A method for constructing an evaluation model of mass dependence of astrocyte-to-neuron mitochondrial transfer and application thereof

Abstract

The present application belongs to the technical field of biomedical experimental models, and particularly relates to a method for constructing an evaluation model for mass dependence of astrocyte-to-neuron mitochondrial transfer and application thereof. The present application first constructs an in vivo quantitative model capable of systematically evaluating the mass dependence of astrocyte-to-neuron mitochondrial transfer (AMT) in the process of AMT, breaking through the limitation of the prior art that only focuses on the number of transfer and ignores the functional state of mitochondria. The model integrates specific cell markers, controllable neuron pathological induction, in vivo dynamic imaging, cross-scale mitochondrial mass quantification and gene regulation, and other multi-dimensional technologies, and realizes precise and dynamic observation and correlation analysis of AMT events and their biological consequences. Through the model, it is determined that the “mass” of astrocyte-derived mitochondria is a key factor determining whether it can effectively protect neurons, rather than simply the number of transfer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical experimental model technology, and in particular relates to a method for constructing a quality-dependent evaluation model of astrocyte-to-neuronal mitochondrial transfer and its application. Background Technology

[0002] Astrocytes play a crucial role in maintaining nervous system homeostasis, regulating neuronal survival through metabolic coupling. Recent studies have shown that astrocytes can provide metabolic support to neurons by transferring healthy mitochondria (AMTs) to them. However, existing research mainly focuses on the quantity of AMTs, while systematic studies on the decisive impact of mitochondrial 'quality' on neurons are lacking. Existing technologies have the following shortcomings: (1) lack of tools for tracking AMTs in vivo or quasi-in vivo; (2) inability to distinguish the effects of mitochondria of different quality states after entering neurons; and (3) lack of AMT quality-dependent quantitative models that can be used for drug screening.

[0003] In summary, the shortcomings of existing technologies in AMT tracking tools, quality effect differentiation, and quantitative evaluation models make it difficult to conduct in-depth research on the quality-dependent regulatory mechanism of AMT, and thus cannot meet the needs of related basic research and drug development. Therefore, constructing an evaluation model that can accurately assess the impact of AMT quality on neurons has become an urgent technical problem to be solved in this field. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for constructing a quality-dependent evaluation model of astrocyte-derived mitochondrial transfer and its application. This method aims to study the impact of astrocyte-derived mitochondrial quality on neuronal state, thereby achieving regulation of mitochondrial quality, quantitative detection of AMT events, and evaluation of the correlation between mitochondrial quality and neuronal survival.

[0005] To achieve the above objectives, the technical solution adopted by the present invention includes: In a first aspect, the present invention provides a method for constructing a quality-dependent evaluation model of astrocyte-to-neuronal mitochondrial transfer, comprising the following steps: S1. Constructing a visualization model of astrocyte mitochondria: By injecting a mitochondrial-targeting fluorescent protein viral vector driven by an astrocyte-specific promoter into the target brain region, specific fluorescent labeling of astrocyte mitochondria is achieved. S2. Constructing a neuronal degeneration model: By injecting a viral vector driven by a neuron-specific promoter that can induce the expression of neuronal degeneration proteins into the target brain region, a neurodegenerative disease model is established. S3. Imaging detection of AMT events: Using in vivo imaging technology, fluorescence signals of astrocyte mitochondria and neurons are monitored simultaneously in the same field of view. By analyzing time-series images, the event of astrocyte mitochondria entering neurons is identified and confirmed. S4. Evaluate neuronal status: Record and analyze the morphological characteristics, fluorescence signal changes, and survival status of neurons; S5. Quantify mitochondrial quality parameters: Assess mitochondrial morphological parameters through in vivo imaging and / or assess mitochondrial ultrastructural parameters through transmission electron microscopy. S6. Regulating the quality of astrocyte mitochondria: By altering the expression levels of mitochondrial-related genes in astrocytes through gene manipulation, the quality status of astrocyte mitochondria can be regulated in vivo. S7. Based on the results of steps S3-S6, evaluate the correlation between mitochondrial quality transmitted by astrocytes and neuronal survival status.

[0006] Preferably, in step S1, the astrocyte-specific promoter is the GFAP promoter, the fluorescent protein is EGFP, and the viral vector is an adeno-associated virus vector.

