Method for high-resolution visual observation of dynamic interaction of proteins in depression-related brain region neurons

Abstract

The invention discloses a method for high-resolution visual observation of dynamic interaction of proteins in depression-related brain region neurons. According to the method, virus delivery and PLA proximity ligation detection are combined; and a confocal microscope is used for shooting a fluorescent layer scanning picture, Image J is used for carrying out PLA signal point density statistics, and the dynamic interaction process of the proteins in the cerebral region neurons related to depression is observed. According to the invention, AAV virus delivery and PLA proximity ligation detection are combined for the first time, so that the problem of insufficient expression of target protein in brain tissues can be solved; the method is expected to break through the limitation of the prior art, a brand new tool is provided for accurate diagnosis and treatment of depression, and a new method is provided for visual observation of protein interaction of multiple brain regions and multiple types of cells in the brain region.

Description

Technical Field

[0001] This invention belongs to the fields of neurobiology and molecular imaging technology, specifically relating to a visualization method combining viral vector injection and adjacent linkage technology for in situ detection of protein interactions in brain cells of a mouse model of depression. Background Technology

[0002] Depression is a complex mental illness, and its pathological mechanisms have not yet been fully elucidated. In particular, the dynamic changes in protein interactions within neurons in specific brain regions (such as the hippocampus and prefrontal cortex (PFC)) have long lacked high-resolution, real-time visualization methods, which has severely limited a deeper understanding of its molecular mechanisms.

[0003] Existing research indicates that the hippocampus, as a key hub for emotional memory and stress response, is considered an important pathological feature of depression due to neurogenesis disorders and dysregulation of synaptic protein expression. However, traditional research techniques (such as Western blotting and immunohistochemistry) can only provide static information on protein expression levels and cannot capture the spatiotemporal dynamic changes in protein-protein interactions within neurons, let alone achieve high-resolution observations under in-situ conditions.

[0004] Currently, although advancements in techniques such as fluorescent labeling (e.g., FRET, fluorescent protein fusion) and super-resolution microscopy (e.g., STORM, PALM) have provided new tools for protein-protein interaction research, these methods still face challenges in deep brain tissue imaging, including insufficient penetration depth, high phototoxicity, and low signal-to-noise ratio. They are particularly difficult to implement long-term, high-sensitivity dynamic monitoring in specific brain regions of animal models of depression. Furthermore, intraneuronal protein-protein interaction networks are highly complex and microenvironment-dependent, and existing technologies lack the ability to provide multi-dimensional analysis of key protein interactions in specific signaling pathways (e.g., the IP3R-GRP75-VDAC pathway).

[0005] Therefore, developing a new technique capable of high-resolution visualization of dynamic protein interactions within neurons in depression-related brain regions is of great significance for elucidating the molecular mechanisms of depression and discovering novel drug targets. This patent proposes an innovative method based on a combination of AAV-specific neuronal viruses and adjacent linking technology, aiming to overcome the limitations of existing technologies and provide a new tool for the precise diagnosis and treatment of depression. Summary of the Invention

[0006] Existing protein-protein interaction research techniques (such as immunoprecipitation, fluorescence resonance energy transfer (FRET), and super-resolution microscopy) have limitations in the dynamic monitoring of neurons in depression-related brain regions (such as the prefrontal cortex and hippocampus), including insufficient spatial resolution, poor brain tissue penetration, and limited multi-target detection capabilities. To overcome these shortcomings, this invention combines viral delivery with PLA to address the problem of insufficient expression of target proteins in brain tissue, achieving the following breakthroughs:

[0007] 1. Combining AAV virus delivery with PLA technology: addressing the problem of insufficient expression of target proteins in brain tissue;

[0008] 2. Specific steps and procedures for data analysis of protein interactions in the brain cells of a mouse model of depression.

