the brain over time, it remained relatively high in all brain regions at 120 minutes. At 120 minutes, the regional uptake in the cortex, striatum, hippocampus, thalamus, cerebellum, and brainstem was 1.702±0.233, 1.435±0.180, 1.615±0.172, 1.572±0.138, 1.607±0.215, and 2.174±0.281, respectively.In the blocking study, compared with the 30-minute control group without blocking compounds, the total brain uptake of rats pretreated with 1 mg / kg SA4503 increased from 1.627±0.097 to 1.659±0.063, among which the uptake in the cortex, hippocampus, striatum and thalamus increased, especially the uptake in the cortex increased significantly from 1.723±0.168 to 2.149±0.094 (p<0.01), and the uptake in the hippocampus increased significantly from 1.551±0.137 to 1.741±0.069 (p<0.05). This may be because SA4503 affects the internalization process of Sig-1R, increases the availability of Sig-1R in these brain regions, and competitively binds to Sig-1R. The affinity of the probe for Sig-1R (Ki(+)-TZ3108-Sig-1R=0.14nM) was better than that for SA4503 (KiSA4503-Sig-1R=4.6nM); however, the uptake in the cerebellum and brainstem was reduced, with the uptake in the cerebellum significantly decreasing from 1.736±0.099 to 1.389±0.109 (p<0.01), and the uptake in the brainstem significantly decreasing from 1.900±0.233 to 1.415±0.081 (p<0.01). This may be due to the wider distribution of Sig-1R in the cerebellum and brainstem, suggesting that the probe binds more specifically to Sig-1R in these brain regions.

[0031] Consistent with the distribution of Sig-1R in peripheral tissues, the initial uptake of (+)-[18F]YL1 in the lungs was the highest, i.e., 16.620±1.778 at 5 minutes after injection; by 30 minutes, the activity in the lungs had dropped to 5.971±1.671, and at 120 minutes, the activity in the lungs had dropped to 2.165±0.347. The activity in blood was very low (0.042 ± 0.008 after 5 minutes, decreasing to 0.018 ± 0.004 after 60 minutes and 0.016 ± 0.007 after 120 minutes); the activity in the liver increased from 1.778 ± 0.374 after 5 minutes to 3.501 ± 0.335 after 120 minutes, indicating that the probe was mainly metabolized and eliminated from the body by the liver. No in vivo defluorination was observed: the bone resorption rate was 0.690 ± 0.177 at 5 minutes and 0.551 ± 0.076 at 120 minutes; the total bone resorption was low, and the change from 5 minutes to 120 minutes was negligible. Liver defluorination was observed at all time points. , lungs, kidneys, spleen, and pancreas. In the blocking study, the uptake of the probe in the pancreas, kidneys, and lungs of rats pretreated with 1 mg / kg SA4503 was reduced compared to the 30-minute control group without blocking compounds. In particular, the uptake in the pancreas was significantly reduced from 2.002±0.196 to 1.030±0.125 (p<0.001). This is consistent with the distribution of Sig-1R in these peripheral organs. The increased uptake observed in the liver (from 2.145±0.660 to 3.225±0.232) may reflect the increased clearance when target tissue uptake is blocked.

[0032] The observed distribution of (+)-[18F]YL1 in the rat brain and peripheral organs is consistent with the reported distribution of Sig-1R. Together, these data demonstrate that (+)-[18F]YL1 can cross the blood-brain barrier (BBB) ​​and accumulate in brain regions known to have high Sig-1R expression. Furthermore, (+)-[18F]YL1 is rapidly cleared from the blood, exhibits specific uptake in Sig-1R-rich tissues, and exhibits no significant defluorination in vivo.

