Introduction: Transcription factor EB (TFEB), a key regulator of autophagy and lysosomal biogenesis, has diverse roles in various physiological processes. Enhancing lysosomal function by TFEB activation has recently been implicated in restoring neural stem cell (NSC) function. Overexpression of TFEB can inhibit the cell cycle of newborn cortical NSCs. It has also been found that TFEB regulates the pluripotency transcriptional network in mouse embryonic stem cells independent of autophagy and lysosomal biogenesis. This study aims to explore the effects of TFEB activation on neurogenesis in vivo through transgenic mice. Methods: We developed a glial fibrillary acidic protein (GFAP)-driven TFEB overexpression mouse model (TFEB GoE) by crossing the floxed TFEB overexpression mice and hGFAP-Cre mice. We performed immunohistochemical and fluorescence staining on brain tissue from newborn mice to assess neurogenesis changes, employing markers such as GFAP, Nestin, Ki67, doublecortin (DCX), Tbr1, and NeuN to trace different stages of neural development and cell proliferation. Results: TFEB GoE mice exhibited premature mortality, dying 10–20 days after birth. Immunohistochemical analysis revealed significant abnormalities, including disrupted hippocampal structure and cortical layering. Compared to control mice, TFEB GoE mice showed a marked increase in radial glial cells (RGCs) in the hippocampus and cortex, with Ki67 staining indicating these cells were predominantly in a quiescent state. This suggests that TFEB overexpression suppresses RGC proliferation. Additionally, abnormal distributions of migrating neurons and mature neurons were observed, highlighted by DCX, Tbr1, and NeuN staining, indicating a disruption in normal neurogenesis. Conclusion: This study, using transgenic animals in vivo, revealed that GFAP-driven TFEB overexpression leads to abnormal neural layering in the hippocampus and cortex by dysregulating neurogenesis. Our study is the first to discover the detrimental impact of TFEB overexpression on neurogenesis during embryonic development, which has important reference significance for future TFEB overexpression interventions in NSCs for treatment.

Transcription factor EB (TFEB) is a transcription factor that belongs to the MiTF/TFE family and is involved in regulating many important physiological and pathological processes, such as embryonic angiogenesis, autophagy, and lysosomal function, particularly playing a crucial role in the regulation of autophagolysosome biogenesis [1‒3]. Under normal conditions, TFEB is localized in the cytoplasm. However, in the presence of cellular stressors such as starvation or injury, TFEB translocates to the nucleus, promoting the expression of genes related to autophagolysosomes [4, 5]. Dysregulation of TFEB has been linked to neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease, which are characterized by neuronal loss. Activation or overexpression of TFEB has been shown to promote the clearance of toxic proteins and neuronal survival, suggesting a potential therapeutic role for TFEB in these conditions [6‒9].

Research has shown that quiescent neural stem cells (NSCs) contain large numbers of lysosomes, in which protein aggregates accumulate, and that activation of lysosomes can clear aggregates and enhance the activation of quiescent NSCs in aged mice, suggesting that lysosomes keep NSCs young [10]. Another study also found that enhancing lysosomal degradation could maintain the quiescent state of NSCs [11]. NSCs are a type of stem cells in the central nervous system (CNS) and have the remarkable ability to self-renew and differentiate into various types of neural cells, including neurons, astrocytes, and oligodendrocytes [12]. In the adult brain, NSCs primarily reside in two regions: the hippocampal dentate gyrus (DG) and the subventricular zone of the lateral ventricle [13]. Quiescent NSCs are NSCs that remain in the G0 phase, and upon receiving activation signals, they reenter the cell cycle and differentiate into mature neurons. Quiescence is essential to avoid the precocious exhaustion of NSCs, ensuring a sustainable source of available stem cells in the brain [14].