[0007] More preferably, the adeno-associated virus vector is AAV-GFAP-mito-EGFP.

[0008] Preferably, in step S1, the viral suspension is injected into the target region of the mammalian cerebral cortex via stereotactic injection; the mammal includes C57BL / 6 mice.

[0009] Preferably, in step S2, the neuron-specific promoter is the CaMKIIα promoter, the neuronal degeneration-inducing protein is ΔNLS-TDP-43, and the viral vector is an adeno-associated virus vector.

[0010] More preferably, in step S2, the neurodegenerative disease model is achieved using the AAV-CaMKIIα-ΔNLS-TDP-43-mScarlet viral vector, which is stereotactically injected into the mammalian cerebral cortex and specifically expressed in excitatory neurons.

[0011] Preferably, in step S3, the live imaging technology is two-photon microscopy.

[0012] Preferably, in step S5, the morphological indicators include mitochondrial length and / or fragmentation ratio; the ultrastructural indicators include mitochondrial cristae structural integrity and / or membrane integrity.

[0013] Preferably, in step S6, the gene manipulation includes knocking down or eliminating the mitochondrial deacetylase Sirt3 gene using an astrocyte-specific promoter-driven short hairpin RNA (shRNA) or a gene editing tool.

[0014] Preferably, in step S7, the evaluation correlation includes: comparing the survival time, structural degeneration rate, or cumulative mortality rate of neuronal populations that received mitochondria from astrocytes of different quality; and / or, performing a correlation analysis on the quality parameters of mitochondria derived from astrocytes within neurons and the survival status of the neurons.

[0015] Secondly, the present invention provides the application of the method in screening for drugs or gene targets that can improve or regulate the quality of astrocyte mitochondria.

[0016] Thirdly, this invention provides the application of the method in studying the interaction mechanism between astrocytes and neurons in neurodegenerative diseases.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention is the first to construct an in vivo quantitative model that can systematically assess the mitochondrial quality dependence during astrocyte-to-neuronal mitochondrial transfer (AMT), overcoming the limitation of existing technologies that only focus on the number of transfers while ignoring the functional state of mitochondria. This model integrates multi-dimensional technologies such as specific cell markers, controllable neuronal pathological induction, in vivo dynamic imaging, cross-scale mitochondrial quality quantification, and gene regulation, enabling precise and dynamic observation and correlation analysis of AMT events and their biological consequences. Through this model, it is clearly established in vivo that the "quality" of astrocyte-derived mitochondria is the key factor determining whether they can effectively protect neurons, rather than simply the number of transfers.