[0033] Table 1 shows the results of the biodistribution experiment of (+)-[18F]YL1 in rats:

[0034]

[0035] 4. Metabolic stability experiment

[0036] Experimental methods:

[0037] (1) In vitro metabolic stability test:

[0038] 1-5 μL of the tracer (+)-[18F]YL1 was diluted 1000-fold with the mobile phase and allowed to stand at room temperature for 60 minutes. 50 μL was then injected onto an analytical HPLC column (mobile phase A: deionized H2O containing 0.1% trifluoroacetic acid, phase B: acetonitrile, 25% acetonitrile + 75% H2O; flow rate 1 mL / min; UV wavelength: 254 nm). The HPLC-grade flowthrough was collected at 1-minute intervals per tube for 15 minutes. Each tube was read for 30 seconds using a gamma counter. The counts for each tube were decay-corrected to the injection time and concatenated to form a curve. The ratio of the peak area of ​​the product and metabolic peaks to the total area under the curve was calculated using Origin 2022 software (OriginLab, Northampton, Massachusetts, USA). This data represents the in vitro control group for the 60-minute injection of in vivo metabolic stability.

[0039] (2) In vivo metabolic stability experiment:

[0040] ① Experimental Mouse Treatment: Adult male Sprague-Dawley rats weighing approximately 200 g were injected with (+)-[18F]YL1 probe (30-37 MBq) via the tail vein. Five, 30, and 60 minutes later, the rats were sacrificed by cervical dislocation under deep anesthesia with 2-3% isoflurane. The brains were then rapidly removed by thoracotomy.

[0041] ② Brain sample processing: Weigh, mince, and place in a mortar. Add an appropriate amount of liquid nitrogen to the sample and grind. Continue grinding by adding 1-2 mL of phosphate buffered saline (PBS) containing protease inhibitors. Transfer the homogenate to a 2 mL centrifuge tube and centrifuge in a 4°C refrigerated centrifuge for 5 minutes (10,000 g / min). Separate the supernatant and precipitate. Mix the supernatant with cold acetonitrile in a 2:3 volume ratio by vortexing and centrifuge at high speed for 5 minutes (10,000 g / min). Mix 100 μL of the supernatant with 100 μL of mobile phase in a 1:1 ratio. Filter through a 0.22 μm filter membrane, and apply 50 μL to the HPLC.

[0042] ③ Reading on the machine: After the supernatant was injected into an analytical HPLC column (mobile phase A: deionized H2O containing 0.1% trifluoroacetic acid, phase B: acetonitrile, V (acetonitrile): V (H2O) = 25:75; flow rate, 1 mL / min; UV wavelength: 254 nm), the HPLC-grade flow-through was collected at intervals of 1 minute / tube for 15 minutes. Each tube was read for 30 seconds using a gamma counter. The counts (counts) of each tube were decay-corrected to the injection time and connected to form a curve. The ratio of the peak area of ​​the product peak and the metabolic peak to the total area under the curve was calculated using the software Origin 2022 (OriginLab, Northampton, Massachusetts, USA).

[0043] Experimental results: The metabolic stability of the probe is as follows Figure 4 . The in vitro stability results of the tracer (+)-[18F]YL1 suggest that its properties remain quite stable even after 60 minutes of incubation in the mobile phase. According to the previous quality control analysis results of the probe, the peak that appears at 9 minutes is the product peak, namely (+)-[18F]YL1, and the peaks that appear at the rest of the time are metabolite peaks. In rat brain tissue samples, the percentage of product peaks was 94.23%, 90.09% and 90.74% 5, 30 and 60 minutes after injection of (+)-[18F]YL1, respectively, showing a slowly decreasing trend, which indicates that the probe is relatively stable in in vivo imaging.

[0044] 5. Behavioral assessment

[0045] Because the symptoms of Fragile X syndrome are diverse, we studied the manifestations of Fragile X syndrome from three main aspects: cognition, social interaction, and stereotyped repetition.

[0046] (1) Open field test

[0047] Experimental Methods: To assess locomotor activity, anxiety, and spatial cognition in WT and Fmr1 KO mice, we conducted an open-field test in a black, opaque, acrylic cube (50 cm × 50 cm × 40 cm). Mice were placed in the center of the open field, and their behavioral profiles were automatically tracked for 10 minutes using VisuTrack software. The total distance traveled, the time spent immobile, and the proportion of total time spent in the center area were measured. After each trial, the open-field surface was wiped with 75% ethanol to prevent odor carryover between trials.