During embryonic development, NSCs play a crucial role in the formation of the nervous system. They are involved in a series of complex processes to produce all the various types of neurons, known as neurogenesis [15]. The cerebral cortex and hippocampus are two of the most active regions in the developing brain [16]. The cerebral cortex is responsible for higher cognitive functions and plays crucial roles in sensory perception, motor control, language, and memory. This unique layered structure is achieved by the proliferation and differentiation of radial glial cells (RGCs). In the early stage of cortical development, RGCs in the ventricular zone (VZ) serve as the primary NSCs. They undergo self-renewal to generate new RGCs, leading to the expansion of the stem cell pool. Subsequently, RGCs divide into either RGCs and neurons or neuronal progenitor cells through asymmetric division. Additionally, RGCs extend long radial fibers from the inner surface of the VZ to the outer surface of the marginal zone (MZ), forming a scaffold that guides neuronal migration [17]. Once neurons are generated, they leave the inner regions of the VZ, migrate outward along the radial glial scaffold through the intermediate zone (IZ), and finally reach the cortical plate (CP). The CP is formed in six distinct layers (Ⅰ to Ⅵ) in an inside-out pattern, with deep layers (Ⅵ) being generated earlier and superficial layers (Ⅰ) being generated later. During this process, neurons undergo plasticity changes, establish synaptic connections with other neurons, slide along the radial fibers, and gradually move outward along the fibers’ paths until they reach their appropriate layers in the cortex, ultimately giving rise to neural functionality [18, 19]. During the late stage of embryogenesis, RGCs proliferate and give rise to oligodendrocytes and astrocytes after neurogenesis is complete [20, 21]. The hippocampus is a region of the brain involved in learning, memory, and spatial navigation. It consists of several subregions, including the DG and the cornu ammonis (CA), which is further divided into the CA1, CA2, and CA3 regions. Each of these subregions has specific patterns of neuronal patterning and lamination.

To elucidate the role of the TFEB gene in brain development in vivo, we generated transgenic mice with overexpression of TFEB (TFEB GoE) through the breeding of TFEB-overexpressing floxed mice with hGFAP-Cre mice (expressing Cre recombinase under the control of the human glial fibrillary acidic protein [hGFAP] promoter) [22]. We observed that during embryonic development, TFEB overexpression in the CNS resulted in disrupted cortical layering and hippocampal structural abnormalities. These results provide in vivo evidence that embryonic TFEB overexpression inhibits neurogenesis, dysregulates neuronal migration during development, and subsequently causes neuronal lamination defects in select brain regions. These results indicate that TFEB plays a critical role in regulating NSC proliferation, differentiation, migration, and ultimately neuronal patterning in the CNS in a temporal, spatial, and cell type-specific manner.

Mice

To generate TFEB-overexpressing mice, hGFAP-Cre mice were crossed with TFEBloxp/loxp mice. TFEBloxp/loxp mice were obtained by inserting CAG Pr-loxP-Stop-loxP-TFEB CDS-WPRE-pA into the Rosa26 locus using the EGE system based on CRISPR/Cas9 technology produced by Biocytogen. hGFAP-Cre mice were obtained from Jackson Laboratory (Strain #: 004600). Heterozygous TFEBloxp/+; GFAP-Cre+ mice (TFEB GoE) served as the experimental group, and TFEBloxp/+; GFAP-Cre- mice served as the control group. Genotyping of newborn mice was performed on the day of birth, and brain tissues were collected after anesthesia for histological staining. The mice were housed in the laboratory of Lanzhou University under controlled conditions of 45–60% humidity, 20–25°C temperature, and a 12-hour light-dark cycle.

Immunohistochemical Staining

Hematoxylin and eosin staining was performed to evaluate the tissue morphology and cellular details. Following overnight postfixation in a 4% paraformaldehyde solution at 4°C, mouse brain tissues were embedded in paraffin wax and sectioned coronally at a thickness of 5 μm. Paraffin-embedded tissue sections were deparaffinized in xylene for 20 min, followed by rehydration through a series of graded alcohols (100%, 95%, and 70%) for 5 min each. The sections were then immersed in a Harris hematoxylin solution for 5 min to stain the nuclei. After rinsing with running tap water, the sections were differentiated in 1% hydrochloric acid ethanol for 10 s and washed again. Subsequently, the sections were counterstained with eosin Y solution for 5 min to stain the cytoplasm. After a brief rinse in distilled water, the sections were dehydrated through a series of graded alcohols (70, 95, and 100%) for 5 min each. Finally, the sections were cleared in xylene for 10 min and mounted with a coverslip using a mounting medium. The stained sections were examined under a light microscope for histological analysis, and images were captured for further analysis.

Immunofluorescence Staining

After deparaffinization and rehydration of the paraffin-embedded tissue sections, antigen retrieval was performed using 10 mm sodium citrate buffer (pH 6.0) at 95°C for 10 min. After cooling and rinsing with 0.01 m PBST, the sections were permeabilized with 0.5% Triton X-100 in phosphate-buffered saline (PBS) for 10 min. Sections were rinsed with 0.01 m PBST and then blocked with 10% goat serum in PBS for 1 h at room temperature to reduce nonspecific binding. Sections were incubated at 4°C overnight with the following primary antibodies: GFAP (1:800; GTX108711 GeneTex), NeuN (1:800; GTX132974 GeneTex), doublecortin (DCX) (1:300; ab18723 Abcam), Nestin (1:200; GB115685 Servicebio), and Ki67 (1:200; ab15580 Abcam) in antibody dilution buffer (0.01 m PBS, 1% goat serum, and 3% Triton X-100). The sections were then rinsed with 0.01 m PBS and incubated with goat anti-rabbit Alexa Fluor 594 (1:200; Abbkine) at 37°C for 1 h in the dark. After washing with PBS, the sections were counterstained with 4′,6-diamidino-2-phenylindole for nuclear staining. Finally, the sections were mounted with 50% glycerol for imaging. Images were acquired with an inverted microscope and were taken under identical exposures and conditions. For the quantification of positive staining, three adjacent sections were acquired from each mouse. The number of positive cells was calculated using ImageJ software.