[0018] (2) The evaluation system established by this invention is stable, reliable and highly reproducible. It provides a new standardized research platform and screening tool for screening drugs that can improve the quality of astrocyte mitochondria to enhance neuroprotective effects or for discovering related regulatory gene targets. It is of great value for a deeper understanding of the intercellular interaction mechanism of neurodegenerative diseases and the development of new intervention strategies. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of astrocyte mitochondrial labeling and neuronal degeneration model; mScarlet is used to label neurons, while mito-EGFP is used to label mitochondria of astrocytes; Figure 2 The results of the method for recognizing mitochondria from astrocytes entering neurons are shown in the figure. Figure 3This is a graph showing the neuron state evaluation results; where, Figure 3 Figure 'a' shows a time-series image of the degenerative changes in neurons over time, with and without AMT. Figure 3 In sections bc and e, the cell body size, survival time, and cumulative mortality over time of neurons containing or without astrocyte-derived mito-EGFP were compared. Figure 3 The morphological differences of transferred mitochondria in surviving and degenerated neurons are further demonstrated in d. Figure 4 This is a graph showing the results of the mitochondrial mass quantification method; where, Figure 4 The results in ab represent two-photon imaging, showing the changes in mitochondrial density in astrocytes over time in the OE-TDP-43 group and the control group (8 fields of view from 3 mice in each group). Figure 4 The CD in the image shows a comparison of mitochondrial fragmentation in two groups of astrocytes (4 mice in each group). Figure 4 In Figure 'e', ​​the results of a comparison of the relative levels of mitochondrial fragmentation in the two groups of astrocytes are shown. Figure 4 f in the image represents transmission electron microscopy images of the ultrastructure of mitochondria in two groups of astrocytes; Figure 4 In the figure, g represents the comparison results of the number of mitochondria and the proportion of mitochondrial fragmentation in the two groups of astrocytes (each group was derived from 8 tissue sections from 3 mice). Figure 5 This study demonstrates the impact of SIRT3 gene regulation in astrocytes on mitochondrial quality; among other things, Figure 5 In the figure, ab represents the confocal imaging verification results of the SIRT3 knockdown effect in astrocytes, with shRNA-Ctrl as the control (4 mice in each group). Figure 5 The cd in the image represents two-photon imaging images of the mitochondrial morphology changes over time in two groups (OE-TDP-43+shRNA-Ctrl, OE-TDP-43+shRNA-Sirt3) of astrocytes, as well as the statistical results of the proportion of mitochondrial fragmentation (each group is from 12 fields of view of 3 mice). Figure 5 In the figure, 'e' represents the statistical comparison of the proportion of mitochondrial fragmentation in each group (Control+shRNA-Ctrl, OE-TDP-43+shRNA-Ctrl, Control+shRNA-Sirt3, OE-TDP-43+shRNA-Sirt3) under confocal imaging (8 fields of view for each group). Figure 5 fg in the figure represents the transmission electron microscopy images of mitochondria in astrocytes of the SIRT3 knockdown group and the control group, as well as the statistical results of the proportion of damaged mitochondria (3 mice in each group, 4 tissue sections were taken from each mouse). Figure 6The figure shows the results of the correlation assessment between mitochondrial quality and neuronal survival in astrocytes; among them, Figure 6 The left side of 'a' in the figure shows the adeno-associated virus (AAV) combination used, which includes AAV9-hGFAP-shRNA-Sirt3 for achieving astrocyte-specific SIRT3 gene knockdown and AAV9-CaMKIIα-TDP-43(ΔNLS)-mScarlet for achieving neuron-specific TDP-43(ΔNLS) overexpression. The corresponding knockdown control vector AAV9-hGFAP-shRNA-Ctrl is also shown. Figure 6 The right side of 'a' in the figure shows representative time-series imaging results used to show the process by which neurons gradually acquire astrocyte-derived mito-EGFP signals at multiple time points after surgery. The images shown are all maximum intensity projections of the z-stack in each field of view. Figure 6 bc in the figure shows the changes in cell body size and survival rate of astrocyte-derived mito-EGFP positive neurons in different treatment groups. The cell body size measurement was based on 8 neurons from 4 mice in each group, and the survival rate measurement was based on 30 or 22 neurons from 4 mice in each group. Figure 6 d in the figure shows the decrease over time in the number of neurons that initially exhibited mitochondrial transfer (AMT) during neurodegeneration in each group (4 mice per group). Figure 6 e in the figure shows the temporal variation in the number of astrocyte-derived mito-EGFP-positive mitochondria inside a single neuron in each group during neurodegeneration (8 neurons from 4 mice in each group). Figure 6 f in the image represents an immunoelectron microscope image used to show the morphological and structural features of astrocyte-derived mito-EGFP-positive mitochondria inside neurons of different treatment groups. Figure 6 The figure 'g' shows the statistical results of the comparison of morphological characteristics of mito-EGFP-positive mitochondrial fragmentation from astrocytes within neurons of different groups (3 mice per group). Detailed Implementation

[0020] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0021] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0022] Example 1. Construction of an astrocyte mitochondrial-specific marker model (AAV-GFAP-mito-EGFP) This embodiment aims to establish an in vivo model of astrocyte mitochondria-specific fluorescent labeling, providing a visualization tool for subsequent observation of mitochondrial transcellular transfer (AMT). The specific steps are as follows: Healthy adult C57BL / 6 mice were selected, and all procedures were performed according to protocols approved by the animal ethics committee. During the experiment, mice were fixed in a small animal stereotaxic apparatus under appropriate anesthesia, and the target injection site in the cerebral cortex was determined based on a stereotaxic atlas. After routine scalp incision and skull exposure, a bone window was prepared at the predetermined location, and AAV-GFAP-mito-EGFP suspension was slowly injected using a microinjection system to distribute viral particles to the target cortical region. After injection, the needle was slowly withdrawn, and the wound was rinsed and sutured. Mice were returned to their cages and housed individually or in pairs, and received routine postoperative monitoring and care. Fifteen days after viral injection and recovery, mice were euthanized or, before in vivo imaging, their mitochondrial signals in the cerebral cortex were observed and verified using fluorescence microscopy, confocal imaging, or two-photon imaging. By co-staining with astrocyte markers (such as GFAP) and neuronal markers, the specific distribution of mito-EGFP signal in astrocyte mitochondria can be further confirmed, thereby obtaining an in vivo model of astrocyte mitochondrial-specific markers.