[0048] Results: Several specific behaviors of mice in the open field were evaluated, including the total distance moved, average speed of movement, time spent motionless during the test, and the proportion of time spent in the central area of ​​the open field to the total test time. Compared with WT littermates, Fmr1 KO mice moved a significantly shorter total distance during the same test time (WT: 5483.34±344.95 cm; Fmr1 KO: 4390.21±202.76 cm; p<0.05) ( Figure 5 (A), the immobility time was longer (WT: 123.13 ± 11.30 s; Fmr1 KO: 180.21 ± 15.14 s; p < 0.01) ( Figure 5 B in the figure) indicates that Fmr1 KO mice have reduced exploratory behavior in the open field and are less anxious about being exposed to an open environment. In addition, mice will naturally approach the edge of the field in a novel environment, which is called tactility. This characteristic protects mice from the invasion of outsiders. Therefore, the time spent in the central area is a reflection of the spatial cognitive ability of mice. Normal mice will avoid the open environment, quickly leave the central area, and move around the periphery. In this experiment, Fmr1 KO mice spent more time in the central area of ​​the open field than WT mice (WT: 7.82±0.78%; Fmr1 KO: 12.58±1.31%; p<0.01) ( Figure 5 C), indicating that Fmr1 KO mice have poor cognitive ability in novel environments and may have spatial cognition impairment.

[0049] (2) New object recognition experiment

[0050] Experimental Methods: Memory and learning abilities of WT and Fmr1 KO mice were assessed using a novel object recognition test. The day before the experiment (habituation phase), mice were placed in a 35 cm × 35 cm × 25 cm object-free test box and allowed to freely explore for 30 minutes to familiarize themselves with the environment. On the first training day (familiarization phase), two identical blocks (designated Familiar A and Familiar A′) were placed on one side of the test box. Mice were placed in the test box from the other side, facing away from the blocks, and allowed to freely explore for 10 minutes. Twenty-four hours later, the recognition test began. Block B was replaced with a novel block (designated Novel B) that the mice had never encountered before. The mice were then placed in the test box again in the same location and explored for 10 minutes. VisuTrack software was used to record and analyze the amount of time the mice spent sniffing the two blocks during both the familiarization and test phases.

[0051] Calculate the preference ratio of mice to different building blocks:

[0052] Novelty preference = sniffing time on the new block ÷ sum of sniffing time on the new block and familiar block × 100

[0053] Familiarity preference = sniffing time on familiar blocks ÷ sum of sniffing time on novel and familiar blocks × 100

[0054] Calculate the discrimination ratio of mice to different blocks:

[0055] Discrimination index = (sniffing time for the new block - sniffing time for the familiar block) ÷ sum of sniffing time for the new block and the familiar block × 100

[0056] Experimental results: To further explore the cognitive ability of Fmr1 KO mice, we conducted a novel object recognition test based on the spontaneous tendency of mice to spend more time exploring novel objects rather than familiar objects. During the familiarization phase, neither Fmr1 KO mice nor their WT littermates showed a significant preference for the blocks placed in the test box (WTObjectApreference: 56.35±2.35; KOobjectApreference: 57.20±3.29; p=0.8383) ( Figure 5 However, as shown in the figure, in the test phase, when one of the blocks was replaced by a new block, the discrimination index of the Fmr1 KO mouse group was significantly lower than that of the WT group (WT Novelpreference: 77.77±2.68; KO Novelpreference: 61.07±3.71; p<0.05; WT Familiarpreference: 22.23±2.68; KO Novelpreference: 38.93±3.71; p<0.05; WT Discrimination Index: 55.54±5.35%; KO Discrimination Index: 22.14±7.41%, p<0.05) ( Figure 5 EF in ( ), indicating that Fmr1 KO mice have impaired discrimination between familiar and novel objects compared with WT mice

[0057] (3) Three-box social experiment

[0058] Experimental Methods: To investigate the social behavior and cognitive abilities of WT and Fmr1 KO mice, we conducted a three-chamber socialization experiment in a rectangular testing box (60 cm × 45 cm × 22 cm). To eliminate the influence of visual orientation, two black cylindrical social cages with longitudinal transparent plastic bars were placed diagonally on either side of the chamber.