Double Immunofluorescence Staining

For double immunofluorescence, deparaffinized tissue sections were subjected to antigen retrieval, permeabilization, and blocking with 10% goat serum. NeuN primary antibody (1:500; GB15138; Servicebio) and Tbr1 primary antibody (1:1000; GB111317; Servicebio) were incubated overnight at 4°C. The sections were rinsed with 0.01 m PBS and incubated with goat anti-mouse Alexa Fluor 594 (1:200; Abbkine) and goat anti-rabbit Alexa Fluor 488 (1:200; Abbkine) for 1 h at 37°C in the dark. Then, the sections were rinsed again with 0.01 m PBS and the nuclei were counterstained with 4′,6-diamidino-2-phenylindole. Fluorescence signals were detected using an LSM 900 confocal scanning system (Carl Zeiss Ltd.).

Statistical Analysis

All statistical analyses were performed with PRISM 8 software and SPSS Statistics 17.0 using unpaired Student’s t tests between two groups and one-way ANOVAs among multiple groups. Data are shown as the mean ± SEM. Groups were considered significantly different when p < 0.05 (*p < 0.05, **p < 0.01, and ***p < 0.001).

The hGFAP Promoter Directs TFEB Overexpression in the CNS

The TFEB gene (Gene ID: 21425) is on the forward strand of chromosome 17 with a full length of 55.4 kb. Using CRISPR/Cas9 technology and based on the characteristics of the Rosa26 gene, a targeting vector for the TFEB gene knock-in loxp system was constructed. The targeting vector for the Rosa26 gene knock-in consisted mainly of the following functional elements: a CAG promoter, a loxP-Stop-loxP regulatory element, a TFEB CDS-WPRE-pA expression element, and approximately 2 kb homology arms at the 5′ and 3′ ends (Fig. 1a). The primers were synthesized according to the designed sgRNA sequence and ligated into the pCS (puro) vector (Fig. 1b) through annealing and polymerization. After the ligation product was transformed, samples were sent for sequencing to verify that they were correct. The sgRNA with the highest activity was connected to the plasmid vector with a T7 promoter and transcribed in vitro to determine the RNA for microinjection. The Cas9/sgRNA targeting vector was microinjected into mouse embryos, and the offspring TFEB gene knock-in loxp mice (TFEBloxp/loxp) were identified (Fig. 1c). To investigate the effects of TFEB overexpression in the CNS, we generated compound mice by crossing TFEBloxp/loxp mice with transgenic mice bearing hGFAP-Cre (Fig. 1d).

Fig. 1.

hGFAP-targeted TFEB overexpression in the mouse brain. a Schematic diagram of the targeting vector for TFEB gene overexpression. The targeting vector for Rosa26 gene knock-in mainly consists of the following functional elements: a CAG promoter, a loxP-Stop-loxP regulatory element, a TFEB CDS-WPRE-pA expression element, and approximately 2 kb homology arms at the 5′ and 3′ ends. b Cas9/sgRNA targeting vector map. c Schematic diagram of the primers designed for genotype detection and primer sequences. The genomic structure of the TFEB conditional allele with a targeting construct and the structure of the TFEB allele after Cre-lox recombination. The arrows represent the primers used for PCR analysis of the genomic DNA. The dashed lines show the amplified fragment length. d Schematic diagram of mouse breeding and PCR genotyping results. GFAP, glial fibrillary acidic protein.

Fig. 1.

hGFAP-targeted TFEB overexpression in the mouse brain. a Schematic diagram of the targeting vector for TFEB gene overexpression. The targeting vector for Rosa26 gene knock-in mainly consists of the following functional elements: a CAG promoter, a loxP-Stop-loxP regulatory element, a TFEB CDS-WPRE-pA expression element, and approximately 2 kb homology arms at the 5′ and 3′ ends. b Cas9/sgRNA targeting vector map. c Schematic diagram of the primers designed for genotype detection and primer sequences. The genomic structure of the TFEB conditional allele with a targeting construct and the structure of the TFEB allele after Cre-lox recombination. The arrows represent the primers used for PCR analysis of the genomic DNA. The dashed lines show the amplified fragment length. d Schematic diagram of mouse breeding and PCR genotyping results. GFAP, glial fibrillary acidic protein.