[0023] like Figure 1 As shown, this invention constructs an AAV combination capable of visualizing specific cell types, wherein mito-EGFP is used to label the mitochondria of astrocytes. Model validation results show that 15 days after viral injection, a clear green fluorescent signal (mito-EGFP) can be detected in the target region of the cerebral cortex, and this fluorescent signal is highly colocalized with the immunofluorescence signal of the astrocyte marker GFAP, indicating that mito-EGFP is specifically expressed in the mitochondria of astrocytes, successfully obtaining an in vivo model of mitochondrial-specific labeling in astrocytes.

[0024] Example 2. Constructing a neuronal degeneration model (AAV-CaMKIIα-ΔNLS-TDP-43-mScarlet) and performing two-photon imaging. This embodiment aims to establish a stable model of neurodegenerative diseases and, combined with in vivo two-photon imaging technology, achieve long-term dynamic tracking of the pathological process of single neurons. The specific steps are as follows: Adult C57BL / 6 mice were used, and all experiments were conducted after obtaining animal ethics approval. Mice were placed on a small animal stereotaxic apparatus after general anesthesia, and their head positions were adjusted. Injection sites in the cerebral cortex were determined using standard brain atlases. The scalp was routinely disinfected and incised. Local connective tissue was gently removed to expose the skull, and a micro-opening was drilled in the predetermined cortical region. Subsequently, the pre-prepared AAV-CaMKIIα-ΔNLS-TDP-43-mScarlet viral vector was injected into the target cortical layer via a micro-dose delivery device, allowing it to be expressed in a local population of excitatory neurons. After injection, the needle was held briefly to reduce reflux before being slowly withdrawn. The surgical area was cleaned and the scalp sutured. Post-operatively, the animals were returned to their cages and housed in a constant environment for necessary recovery observation. After a certain expression period, cortical imaging windows were prepared in some mice, and repeated in vivo imaging of the same cortical region was performed using two-photon microscopy. Using neurons labeled with mScarlet fluorescent signals, we can observe the morphological changes, abnormal fluorescence distribution, and potential degenerative features of neurons induced by ΔNLS-TDP-43 expression, and evaluate the occurrence and progression of neuronal degenerative changes through time series analysis.

[0025] Two-photon imaging results show ( Figure 2 At different postoperative time points (15, 17, 19, 21, 23, and 25 days), clear red fluorescent signals (mScarlets) were detected in the target cortical region, indicating that AAV-CaMKIIα-ΔNLS-TDP-43-mScarlet was successfully expressed in excitatory neurons of the cortex. Over time, neurons expressing ΔNLS-TDP-43 exhibited significant morphological changes, including cell body shrinkage, reduced dendritic branching, and uneven distribution of fluorescent signals, demonstrating a progressive worsening of these pathological changes. Time-series analysis showed that neuronal degenerative changes gradually appeared from around 17 days postoperatively, with the degree of degeneration intensifying by 25 days. This successfully established a neuronal degenerative disease model, providing a foundational model for subsequent joint studies with AMT interventions.