[0059] The experiment was conducted in three phases. During the habituation phase (10 minutes), the test mice were placed in the central chamber with the cage door open and allowed to freely explore the three-chamber apparatus in an empty cage. Then, a sociability test phase (10 minutes) was conducted, in which a stimulus mouse (Stranger A) of the same strain as the test mouse, with no previous interaction, was placed in one of the social cages, while the other cage remained empty. During this phase, the test mouse was placed in the central chamber with the cage door open and allowed to move freely and interact with the stimulus mouse. Finally, a social novelty preference test (10 minutes) was conducted, in which another novel stimulus mouse (Stranger B) was placed in the empty social cage from the previous phase. The remaining procedures were the same as those for the sociability test phase. After each phase, the test box and social cage were wiped clean with 75% ethanol. VisuTrack software was used to measure the number and duration of contacts between the test mice and the empty cage, Stranger A, or B, and each other. The contact range was defined as 4 cm around the social cage.

[0060] Calculate the social preference of mice during the sociability test phase:

[0061] Sociability index = time spent exploring the stimulus mouse or the empty cage / total time spent exploring the stimulus mouse and the empty cage × 100

[0062] Calculate the social novelty preference of mice in the social novelty preference test phase:

[0063] Social novelty preference index = the time the mouse spends exploring the new stimulus mouse or the familiar stimulus mouse / the total time the mouse spends exploring the new and familiar stimulus mice × 100

[0064] Experimental results: To test the social interest and social memory of WT and Fmr1 KO mice, we conducted a three-box social experiment. In the sociability test phase, by comparing the exploration time of experimental mice with unfamiliar mice and empty cages, we found that Fmr1 KO mice, like WT mice, preferred to interact with unfamiliar mice rather than empty cages ( Figure 5 GH in the social novelty preference test phase). Subsequently, the exploration time of the experimental mice with the familiar stranger mice and the new stranger mice was compared in the previous phase. The results showed that WT mice preferred to explore the new stranger mice, while Fmr1 KO mice explored the new stranger mice significantly less than the familiar mice (p<0.05) ( Figure 5 These results indicate that Fmr1 KO mice exhibit social cognitive impairment.

[0065] (4) Buried beads experiment

[0066] Experimental Methods: We performed a bead-burying test to investigate the presence of stereotypic and compulsive behaviors in WT and Fmr1 KO mice. A new home cage was filled with 6 cm thick corncob bedding. Twelve 20 mm diameter glass marbles were evenly spaced in a 3 x 4 pattern on the flattened bedding, with each marble spaced 5 cm apart. Mice were individually placed in their cages, and the number of marbles buried within 30 minutes was recorded. A marble was considered buried if two-thirds or more of its volume was covered by the bedding.

[0067] Experimental results: To explore the stereotypic and compulsive behaviors of Fmr1 KO mice, we conducted a bead burying test. The cage was padded with 6 cm deep corn cob bedding, on which 12 glass marbles were placed at equal distances. The mice were allowed to freely explore the new cage without any interference. After 30 minutes, the number of marbles buried by WT mice and Fmr1 KO mice was significantly different by unpaired t-test (WT: 5.00 ± 0.89; KO: 8.16 ± 0.8; p < 0.05) ( Figure 5 KL in (Fig. 3), indicating that Fmr1 KO mice exhibit repetitive and stereotyped behaviors.

[0068] 6. Micro MRI Scan

[0069] Mice underwent magnetic resonance imaging to determine the anatomical location of brain regions in PET images. MRI was performed on a 9.4 Tesla miniature MRI scanner (BioSpec 94 / 30USER, Bruker BioSpin MRI GmbH, Germany) using Paravision 5.1 software (www.bruker.com). Structural imaging data were acquired using a two-dimensional T2-weighted (T2W) TurboRapid Acquisition with Refocused Echoes (TurboRARE) sequence with the following parameters: matrix size = 150 × 150, field of view (FOV) = 15 × 15 mm, number of consecutive slices = 55, slice thickness = 0.3 mm; effective spatial resolution = 0.1 × 0.1 × 0.3 mm, repetition time (TR) = 6200 ms, echo time (TE) = 22.5 ms, averaging = 6, and RARE factor = 8. The resulting MRI images are then re-fused with the PET / CT images to assist with positioning, calibration, and VOI delineation.