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GFAP-Driven TFEB Overexpression Leads to Disordered Neuronal Patterning and Lamination in the Hippocampus and Cortex

The heterozygous TFEB GoE mice were viable and showed lower body weight compared to the control mice at birth (p < 0.05, Fig. 2a). The TFEB GoE mice died prematurely between 10 and 20 days after birth. Hematoxylin and eosin histological analysis at postnatal day 0, a stage when cortical neurogenesis finishes, revealed the abnormal hippocampal structure and cortical layering (Fig. 2d). The hippocampal neuronal pattern in TFEB GoE mice was completely disrupted, and these mice lacked the typical CA1, CA2, CA3, and DG structures observed in the control mice. All TFEB GoE mice exhibited clustered aggregates of pyramidal neurons and granule cells in the DG, which cannot be distinguished from the densely arranged laminar cells in the hippocampal structure. In the cortex, TFEB GoE mice exhibited increased cortical thickness (p < 0.01, Fig. 2b) and differences in cortical sublayer thickness proportions compared to the control group, particularly evident in the thickening of the VZ (p < 0.05, Fig. 2c).

Fig. 2.

TFEB overexpression results in abnormal hippocampal structure and cortical layering at postnatal day 0. a TFEB GoE mice showed lower body weight compared to the control mice (n = 14 mice per control group; n = 13 mice per TFEB GoE group). b-d H&E immunohistochemistry revealed abnormal structure in the hippocampus (middle column); TFEB GoE mice lacked the typical CA1, CA2, CA3, and DG structures observed in the control mice. The cortex, especially the VZ sublayer (right column), was thickened in TFEB GoE mice. b, c Quantification of cortex and cortical sublayer thicknesses (n = 4 mice per group). Both solid and dashed black boxes show higher magnification views. *p < 0.05, **p < 0.01 compared to control mice. Data are represented as the mean ± SEM. H&E, hematoxylin and eosin; DG, dentate gyrus; CA, cornu ammonis; MZ, marginal zone; CP, cortical plate, IZ, intermediate zone; VZ, ventricular zone.

Fig. 2.

TFEB overexpression results in abnormal hippocampal structure and cortical layering at postnatal day 0. a TFEB GoE mice showed lower body weight compared to the control mice (n = 14 mice per control group; n = 13 mice per TFEB GoE group). b-d H&E immunohistochemistry revealed abnormal structure in the hippocampus (middle column); TFEB GoE mice lacked the typical CA1, CA2, CA3, and DG structures observed in the control mice. The cortex, especially the VZ sublayer (right column), was thickened in TFEB GoE mice. b, c Quantification of cortex and cortical sublayer thicknesses (n = 4 mice per group). Both solid and dashed black boxes show higher magnification views. *p < 0.05, **p < 0.01 compared to control mice. Data are represented as the mean ± SEM. H&E, hematoxylin and eosin; DG, dentate gyrus; CA, cornu ammonis; MZ, marginal zone; CP, cortical plate, IZ, intermediate zone; VZ, ventricular zone.

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GFAP-Driven TFEB Overexpression Maintains the Quiescent State of RGCs

To investigate the impact of TFEB overexpression on RGCs in postnatal mouse brains, we checked the expression of GFAP (a marker for both activated astrocytes and RGCs) by immunostaining at postnatal day 0. In control mice, GFAP+ cells primarily exhibited an activated astrocyte-like morphology and were uniformly distributed in the hippocampal CA area, with a higher number in the DG area, mainly located in the periphery of granule cells. However, in TFEB GoE mice, GFAP+ cells mainly displayed an RGC-like morphology with apparent radial fibers that were densely distributed in both the hippocampal CA and DG areas (Fig. 3a), and their numbers were significantly increased (p < 0.05, Fig. 3c). In the cortex of control mice, GFAP+ cells also exhibited an activated astrocyte-like morphology, evenly distributed across layers, with a slightly pronounced presence in the VZ. In TFEB GoE mice, GFAP+ cells displayed an RGP-like morphology, extending from the VZ surface to the MZ surface (Fig. 3b), and GFAP+ cells also increased (p < 0.001, Fig. 3d), particularly in the CP, IZ, and VZ (p < 0.01, Fig. 3e). To further clarify the expression of RGCs, we observed with Nestin staining (a NSC marker) and found the same results. In TFEB GoE mice, Nestin+ cells were increased in both the hippocampus (p < 0.001, online suppl. SFig. 1a, c; for all online suppl. material, see https://doi.org/10.1159/000538656) and cortex (p < 0.01, online suppl. SFig. 1b, d, e). These results showed that TFEB GoE mice had a significant increase in RGCs, which were mainly distributed throughout the hippocampus and cortical IZ and VZ sublayers, indicating that TFEB overexpression leads to abnormal radial glial patterning.