[0009] This invention provides a novel technique for high-resolution visualization of dynamic protein interactions within neurons of brain regions associated with depression. It proposes an innovative method combining AAV-specific neuronal viruses and adjacent linking technology, aiming to overcome the limitations of existing technologies and provide a new tool for the precise diagnosis and treatment of depression. It also offers a research method for visualizing protein interactions across multiple brain regions and cell types. In a preferred embodiment, by injecting Vglut2-Cre mice with rAAV-CMV-DIO-EGFP virus to label Vglut2 neurons in the hippocampus, combined with PLA technology to label IP3R3-GRP75 or GRP75-VDAC1 proteins, visualization of the IP3R-GRP75-VDAC pathway protein interactions in Vglut2 neurons of the hippocampus of depressed mouse models can be achieved. Attached Figure Description

[0010] Figure 1 This is a schematic diagram of the virus injection and modeling process.

[0011] Figure 2 Image of the CA1 region of the mouse hippocampus using virus-PLA technology.

[0012] Figure 3 Statistical plot of the interaction between IP3R3-GRP75 or GRP75-VDAC1 proteins in Vglut2 neurons. Detailed Implementation

[0013] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the specific material ratios, process conditions, and results described in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0014] Example 1

[0015] A method for high-resolution visualization of dynamic protein interactions within neurons in brain regions associated with depression includes the following steps:

[0016] 1. Virus injection

[0017] When injecting the virus into the hippocampus of mice, the heads of anesthetized and fixed Vglut2-Cre mice were first positioned on a stereotaxic apparatus to expose the skull and determine the hippocampal coordinates (-2.0 mm posterior to the anterior fontanelle, ±1.5 mm lateral to the midline, and -1.8 mm inferior to the skull). After drilling a hole with a micro-drill, 0.5-1 μL of the viral vector carrying the target gene (rAAV-CMV-DIO-EGFP virus) was slowly injected at a rate of 50 nL / min using a microinfusion pump. The needle was left in place for 5-10 minutes after injection to prevent backflow. The wound was sutured postoperatively, and the mice's recovery was monitored. After 2-4 weeks of viral expression, the transfection effect was verified by fluorescence microscopy or immunohistochemistry.

[0018] 2. Depression Model Construction

[0019] A mouse model of depression was established using chronic social frustration stress (CSDS) for 10 days. Each day, the mice were placed in cages with aggressive CD1 mice and subjected to 5 minutes of direct physical aggression, followed by 24 hours of continuous sensory stress (separation only). Depressive phenotypes were screened using the social avoidance test (SIT), and depressed mice significantly avoided interaction with unfamiliar CD1 mice. Decreased sucrose preference and prolonged immobility during tail suspension / forced swimming were used to verify depression-like behaviors.

[0020] 3. PLA Adjacency Connection Detection

[0021] After behavioral tests, mice were anesthetized with 1% sodium pentobarbital, and the heart tissue was dissected. 20 ml of physiological saline and 15 ml of 4% paraformaldehyde solution were slowly perfused through the apex of the heart to dissect the whole brain of the mice. The brain was then placed in a gradient sucrose solution for dehydration preservation (4% paraformaldehyde solution 24h → 20% sucrose paraformaldehyde solution 24h → 30% sucrose aqueous solution 24h). After OCT embedding, 20 μm brain slices were prepared using a cryostat and placed in PBS solution at 4℃.

[0022] Next, approximately 40 μL of blocking solution was added to each brain slice. This covered the entire slice, and the slice was incubated in a humidified incubator at 37°C for 60 minutes. The blocking solution was then removed from the slide, and primary antibody solutions (IP3R and GRP75, GRP75 and VDAC1) were added to each slice. The slides were then placed in a 4°C refrigerator overnight. The next day, the primary antibody solution was removed from the slide, and the slide was washed with 1x wash buffer A at room temperature. Then, PLA probe solution was added, and the slices were incubated in a humidified incubator at 37°C for 1 hour. The slides were then washed with 1x wash buffer A at room temperature, and ligation solution was added. The slices were then incubated in a humidified incubator at 37°C for 30 minutes. Finally, amplification solution was added. The slices were incubated in a humidified incubator at 37°C for 100 minutes, washed with 0.01x wash buffer B, and blocked with blocking solution containing DAPI. After 15 minutes, the slices were observed under a fluorescence microscope. If the distance between the two target proteins is less than 40 nm, the probe DNA loop will close and serve as a template for rolling circle amplification, eventually being detected by fluorescently labeled complementary nucleotides.