[0070] 7. PET / CT imaging acquisition

[0071] PET / CT images were acquired using a small animal microPET / CT imaging system (nanoScan PET / CT 82s). Pre-imaging mice were weighed and anesthetized with isoflurane (2% for induction, 1%-1.5% for maintenance). A homemade indwelling catheter was placed in the tail vein, and the mice were placed in a prone position on the scanning bed. Throughout the imaging process, the animals' vital physiological parameters were continuously monitored and recorded using a physiological monitoring system. The scanning room temperature was controlled at 22-24°C. After acquiring a CT scouting image to assist in planning the scanning area for PET / CT acquisition, the radiotracer (+)-[18F]YL1 (10-15 μCi / g) was injected into the tail vein via the indwelling catheter, and dynamic PET / CT images were immediately acquired for 30 minutes. A spiral CT scan was then performed for image fusion and attenuation correction. Finally, the 3DOSEM (3-dimensional ordered subsets expectation maximization) algorithm was used to reconstruct the acquired PET raw data into 27 frames (3 × 10 s; 4 × 60 s; 10 × 150 s; and 10 × 3 s). The main parameters are as follows:

[0072] ①CT scout scan parameters: tube voltage 35kVp, tube current 980uA, exposure time 170ms;

[0073] ② CT spiral scanning parameters: tube voltage 50 kVp, tube current 980 μA, exposure time 300 ms, pitch 1.0, number of projections 20, binning 1:4;

[0074] ③ CT real-time reconstruction parameters: small voxel thin-layer reconstruction, filter type butter worth, filter cutoff 100;

[0075] ④PET scan parameters: 1 FOV, field of view 98.5 mm, consistent with modes 1-5;

[0076] ⑤PET reconstruction parameters: conformance to modes 1-5, voxel size 0.4, reconstruction method Tera-Tomo 3D (i.e., 3DOSEM), 4 iterations, 4 subsets, random, scatter, and attenuation corrections.

[0077] 8. PET-MRI imaging data analysis

[0078] Analytical Methods: Dynamic PET / CT data were analyzed using PMOD software (version 4.203). Brain volumes of interest (VOIs) were delineated, and raw time activity curves (TACs) and semiquantitative standardized uptake values ​​(SUVs) were obtained. The dynamic data of (+)-[18F]YL1 were fitted with a Logan AIF model using the Kinetic module within PMOD software, using the image-derived input function (IDIF) of the aortic arch as the blood input function, to obtain the fully quantitative pharmacokinetic parameter, total distribution volume (VT).

[0079] Experimental results: The brain regions of interest of mice were delineated and SUV semi-quantitatively analyzed using PMOD software. The preliminary TAC of different brain regions was compared and it was found that almost all brain regions showed differential uptake ( Figure 6 AB in the figure), indicating that Fmr1 gene silencing induced an increase in (+)-[18F]YL1 uptake in most brain regions. Combined with the (+)-[18F]YL1 TAC curves in different brain regions, AUC also showed that the radioactive uptake of (+)-[18F]YL1 in the brain regions of Fmr1 KO mice was increased compared with that in WT mice ( Figure 6 C).

[0080] SUV is only a semi-quantitative method, so we further used the LoganAIF model, a fully quantitative non-compartmental model of the reversible receptor system, to analyze the raw TAC data of (+)-[18F]YL1 imaging. Using the aortic arch TAC as the input function, LoganAIF graphical analysis of the raw (+)-[18F]YL1 TAC data showed that the VT value of the brain region of Fmr1 KO mice was significantly higher than that of WT mice ( Figure 6 D), which further indicates that Sig-1R expression in the brain increases when the Fmr1 gene is silenced and FMRP is lost.