Fig. 3.

TFEB overexpression causes abnormal morphology and distribution of RGCs. Representative immunofluorescence labeling of GFAP in the hippocampus (a) and cortex (b) of the TFEB GoE mice and littermate controls at P0. In control mice, GFAP+ cells primarily exhibited activated astrocytic morphology, mainly distributed in the hippocampal DG region and cortical VZ region. However, in TFEB GoE mice, there was an increased number of GFAP+ cells (c-e), predominantly displaying RGC-like morphology characterized by prominent radial fibers. These cells were densely distributed throughout the entire hippocampus and cortical IZ and VZ regions. The irregular white boxes show the hippocampal structure. The rectangular white boxes show higher-magnification views. n = 4 mice per group. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to control mice. Data are represented as the mean ± SEM. GFAP, glial fibrillary acidic protein; DG, dentate gyrus; CA, cornu ammonis; MZ, marginal zone; CP, cortical plate, IZ, intermediate zone; VZ, ventricular zone; P0, postnatal day 0.

Fig. 3.

TFEB overexpression causes abnormal morphology and distribution of RGCs. Representative immunofluorescence labeling of GFAP in the hippocampus (a) and cortex (b) of the TFEB GoE mice and littermate controls at P0. In control mice, GFAP+ cells primarily exhibited activated astrocytic morphology, mainly distributed in the hippocampal DG region and cortical VZ region. However, in TFEB GoE mice, there was an increased number of GFAP+ cells (c-e), predominantly displaying RGC-like morphology characterized by prominent radial fibers. These cells were densely distributed throughout the entire hippocampus and cortical IZ and VZ regions. The irregular white boxes show the hippocampal structure. The rectangular white boxes show higher-magnification views. n = 4 mice per group. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to control mice. Data are represented as the mean ± SEM. GFAP, glial fibrillary acidic protein; DG, dentate gyrus; CA, cornu ammonis; MZ, marginal zone; CP, cortical plate, IZ, intermediate zone; VZ, ventricular zone; P0, postnatal day 0.

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To clarify whether these RGCs are in a quiescent state, we measured the expression of Ki67, a proliferation marker. Proliferating RGCs express Ki67, but quiescent RGCs are Ki67 negative. In the hippocampus, the DG region of the TFEB GoE mice showed only a few scattered Ki67+ cells, in contrast to the abundant distribution of Ki67+ cells in the DG region of the control group (Fig. 4a). In the cortex, the control group exhibited a regular distribution of Ki67+ cells in the MZ and VZ regions. However, in TFEB GoE mice, Ki67+ cells were mainly distributed in the IZ region, with very few cells observed in the MZ (Fig. 4b). Compared to the control group, the number of Ki67+ cells decreased in both the hippocampus (p < 0.01, Fig. 4c) and the cortex (p < 0.05, Fig. 4d, e) in TFEB GoE mice. These results indicate that TFEB overexpression maintains the quiescent state of RGCs.

Fig. 4.

TFEB overexpression maintains the quiescent state of RGCs. Representative immunofluorescence labeling of Ki67 in the hippocampus (a) and cortex (b). In the hippocampus, TFEB GoE mice showed a sparse and decreased distribution of Ki67+ cells compared to the control group (c). In the cortex, the control mice exhibited a predominant distribution of Ki67+ cells in the MZ and VZ regions. However, in TFEB GoE mice, Ki67+ cells of the cortex were decreased (d) and were mainly distributed in the IZ sublayer (e). The irregular white boxes show the hippocampal structure. The rectangular white boxes show higher-magnification views. n = 4 mice per group. *p < 0.05 and **p < 0.01 compared to control mice. Data are represented as the mean ± SEM. DG, dentate gyrus; CA, cornu ammonis; MZ, marginal zone; CP, cortical plate, IZ, intermediate zone; VZ, ventricular zone.

Fig. 4.

TFEB overexpression maintains the quiescent state of RGCs. Representative immunofluorescence labeling of Ki67 in the hippocampus (a) and cortex (b). In the hippocampus, TFEB GoE mice showed a sparse and decreased distribution of Ki67+ cells compared to the control group (c). In the cortex, the control mice exhibited a predominant distribution of Ki67+ cells in the MZ and VZ regions. However, in TFEB GoE mice, Ki67+ cells of the cortex were decreased (d) and were mainly distributed in the IZ sublayer (e). The irregular white boxes show the hippocampal structure. The rectangular white boxes show higher-magnification views. n = 4 mice per group. *p < 0.05 and **p < 0.01 compared to control mice. Data are represented as the mean ± SEM. DG, dentate gyrus; CA, cornu ammonis; MZ, marginal zone; CP, cortical plate, IZ, intermediate zone; VZ, ventricular zone.