[0023] 4. Data Analysis

[0024] Fluorescence tomography images were captured using a confocal microscope, and PLA signal point density was statistically analyzed using ImageJ.

[0025] (1) Image acquisition specifications: Use a confocal microscope (Zeiss LSM 880) to acquire Z-stack images (0.5μm step, covering the entire cell layer), set fixed parameters as objective lens 63× / 1.4NA, resolution 1024×1024 pixels, PLA channel (usually 647nm), and save as 16-bit TIFF format (to avoid compression loss).

[0026] (2) ImageJ preprocessing: Z-stack is compressed into a 2D image, retaining all PLA signal points; the rolling ball radius is adjusted to approximately 50 pixels (≈5μm, adjusted according to cell size) to eliminate nonspecific background fluorescence.

[0027] (3) PLA signal point detection, density calculation and standardization:

[0028] ① In ImageJ software, select 'Analyze' > 'Tools' > 'ROI Manager' in the menu bar, and then click the 'Add' button to add the currently selected area to the ROI Manager, thereby obtaining the corresponding ROI (Region of Interest);

[0029] ② Perform thresholding on the selected ROI region, and use Image>Adjust>Threshold (ImageJ method) for binarization to distinguish signal points from the background;

[0030] ③ Perform morphological filtering on the selected ROI region, with a screening area of ​​approximately 0.05–1 μm. 2 (Excluding excessively small noise or excessively large clusters) The signal is processed by calculating the pixel area of ​​each connected region and converting it to the actual area (based on image resolution, such as μm). 2 / pixel), while removing areas <0.05μm. 2 (Possibly noise) or Area > 1μm 2 (Possibly non-specific aggregation), the roundness screening range is 0.6–1.0 (to ensure that the signal points are close to a circle);

[0031] ④ Perform mean intensity statistics on the selected ROI region and measure the average gray value of each signal point on the original image;

[0032] ⑤ Data Export: The final export includes Count (number of valid signal points) and Area (area of ​​each signal point, μm). 2 ) and Mean Intensity (average fluorescence intensity per signal point).

[0033] Finally, the PLA dot density is calculated using the formula:

[0034] PLADensity=Mean Intensity / Area(μm 2 )×Count

[0035] like Figure 2 and Figure 3 The results show that: Figure 2 To transmit the constructed rAAV-CMV-DIO promoter (containing a Dnm1l knockdown virus) to Vglut2 neurons labeled with Drp1 protein knockdown in vglut2-cre mice, rAAV-CMV-DIO-EGFP was used as a control virus. After harvesting frozen brain sections, PLA experiments were performed, and confocal imaging was used to observe the interactions between IP3R and GRP75, and between GRP75 and VDAC1 proteins in Vglut2 neurons. Figure 3 for Figure 2Statistical analysis of the results showed the average number of PLA dots around each cell nucleus in each group, and the percentage of Vglut2 neurons containing PLA dots out of the total number of neurons. This invention, by integrating viral delivery technology with PLA (ortho-adjacent connectivity detection) methods, achieves high-resolution, targeted dynamic observation of protein-protein interactions in depression research. It overcomes the technical bottleneck of insufficient target protein expression and enables high-resolution, cell-specific visualization of protein interactions, providing a new tool for the study and treatment of depression mechanisms. The technology is also universally applicable, suitable for research in multiple brain regions such as the cortex, striatum, and amygdala. Alternatively, by changing the Cre mouse strain (e.g., GAD2-cre inhibitory neurons), it can be extended to other nerve cell types. Furthermore, it can be adapted to different antibody combinations for flexible research on various protein pairs.

[0036] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for high-resolution visualization and observation of dynamic protein interactions within neurons of brain regions associated with depression, characterized in that, This method combines viral delivery with PLA adjacent connectivity detection; it uses confocal microscopy to capture fluorescence tomography images and ImageJ to perform PLA signal point density statistics to observe the dynamic protein interaction process in neurons of depression-related brain regions.