[0026] Example 3. Two-photon imaging to track AMT events and neuronal survival After completing astrocyte mitochondrial-specific labeling (AAV-GFAP-mito-EGFP) and establishing a neurodegenerative disease model (AAV-CaMKIIα-ΔNLS-TDP-43-mScarlet), this embodiment utilizes two-photon microscopy to monitor mitochondrial transport events (AMT) from astrocytes to neurons in real-time in vivo and assess the impact of transport on neuronal fate. After the animals recovered and both viral types were adequately expressed, clear cortical imaging windows were established in selected mice to facilitate repeated observations of the same cortical region. During two-photon imaging, mito-EGFP and mScarlet signals were acquired using different excitation wavelengths to clarify the entry, residence, and dynamic changes of astrocyte mitochondria in neuronal structures. Through continuous temporal imaging, neurons undergoing AMT can be identified at the single-cell level, and controls can be established with cells from which astrocyte mitochondria were not obtained. Under a long-term in vivo imaging framework, the morphological characteristics, fluorescence signal changes, and survival status of each neuron were recorded daily or tracked. Based on the timing of mitochondrial transport, neurons were divided into two categories: those that acquired astrocytes and those that did not. Their survival time, degeneration rate, and structural changes were compared. Simultaneously, the morphological characteristics of the astrocytes entering the neurons were used to indirectly assess their "quality," and the correlation between mitochondrial quality and neuronal survival was further analyzed. Through the aforementioned imaging and tracking strategies, a time-series relationship between AMT events and neuronal fate can be established.

[0027] Two-photon imaging results show ( Figure 2 During the follow-up period of 15 to 25 days post-surgery, astrocyte-derived mitochondrial signals were observed in some neurons (simultaneously detected with mito-EGFP green fluorescence and mScarlet red fluorescence), confirming the occurrence of AMT events. Furthermore, in both the OE-TDP-43 treatment group and the control group, neurons gradually acquired mito-EGFP signals from astrocytes over time. Quantitative analysis of the proportion of neurons containing astrocyte-derived mito-EGFP showed that this proportion changed over time in both groups (n=3 mice per group). Simultaneously, the number of mito-EGFP dot structures transported by astrocytes within a single neuron also exhibited a changing characteristic over time (n=36 neurons per group, 3 mice per group).

[0028] Further electron microscopy revealed structural evidence of astrocyte mitochondria entering neurons. Astrocyte mitochondria were detectable by gold-labeled green fluorescent protein, and GFP-immunogold-positive mitochondria were also detected within neurons. Figure 2 ).

[0029] The results of neuronal survival and degeneration analysis are as follows: Figure 3 As shown, compared with neurons that did not acquire astrocytes, neurons that acquired astrocytes had significantly longer survival times, significantly lower cell body size reduction rates and dendritic branch loss rates, and slower degeneration rates. Furthermore, the transferred mitochondria were mostly tubular (high-quality) in surviving neurons, while they were mostly fragmented (low-quality) in degenerating neurons, indicating a close correlation between mitochondrial quality and neuronal survival.

[0030] Example 4. Gene Regulation of Astrocyte Mitochondrial Quality This embodiment aims to alter the biological state of astrocyte mitochondria through gene regulation strategies, thereby obtaining astrocyte mitochondria of varying quality for subsequent functional analysis. To this end, regulatory nodes closely related to mitochondrial homeostasis are selected for intervention. In vivo regulation of astrocyte mitochondrial quality is achieved by delivering specific regulatory tools targeting specific genes into astrocytes. Using the mitochondrial deacetylase Sirt3 gene as the primary regulatory target, an astrocyte-specific Sirt3 deletion model is constructed and injected. Against the backdrop of the existing astrocyte mitochondrial labeling system (AAV-GFAP-mito-EGFP), morphological changes in astrocyte mitochondria after Sirt3 deletion can be directly observed using two-photon microscopy. Two-photon imaging allows these changes to be recorded in vivo and coherently, facilitating the analysis of their dynamic characteristics. To further obtain higher-resolution structural information, this embodiment combines transmission electron microscopy (TEM) technology to observe the ultrastructure of astrocytes and their mitochondria in the cortical region. At the electron microscopy level, parameters such as mitochondrial cristae structure, membrane integrity, and autophagy structure can be assessed, thus more accurately characterizing the quality status of astrocytes under different gene regulatory backgrounds. By combining the in vivo dynamic information obtained from two-photon imaging with the fine structural data provided by electron microscopy, the impact of Sirt3 deficiency on astrocyte quality can be comprehensively evaluated.

[0031] Two-photon imaging results are as follows Figure 4 As shown, compared with the control group (Control+shRNA-Ctrl), the Sirt3 knockdown group (Control+shRNA-Sirt3) showed significantly shorter mitochondrial length and significantly increased fragmentation rate in astrocytes; while the TDP-43 overexpression control group (TDP-43+shRNA-Ctrl) showed increased mitochondrial fragmentation compared with the control group, and the TDP-43 overexpression + Sirt3 knockdown group (TDP-43+shRNA-Sirt3) had the highest mitochondrial fragmentation rate and the shortest mitochondrial length.