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GFAP-Driven TFEB Overexpression Negatively Affects Neuronal Migration and Lamination

To investigate whether TFEB overexpression affects the migration function of neural precursor cells, we stained for the migrating neuron marker DCX. DCX is a microtubule-associated protein involved in the reorganization of the cellular cytoskeleton and microtubule stability during neuronal migration. It plays a role in facilitating the migration of newly generated neurons and the establishment of proper connections. In the hippocampus of the control group, DCX+ cells were evenly distributed in the granule cell and pyramidal cell layers of the CA and DG areas. However, in TFEB GoE mice, DCX+ cells were disorganized, showed irregular migration patterns (Fig. 5a), and were noticeably reduced in number compared with the control group (p < 0.001, Fig. 5c). For the cortex, in TFEB GoE mice, DCX+ cells were mainly located in the MZ and IZ regions. In the MZ, DCX+ cells exhibited a migration pattern toward the MZ surface of the brain, while in the IZ, a tangential migration pattern was observed (Fig. 5b). Although the total number of DCX+ cells in the cortex showed no statistical difference compared to the control mice (p > 0.05, Fig. 5d), sublayer analysis revealed a decrease in the CP sublayer and an increase in the IZ sublayer (p < 0.01, Fig. 5e). These results suggested that TFEB overexpression inhibited normal neuronal migration.

Fig. 5.

TFEB overexpression negatively affects neuronal migration. Representative immunofluorescence labeling of DCX in the hippocampus (a) and cortex (b). In the hippocampus of the TFEB GoE mice, DCX+ cells exhibited a disorganized and irregular migration pattern (a). The number of DCX+ cells was significantly reduced (c). In the cortex of TFEB GoE mice, the number of DCX+ cells did not show significant changes (d), characterized by fewer cells in the CP sublayer and an increase in the IZ sublayer, compared to the control group (e). The irregular white boxes show the hippocampal structure. The rectangular white boxes show higher-magnification views. n = 4 mice per group. **p < 0.01 and ***p < 0.001 compared to control mice. Data are represented as the mean ± SEM. DG, dentate gyrus; CA, cornu ammonis; MZ, marginal zone; CP, cortical plate, IZ, intermediate zone; VZ, ventricular zone.

Fig. 5.

TFEB overexpression negatively affects neuronal migration. Representative immunofluorescence labeling of DCX in the hippocampus (a) and cortex (b). In the hippocampus of the TFEB GoE mice, DCX+ cells exhibited a disorganized and irregular migration pattern (a). The number of DCX+ cells was significantly reduced (c). In the cortex of TFEB GoE mice, the number of DCX+ cells did not show significant changes (d), characterized by fewer cells in the CP sublayer and an increase in the IZ sublayer, compared to the control group (e). The irregular white boxes show the hippocampal structure. The rectangular white boxes show higher-magnification views. n = 4 mice per group. **p < 0.01 and ***p < 0.001 compared to control mice. Data are represented as the mean ± SEM. DG, dentate gyrus; CA, cornu ammonis; MZ, marginal zone; CP, cortical plate, IZ, intermediate zone; VZ, ventricular zone.

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To assess neuronal differentiation and maturation following TFEB overexpression, we evaluated the expression of NeuN (neuronal marker). In control mice, NeuN+ neurons were densely arranged in the pyramidal layer of the CA area and the granule cell layer of the DG area of the hippocampus, forming a typical structure of the hippocampus, whereas in TFEB GoE mice, NeuN+ neurons did not form the typical structure of the hippocampus, were especially distributed in clusters, and had a disordered structure in the DG area (Fig. 6a). The number of NeuN+ neurons increased significantly (p < 0.01, Fig. 6c). In the cortex of control mice, NeuN+ neurons were regularly distributed in various sublayers, especially in the CP sublayer. In contrast, the number of NeuN+ neurons in the cortex of TFEB GoE mice was significantly increased (p < 0.05, Fig. 6d), mainly in the CP, IZ, and VZ sublayers (p < 0.05, Fig. 6e). Using NeuN and Tbr1 (a marker of layers V and VI) double labeling, we found that both Tbr1+ and NeuN+ neurons were increased in the CP sublayer of TFEB GoE mice, particularly in layers V-VI, and their laminar distribution became irregular, in contrast to control mice, where NeuN+ neurons were primarily distributed in layers II and III of the CP sublayer (p < 0.05, Fig. 7a–c). These results indicate disordered neuronal lamination in TFEB GoE mice.