2. The method according to claim 1, characterized in that, Vglut2 neurons in the hippocampus of Vglut2-Cre mice were labeled by injecting them with rAAV-CMV-DIO-EGFP virus. This was combined with PLA technology to label IP3R3-GRP75 or GRP75-VDAC1 proteins. Confocal microscopy was used to capture fluorescence tomography images, and ImageJ was used to perform PLA signal point density statistics. This enabled the visualization and observation of protein interactions in the IP3R-GRP75-VDAC pathway in Vglut2 neurons of the hippocampus of depressed mouse models.

3. The method according to claim 1, characterized in that, The method of virus delivery includes: when injecting the virus into the hippocampus of mice, the head of anesthetized and fixed Vglut2-Cre mice is first positioned on a stereotaxic instrument to expose the skull and determine the hippocampal coordinates. After drilling a hole with a micro-drill, the viral vector rAAV-CMV-DIO-EGFP carrying the target gene is injected using a microinjection pump.

4. The method according to claim 1, characterized in that, PLA adjacent connectivity detection methods include: Brain slices from virus-injected depressed rats were prepared using a cryostat and placed in PBS solution. Blocking buffer was then added to each slice, covering the entire surface, and incubated in a humidified chamber. The blocking buffer was removed from the slides, and primary antibody solutions IP3R and GRP75, and GRP75 and VDAC1 were added to each slice. The slides were then placed in a refrigerator overnight. The next day, the primary antibody solution was removed from the slides, and the slides were washed with 1x wash buffer A at room temperature. PLA probe solution was then added, and the slices were incubated in a humidified chamber. The slides were washed again with 1x wash buffer A at room temperature, followed by the addition of ligation solution, and then incubated in a humidified chamber. Finally, amplification solution was added, and the slices were incubated in a humidified chamber and washed with 0.01x wash buffer B. The slices were then blocked with blocking buffer containing DAPI and observed under a fluorescence microscope. If the distance between the two target proteins was <40 nm, the probe DNA loop would close and serve as a template for rolling circle amplification, ultimately detected by fluorescently labeled complementary nucleotides.

5. The method according to claim 4, characterized in that, 1x Wash Buffer A is the washing solution used in the PLA probe incubation step; 0.01x Wash Buffer B is the washing solution used in the amplification incubation step.

6. The method according to claim 1, characterized in that, Fluorescence tomography images were captured using a confocal microscope, and PLA signal point density was statistically analyzed using ImageJ. The specific steps included: (1) Image acquisition specifications: Z-stack images were acquired using a confocal microscope with a step size of 0.5 μm to cover the entire cell layer. The fixed parameters were set as follows: objective lens 63× / 1.4NA, resolution 1024×1024 pixels, PLA channel 647nm, and saved in 16-bit TIFF format. (2) ImageJ preprocessing: Z-stack is compressed into a 2D image, retaining all PLA signal points; the sphere radius is adjusted to 50 pixels to eliminate nonspecific background fluorescence; (3) PLA signal point detection, density calculation and standardization: ① In ImageJ software, select 'Analyze' > 'Tools' > 'ROI Manager' in the menu bar, and then click the 'Add' button to add the currently selected area to the ROI Manager, thereby obtaining the corresponding Region of Interest (ROI). ② Perform threshold segmentation on the selected ROI region, and use Image>Adjust>Threshold for binarization to distinguish signal points from the background; ③ Perform morphological screening on the selected ROI region, with a screening area of ​​approximately 0.05–1 μm. 2 This allows for the elimination of excessively small noise or excessively large aggregate signals. By calculating the pixel area of ​​each connected region and converting it to the actual area based on the image resolution, areas <0.05μm are discarded. 2 Noise or Area > 1 μm 2 Non-specific aggregation with a roundness screening range of 0.6–1.0 ensures that the signal points are nearly circular. ④ Perform mean intensity statistics on the selected ROI region and measure the average gray value of each signal point on the original image; ⑤ Data Export: The final export includes Count (number of valid signal points) and Area (area of ​​each signal point in μm). 2 MeanIntensity, which is the average fluorescence intensity of each signal point; Finally, the PLA dot density is calculated using the formula: PLADensity=Mean Intensity / Are×Count.

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