[0032] Transmission electron microscopy results show that ( Figure 5In the control group, mitochondria were regular in morphology, tubular in shape, with intact and tightly arranged cristae, and good membrane integrity. In the Sirt3 knockdown group, mitochondria were irregular in morphology, with disordered, broken, or even absent cristae, impaired membrane integrity, and some mitochondria were encapsulated by autophagosomes. In the TDP-43 overexpression control group, mitochondrial cristae structure was somewhat disordered, and membrane integrity was slightly reduced. The TDP-43 overexpression + Sirt3 knockdown group showed the most severe ultrastructural damage to mitochondria, with a large number of cristae broken and dissolved, membrane ruptured, and an increase in autophagy-related structures.

[0033] Quantitative statistical results show that ( Figure 5 Sirt3 knockdown significantly reduced the integrity of mitochondrial cristae and membranes, and increased the proportion of damaged mitochondria. This damage effect was more pronounced in the pathological context of TDP-43 overexpression. Astrocyte mitochondria of different quality levels (normal quality, mildly damaged, and severely damaged) were successfully obtained through gene regulation.

[0034] Example 5. Evaluation of the effect of mitochondria from astrocytes of different qualities entering neurons on neuronal survival. Based on the aforementioned astrocyte mitochondrial-specific labeling system, neuronal degeneration model, and long-range two-photon imaging platform, this embodiment aims to compare the effects of astrocyte mitochondria of different quality levels on neuronal structural stability and survival fate after transcellular transport to neurons, thereby clarifying the functional significance of mitochondrial quality in AMT events.

[0035] Under astrocyte-specific gene manipulation conditions (such as the Sirt3 deletion model and normal control group), astrocyte mitochondria with good and damaged quality were obtained, respectively. By combining with the AAV-GFAP-mito-EGFP labeling system, the event of astrocyte mitochondria crossing the cell boundary and entering neurons can be identified and tracked in vivo in real time. In the same neuronal degeneration environment (AAV-CaMKIIα-ΔNLS-TDP-43-mScarlet background), neurons were repeatedly imaged using two-photon imaging to distinguish between neuronal populations that "successfully received astrocyte mitochondria" and those that "did not receive mitochondria." Long-term tracking and analysis of the differences in structure, signal changes, and survival status between the two groups assessed the contribution of mitochondrial acceptance to neuronal fate. To further clarify the structural integrity of the transported mitochondria themselves, this embodiment combined immunoelectron microscopy to observe the ultrastructure of detectable astrocyte-derived mitochondria within neurons. By evaluating the mitochondrial membrane and cristae structure, matrix density, and other structural indicators, the differences in the integrity of mitochondria from Sirt3-deficient astrocytes and normal astrocytes after entering neurons were compared. By correlating mitochondrial quality indicators obtained through electron microscopy with neuronal survival outcomes recorded in two-photon imaging, it is possible to determine whether the quality of astrocytes affects the neuronal resistance to degenerative stress, including the rate of dendritic structure decay, cell body deformation, changes in fluorescence signals, and final survival time.

[0036] Two-photon long-term imaging results are as follows Figure 6 As shown, in the Control+shRNA-Ctrl group (normal quality mitochondria), the proportion of AMT-positive neurons was high, and the cell body size of positive neurons remained stable, exhibiting the highest survival rate and longest survival time. With the decline in mitochondrial quality (Control+shRNA-Sirt3 group, TDP-43+shRNA-Ctrl group, TDP-43+shRNA-Sirt3 group), the proportion of AMT-positive neurons gradually decreased, cell body size shrank significantly, survival rate gradually declined, and survival time shortened. Among these, the TDP-43+shRNA-Sirt3 group (severely damaged mitochondria) showed the most significant decrease in the number of AMT-positive neurons over time, and the number of mito-EGFP-positive mitochondria within a single neuron was also significantly less than in other groups, and they were mostly fragmented. Figure 6 ).