Fig. 6.

TFEB overexpression leads to disordered neuronal lamination. Representative immunofluorescence labeling of NeuN in the hippocampus (a) and cortex (b). In the hippocampus of TFEB GoE mice, NeuN+ neurons were distributed in clusters and had a disordered structure in the DG area (a). The number of NeuN+ neurons was increased in the hippocampus (c). In the cortex of TFEB GoE mice, NeuN+ neurons were increased (d), especially in the CP, IZ, and VZ sublayers (e), and their laminar distribution became irregular (b). The irregular white boxes show the hippocampal structure. The rectangular white boxes show higher-magnification views. n = 4 mice per group. *p < 0.05 and **p < 0.01 compared to control mice. Data are represented as the mean ± SEM. DG, dentate gyrus; CA, cornu ammonis; MZ, marginal zone; CP, cortical plate, IZ, intermediate zone; VZ, ventricular zone.

Fig. 6.

TFEB overexpression leads to disordered neuronal lamination. Representative immunofluorescence labeling of NeuN in the hippocampus (a) and cortex (b). In the hippocampus of TFEB GoE mice, NeuN+ neurons were distributed in clusters and had a disordered structure in the DG area (a). The number of NeuN+ neurons was increased in the hippocampus (c). In the cortex of TFEB GoE mice, NeuN+ neurons were increased (d), especially in the CP, IZ, and VZ sublayers (e), and their laminar distribution became irregular (b). The irregular white boxes show the hippocampal structure. The rectangular white boxes show higher-magnification views. n = 4 mice per group. *p < 0.05 and **p < 0.01 compared to control mice. Data are represented as the mean ± SEM. DG, dentate gyrus; CA, cornu ammonis; MZ, marginal zone; CP, cortical plate, IZ, intermediate zone; VZ, ventricular zone.

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Fig. 7.

TFEB overexpression leads to disrupted cortical layering. Representative double labeling of Tbr1 and NeuN in the cortex (a). In control mice, Tbr1+ cells were primarily distributed in layers V-VI of the CP sublayer, while NeuN+ cells were mainly found in layers II and III. However, in TFEB GoE mice, both Tbr1+ and NeuN+ cells were distributed throughout the entire CP sublayer (a), with an increased number compared to the control group (b, c). n = 4 mice per group. *p < 0.05 compared to control mice. Data are represented as the mean ± SEM. MZ, marginal zone; CP, cortical plate, IZ, intermediate zone; VZ, ventricular zone.

Fig. 7.

TFEB overexpression leads to disrupted cortical layering. Representative double labeling of Tbr1 and NeuN in the cortex (a). In control mice, Tbr1+ cells were primarily distributed in layers V-VI of the CP sublayer, while NeuN+ cells were mainly found in layers II and III. However, in TFEB GoE mice, both Tbr1+ and NeuN+ cells were distributed throughout the entire CP sublayer (a), with an increased number compared to the control group (b, c). n = 4 mice per group. *p < 0.05 compared to control mice. Data are represented as the mean ± SEM. MZ, marginal zone; CP, cortical plate, IZ, intermediate zone; VZ, ventricular zone.

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In this study, we characterized the functions of TFEB overexpression during embryonic development in conditional TFEB transgenic mice. We showed that hGFAP-directed TFEB overexpression resulted in dramatic neuronal layering and patterning defects. TFEB overexpression in the CNS causes the proliferation of RGCs in the hippocampus and cortical areas to be inhibited, preferring to maintain a quiescent, undifferentiated state and aberrantly inducing neuronal migration, ultimately resulting in significant neuronal layering and patterning defects (Fig. 8). Previous studies have observed that activating TFEB through drug intervention or lentivirus-mediated TFEB injection into the DG region of adult mice enhances lysosomal function and maintains the quiescent state of NSCs [11]. In the adult mouse brain, the suppression of NSC differentiation contributes to preserving the stem cell pool and delays the onset of cognitive impairments [23]. Another study utilized electroporation to inject plasmid DNA encoding TFEB into the lateral ventricle of E14.5 mouse embryos and observed that TFEB overexpression inhibited NSC differentiation. The results of that study suggested that TFEB may perform previously overlooked functions to establish or maintain slow-dividing embryonic NSCs [24]. Our study provides in vivo evidence using transgenic mice that TFEB overexpression inhibits NSC proliferation, leading to abnormal neurogenesis and early mortality in mice, even in heterozygous TFEB-overexpressing mice (which exhibit lower levels of TFEB overexpression). Therefore, the consequences of TFEB overexpression specifically in NSCs need to be carefully considered.