[0081] 9. In vitro autoradiography of (+)-[18F]YL1

[0082] Experimental Methods: WT and Fmr1 KO mice were returned to their home cages after 2% isoflurane anesthesia induced by injection of 3.3–3.7 × 10⁴ MBq of the tracer (+)-[⁻¹⁸]YL1 via the tail vein. After a 30-minute wait, the mice were sacrificed by cervical dislocation, and the brains were removed. After embedding in OCT, the brains were sectioned coronally with a cryomicrotome, with a 30 μm interval between sections. Subsequently, the brain slices were exposed to a Beaver micropatterned gas detection system (Ai4R Inc, France) for 3 hours for ex vivo autoradiography. Autoradiographic images were quantified using BeaQuant 3.3 software (Ai4R Inc, France). Radioactivity was quantified (cp / min / mm²) in the cortex (CTX), striatum (STR), hippocampus (HIP), and thalamus (THA). At least eight regions of interest (ROIs) were delineated for each brain region. Data from technical replicates were statistically analyzed.

[0083] Experimental results: (+)-[18F]YL1 autoradiography results are shown in Figure 6 Compared with WT mice, Fmr1 KO mice showed greater uptake of (+)-[18F]YL1 in key brain regions including the cortex, hippocampus, striatum, and thalamus ( Figure 4-8 ), which is consistent with the results of PET / CT, further demonstrating at the microscopic level that Sig-1R expression is upregulated in FXS.

[0084] 10. Immunofluorescence

[0085] Experimental methods: Mice were deeply anesthetized with a 3.0% isoflurane / oxygen mixture and perfused transcardially with normal saline and 4% cold paraformaldehyde. The brains were removed, dehydrated in a sucrose gradient, embedded in OCT, and cut into 5 μm pieces. The mouse brains were fluorescently stained using a primary antibody targeting Sig-1R (rabbit anti-SIGMAR1 polyclonal antibody, 1:300, 15168-1-AP, Proteintech). Slides were incubated with the primary antibody at 4°C overnight. After primary antibody incubation, the slides were washed three times with phosphate-buffered saline (PBS). The primary antibody was labeled with the corresponding secondary antibody (AlexaFluor 488-conjugated goat anti-rabbit IgG, 1:400, GB25303, Servicebio) at room temperature for 1 hour. After secondary antibody incubation, the slides were rinsed three times with PBS and incubated with DAPI (G1012, Wuhan Serwell Biotechnology Co., Ltd.). Images were acquired using a Nikon Eclipse C1 microscope and an upright fluorescence microscope equipped with multiple laser diode modules. DAPI excitation wavelengths were 330–380 nm and emission wavelengths were 420 nm; 488 excitation wavelengths were 465–495 nm and emission wavelengths were 515–555 nm. All images were acquired under identical fluorescence settings, with subsequent adjustments for brightness and contrast to ensure accurate pathological quantification. Slides were scanned and stored using a 3DHISTECH Pannoramic MIDI scanner. Images were viewed, analyzed, and captured using CaseViewer (version 2.4).

[0086] Experimental results: From the obtained immunofluorescence images ( Figure 7 ) showed that the expression of Sig-1R in the cortex and hippocampus of Fmr1 KO mice was higher than that in the control WT mice of the same age. This result was consistent with the previous (+)-[18F]YL1 in vivo PET / CT scan and autoradiography results, and further supported the conclusion that Sig-1R expression was upregulated when Fmr1 gene silencing led to FMRP deficiency.

[0087] 11. Correlation analysis between PET dynamics quantification and different behavioral phenotype results

[0088] Experimental methods: To explore the relationship between PET quantitative results and the phenotypes reflected by different behavioral experiments, we performed Pearson correlation analysis on the pharmacokinetic fitting results (VT) and the open field test (proportion of time spent in the central area), the novel object recognition test (discrimination index), the three-box social test (social novelty preference index) and the bead burying test (number of buried beads).

[0089] Experimental results:

[0090] (1) Correlation analysis between PET dynamics quantitative analysis and open field test results:

[0091] Among all 14 brain regions, there was a strong linear positive correlation between the VT value representing the expression of Sig-1R in the brain region and the parameter representing the proportion of time spent in the central region of spatial cognition ability ( Figure 8 A), the correlation coefficient r was between 0.56-0.65; and except for the cerebellum (p=0.0601), this correlation was statistically significant in other brain regions (p<0.05), which may suggest that the spatial cognitive impairment of FXS may be related to the expression of Sig-1R in the whole brain.