[0037] Immunoelectron microscopy results showed ( Figure 6In the Control+shRNA-Ctrl group, the mitochondrial membranes of neurons derived from astrocytes were intact, and the cristae were intact and tightly arranged. The integrity of the mitochondrial membranes and cristae in the Control+shRNA-Sirt3 group and the TDP-43+shRNA-Ctrl group decreased. The mitochondrial membranes in the TDP-43+shRNA-Sirt3 group were ruptured, with a large number of cristae broken and dissolved, and the matrix density was uneven, resulting in the worst quality.

[0038] Association analysis revealed that the quality of astrocyte-derived mitochondria (membrane integrity, cristae integrity) within neurons was positively correlated with neuronal survival, and negatively correlated with cell body size reduction rate and dendritic structure decay rate. In other words, high-quality mitochondria (normal membrane structure, intact cristae) significantly enhance neuronal resistance to degenerative stress and prolong survival time; while the protective effect of low-quality mitochondria (membrane damage, cristae breakage) is significantly weakened or even absent. This result clarifies that mitochondrial quality, rather than quantity, is the key factor determining the neuronal protective effect in AMT events.

[0039] Finally, it should be noted that the above embodiments are used to illustrate the technical solutions of the present invention and not to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for constructing a quality-dependent evaluation model of astrocyte-to-neuronal mitochondrial transfer, characterized in that, Includes the following steps: S1. Constructing a visualization model of astrocyte mitochondria: By injecting a mitochondrial-targeting fluorescent protein viral vector driven by an astrocyte-specific promoter into the target brain region, specific fluorescent labeling of astrocyte mitochondria is achieved. S2. Constructing a neuronal degeneration model: By injecting a viral vector driven by a neuron-specific promoter that can induce the expression of neuronal degeneration proteins into the target brain region, a neurodegenerative disease model is established. S3. Imaging detection of AMT events: Using in vivo imaging technology, fluorescence signals of astrocyte mitochondria and neurons are monitored simultaneously in the same field of view. By analyzing time-series images, the event of astrocyte mitochondria entering neurons is identified and confirmed. S4. Evaluate neuronal status: Record and analyze the morphological characteristics, fluorescence signal changes, and survival status of neurons; S5. Quantify mitochondrial quality parameters: Assess mitochondrial morphological parameters through in vivo imaging and / or assess mitochondrial ultrastructural parameters through transmission electron microscopy. S6. Regulating the quality of astrocyte mitochondria: By altering the expression levels of mitochondrial-related genes in astrocytes through gene manipulation, the quality status of astrocyte mitochondria can be regulated in vivo. S7. Based on the results of steps S3-S6, evaluate the correlation between mitochondrial quality transmitted by astrocytes and neuronal survival status.

2. The method as described in claim 1, characterized in that, In step S1, the astrocyte-specific promoter is the GFAP promoter, the fluorescent protein is EGFP, and the viral vector is an adeno-associated virus vector.

3. The method as described in claim 1, characterized in that, In step S2, the neuron-specific promoter is the CaMKIIα promoter, the neuronal degeneration-inducing protein is ΔNLS-TDP-43, and the viral vector is an adeno-associated virus vector.

4. The method as described in claim 1, characterized in that, In step S3, the live imaging technology is two-photon microscopy.

5. The method as described in claim 1, characterized in that, In step S5, the morphological indicators include mitochondrial length and / or fragmentation ratio; the ultrastructural indicators include mitochondrial cristae structural integrity and / or membrane integrity.

6. The method as described in claim 1, characterized in that, In step S6, the gene manipulation includes knocking down or eliminating the mitochondrial deacetylase Sirt3 gene using an astrocyte-specific promoter-driven short hairpin RNA or a gene editing tool.

7. The method as described in claim 1, characterized in that, In step S7, the evaluation of correlation includes: comparing the survival time, structural degeneration rate, or cumulative mortality rate of neuronal populations that received mitochondria from astrocytes of different quality; and / or, performing correlation analysis on the quality parameters of mitochondria derived from astrocytes within neurons and the survival status of the neurons.

8. The use of the method according to any one of claims 1-7 in screening for drugs or gene targets that can improve or regulate the quality of astrocyte mitochondria.

9. The application of the method as described in any one of claims 1-7 in the study of the interaction mechanism between astrocytes and neurons in neurodegenerative diseases.

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