Fig. 8.

Schematic illustration of TFEB overexpression causing RGC proliferation to be inhibited, preferring to maintain a quiescent state and aberrantly inducing neuronal migration and differentiation, ultimately resulting in abnormal hippocampal structure and cortical layering.

Fig. 8.

Schematic illustration of TFEB overexpression causing RGC proliferation to be inhibited, preferring to maintain a quiescent state and aberrantly inducing neuronal migration and differentiation, ultimately resulting in abnormal hippocampal structure and cortical layering.

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During mouse brain development, RGCs, as the primary NSCs, not only undergo self-amplification and renewal but also differentiate into neuronal progenitor cells and direct their migration and differentiation as scaffolding guides to establish the correct arrangement of neuronal layers. At the late stage, particularly after embryonic day 17.5, RGCs primarily generate glial cells, including astrocytes and oligodendrocytes [25]. We found that TFEB overexpression increased the number of RGCs and predisposed them to a quiescent state. Abnormalities in RGC migration and differentiation were observed through the distribution of GFAP+, DCX+, and NeuN+ cells. TFEB is a master transcriptional regulator of autophagy and lysosomal biogenesis. We speculate that the effect of TFEB overexpression on RGC function may be mediated through the activation of lysosomal function or may be caused by TFEB regulating other cellular processes and pathways. Perhaps the activation of lysosomes is just a parallel manifestation during this process. Studies have shown that quiescent NSCs have abundant lysosomes, and TFEB activation of lysosomes inhibits NSC differentiation [26]. Additionally, a study conducted in vitro found that TFEB regulates the pluripotency transcriptional network in mouse embryonic stem cells independently of autophagy-lysosomal biogenesis [27]. The specific mechanisms by which TFEB overexpression affects NSC function and neurodevelopment require further investigation.

The functions of TFEB encompass various biological and physiological processes. TFEB was initially found to be highly expressed in renal cell carcinoma [28]. Though TFEB has been primarily studied for its role in promoting autophagy-lysosomal function, studies have also revealed its involvement in maintaining basal homeostasis, vascular development, tumor occurrence, and other cellular processes [29‒31]. In recent years, several studies on NSCs have presented encouraging results regarding the role of TFEB in promoting NSC functional recovery [32‒34]. However, our results raise concerns about the use of TFEB overexpression as an intervention for therapeutic purposes. Our study, conducted using transgenic mice, better reflects the complexity of the organism’s environment compared to in vitro experiments. Furthermore, we observed hippocampal structural abnormalities and disrupted cortical layering in heterozygous mice on the day of birth, indicating the need for caution when considering TFEB overexpression as a therapeutic intervention.

Our study has several limitations. First, we achieved TFEB overexpression under the control of the GFAP promoter, leading to observations limited to RGC overexpression [35]. We observed neonatal death in mice overexpressing TFEB under the control of the endothelial cell promoter, characterized by systemic cyanosis in mice. Subsequent experiments could explore the effects of CMV promoter-controlled TFEB overexpression on a wider range of cell types. Second, while gene function studies generally employ knockout methods for more convincing results, considering the broad and fundamental functions of TFEB, we speculated that TFEB gene knockout would directly result in mouse death. Third, our study did not further investigate the specific mechanisms involved in TFEB overexpression and its effects. Additional experiments are needed to continue investigating its mechanism.

This study suggests that TFEB overexpression plays a critical role in neural development, affecting multiple pathways involved in NSC proliferation, migration, and differentiation. Our findings provide insights for future research on the role of TFEB overexpression in neurodevelopmental disorders.

This study, conducted through in vivo experiments using transgenic mice, revealed that embryonic overexpression of TFEB in the CNS leads to the development of abnormal hippocampal structure and disrupted cortical layering. Our findings also indicated that TFEB overexpression causes aberrant proliferation, migration, and differentiation of NSCs during embryonic development. Our study suggests that TFEB overexpression may play a detrimental role in embryonic neurogenesis.

All experiments with animals were approved by the Animal Ethics Committee (No. D2021-338) of Lanzhou University Second Hospital and carried out according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health.

The authors declare that they have no competing interests.

This study was supported by the Gansu Province Science Foundation for Youths (No. 21JR7RA425).

Lei Wang, Yuhong Jing, and Yonggang Wang contributed to the conception and design of the work. Lei Wang, Jiaxin Cao, Haichao Chen, Yuezhang Ma, Yishu Zhang, and Xiaomei Su contributed to the acquisition, analysis, and interpretation of data. Lei Wang and Jiaxin Cao drafted the manuscript, and all authors critically reviewed the manuscript before submission.

Additional Information

Lei Wang and Jiaxin Cao contributed equally to this work.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

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