[0092] (2) Correlation analysis between PET dynamics quantification and the results of the three-box social experiment

[0093] To explore the relationship between PET quantitative results and social cognitive impairment, we performed Pearson correlation analysis on the pharmacokinetic results VT in different brain regions of the two groups of mice and the results of the three-box social experiment (social novelty preference). The results showed that in all 14 brain regions, there was a strong linear negative correlation between the VT value representing the expression of Sig-1R in the brain region and the social novelty preference representing social cognitive ability ( Figure 8 The correlation coefficients (r) ranged from 0.64 to 0.74, and these correlations were statistically significant (p<0.05), suggesting that social cognitive impairment in FXS may be associated with the expression of Sig-1R in the whole brain.

[0094] (3) Correlation analysis between PET dynamics quantification and buried bead experiment results

[0095] To explore the relationship between PET quantitative results and repetitive stereotypic behaviors, we performed Pearson correlation analysis on the pharmacokinetic results VT and the results of the bead burying experiment (number of marbles buried) in different brain regions of the two groups of mice. The results showed that in all 14 brain regions, there was a strong linear positive correlation between the VT value representing the expression of Sig-1R in the brain region and the number of marbles buried representing repetitive stereotypic behaviors ( Figure 8 C), the correlation coefficients ranged from 0.64 to 0.71, and the correlations were statistically significant (p<0.05), which may suggest that the repetitive stereotyped behaviors of FXS may be related to the expression of Sig-1R in the whole brain.

[0096] (4) Correlation analysis between PET dynamics quantification and novel object recognition experimental results

[0097] Among all 14 brain regions, the correlation between the VT value representing the expression of Sig-1R in the brain region and the discrimination index representing the cognitive ability of object recognition was very weak ( Figure 8The correlation coefficients r ranged from -0.14 to -0.43, and none of these correlations were statistically significant (p>0.05), which may suggest that the object recognition cognitive impairment in FXS may not be significantly correlated with the expression of Sig-1R in the whole brain.

[0098] 12. Statistical processing

[0099] All data were analyzed using an unpaired, two-tailed Student's t-test and presented as mean ± standard error. All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad, La Jolla, CA). To assess correlations, the Pearson correlation coefficient r was calculated. Statistical significance was set at p < 0.05.

Claims

1. A fluorine-18 labeled Sigma-1 receptor targeting compound (+)-[ 18 The application of F]YL1 combined with PET brain region quantification in FXS auxiliary diagnosis is characterized by: The Sig-1R changes in each brain region include: The brain of FXS mice is divided into 14 brain regions, including cortex, striatum, hippocampus, thalamus, cerebellum, basal forebrain, hypothalamus, amygdala, brainstem, central gray matter, superior colliculus, olfactory bulb, midbrain, hypothalamus, (+)-[ 18 F]YL1 PET was quantitatively increased in various brain regions, indicating that Sig-1R expression was upregulated. The structural formula of the receptor-targeting compound is: , fluorine-18 is a radioactive signal group.

2. As claimed in claim 1, the fluorine-18 labeled Sigma-1 receptor targeting compound (+)-[ 18 The application of F]YL1 combined with PET brain region quantification in FXS auxiliary diagnosis is characterized by: The correlation between the Sig-1R expression level in each brain region and the severity of cognitive impairment and stereotyped repetition includes: (1) Spatial cognitive impairment and (+)-[ 18 Correlation analysis of F]YL1 PET quantification showed that there was a positive correlation in 13 brain regions except the cerebellum. The more severe the spatial cognition, the higher the PET quantification. (2) Social cognition and (+)-[ 18 Correlation analysis of F]YL1 PET quantification showed that there was a negative correlation in all 14 brain regions, and the better the social cognition performance, the lower the PET quantification; (3) Severity of stereotyped repetition and (+)-[ 18 Correlation analysis of F]YL1 PET quantification showed that there was a positive correlation in all 14 brain regions. The more severe the stereotypy, the higher the PET quantification. (4) Object recognition cognitive impairment and (+)-[ 18 Correlation analysis of F]YL1 PET quantification showed that there was no correlation in any of the 14 brain regions.

Images