EDTP enhances and protects the fluorescent signal of GFP in cleared and expanded tissues.

Advanced 3D high-resolution imaging techniques are essential for investigating biological challenges, such as neural circuit analysis and tumor microenvironment in intact tissues. However, the fluorescence signal emitted by endogenous fluorescent proteins in cleared or expanded biological samples gradually diminishes with repeated irradiation and prolonged imaging, compromising its ability to accurately depict the underlying scientific problem. We have developed a strategy to preserve fluorescence in cleared and expanded tissue samples during prolonged high-resolution three-dimensional imaging. We evaluated various compounds at different concentrations to determine their ability to enhance fluorescence intensity and resistance to photobleaching while maintaining the structural integrity of the tissue. Specifically, we investigated the impact of EDTP utilization on GFP, as it has been observed to significantly improve fluorescence intensity, resistance to photobleaching, and maintain fluorescence during extended room temperature storage. This breakthrough will facilitate extended hydrophilic and hydrogel-based clearing and expansion methods for achieving long-term high-resolution 3D imaging of cleared biological tissues by effectively safeguarding fluorescent proteins within the tissue.

Morphological analysis of descending tracts in mouse spinal cord using tissue clearing, tissue expansion and tiling light sheet microscopy techniques.

Descending tracts carry motor signals from the brain to spinal cord. However, few previous studies show the full view of the long tracts from a 3D perspective. In this study, we have followed five less well-known tracts that project from midbrain, hindbrain, and cerebellum to the mouse spinal cord, using the tissue clearing method in combination with tiling light sheet microscopy. By tracing axons in spinal cord, we found several notable features: among the five tracts the collateral "sister" branches occurred only in the axons originating from the cerebellospinal tracts; the axons from the spinal trigeminal nucleus crossed the midline of spinal cord to the contralateral side; those arising in the medullary reticular formation ventral part gave many branches in both cervical and lumbar segments; the axons from superior colliculus terminated only at upper cervical but with abundant branches in the hindbrain. Furthermore, we investigated the monosynaptic connections between the tracts and motor neurons in the spinal cord through hydrogel-based tissue expansion, and found several monosynaptic connections between the medullary reticular formation ventral part axons and spinal motor neurons. We believe that this is the first study to show the full 3D scope of the projection patterns and axonal morphologies of these five descending tracts to the mouse spinal cord. In addition, we have developed a new method for future study of descending tracts by three-dimensional imaging.

Dcf1 induces glioblastoma cells apoptosis by blocking autophagy.

BACKGROUND: Dcf1 has been demonstrated to play vital roles in many CNS diseases, it also has a destructive role on cell mitochondria in glioma cells and promotes the autophagy. Hitherto, it is unclear whether the viability of glioblastoma cells is affected by Dcf1, in particular Dcf1 possesses broad localization on different organelles, and the organelles interaction frequently implicated in cancer cells survival. METHODS: Surgically excised WHO grade IV human glioblastoma tissues were collected and cells isolated for culturing. RT-PCR and DNA sequencing assay to estimate the abundance and mutation of Dcf1. iTRAQ sequencing and bioinformatic analysis were performed. Subsequently, immunoprecipitation assay to evaluate the degradation of HistoneH2A isomers by UBA52 ubiquitylation. Transmission electron microscopy (TEM) was applied to observe the structure change of mitochondria and autophagosome. Organelle isolated assay to determine the distribution of protein. Cell cycle and apoptosis were evaluated by flow cytometric assays. RESULTS: Dcf1 was downregulated in WHO grade IV tumor without mutation, and overexpression of Dcf1 was found to significantly regulate glioblastoma cells. One hundred and seventy-six differentially expressed proteins were identified by iTRAQ sequencing. Furthermore, we confirmed that overexpression of Dcf1 destabilized the structure of the nucleosome via UBA52 ubiquitination to downregulate HistoneH2A.X but not macroH2A or HistoneH2A.Z, decreased the mitochondrial DNA copy number and inhibited the mitochondrial biogenesis, thus causing mitochondrial destruction and dysfunction in order to supply cellular energy and induce mitophagy preferentially but not apoptosis. Dcf1 also has disrupted the integrity of lysosomes to block autolysosome degradation and autophagy and to increase the release of Cathepsin B and D from lysosomes into cytosol. These proteins cleaved and activated BID to induce glioblastoma cells apoptosis. CONCLUSIONS: In this study, we demonstrated that unmutated Dcf1 expression is negatively related to the malignancy of glioblastoma, Dcf1 overexpression causes nucleosomes destabilization, mitochondria destruction and dysfunction to induce mitophagy preferentially, and block autophagy by impairing lysosomes to induce apoptosis in glioblastoma.

A preliminary study on abnormal brain function and autistic behavior in mice caused by dcf1 deletion.

Autism is one of the urgent problems in neuroscience. Early research in our laboratory found that dcf1 gene-deficient mice exhibited autistic behavior. Reviewing the literature, we know that the caudate putamen (CPu) brain region is closely related to the occurrence of autism. In this study, we observed that the electrical signal in the abnormal brain region of adult mice was enhanced by using field potential detection for the corresponding brain region. We then used retrovirus markers to track neurons in the CPu brain region and found that there are neural projections in the hippocampus-CPu brain region. Therefore, we selected DREADDs (Designer receptors exclusively activated by designer drugs) to inhibit the abnormal brain region of the mouse and found, through behavioral testing, that this can inhibit the autistic behavior of mice. This research provides new evidence for the understanding of the cause of autism and has accumulated new basis for the treatment of autism. It has theoretical significance and potential application value for the understanding and treatment of autism.

Protocol for constructing a versatile tiling light sheet microscope for imaging cleared tissues.

Here, we describe a protocol to construct, calibrate, and operate a versatile tiling light sheet microscope for imaging cleared tissues. The microscope uses adjustable tiling light sheets to achieve higher spatial resolution and better optical sectioning ability than conventional light sheet microscopes and to image cleared tissues with the cellular to the subcellular spatial resolution. It is compatible with all tissue clearing methods and aligned semiautomatically through the phase modulation of the illumination light. For complete details on the use and execution of this protocol, please refer to Chen et al. (2020).

Deletion of Dcf1 Reduces Amyloid-ß Aggregation and Mitigates Memory Deficits.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disease. One of the pathologies of AD is the accumulation of amyloid-ß (Aß) to form senile plaques, leading to a decline in cognitive ability and a lack of learning and memory. However, the cause leading to Aß aggregation is not well understood. Dendritic cell factor 1 (Dcf1) shows a high expression in the entorhinal cortex neurons and neurofibrillary tangles in AD patients. OBJECTIVE: Our goal is to investigate the effect of Dcf1 on Aß aggregation and memory deficits in AD development. METHODS: The mouse and Drosophila AD model were used to test the expression and aggregation of Aß, senile plaque formation, and pathological changes in cognitive behavior during dcf1 knockout and expression. We finally explored possible drug target effects through intracerebroventricular delivery of Dcf1 antibodies. RESULTS: Deletion of Dcf1 resulted in decreased Aß42 level and deposition, and rescued AMPA Receptor (GluA2) levels in the hippocampus of APP-PS1-AD mice. In Aß42 AD Drosophila, the expression of Dcf1 in Aß42 AD flies aggravated the formation and accumulation of senile plaques, significantly reduced its climbing ability and learning-memory. Data analysis from all 20 donors with and without AD patients aged between 80 and 90 indicated a high-level expression of Dcf1 in the temporal neocortex. Dcf1 contributed to Aß aggregation by UV spectroscopy assay. Intracerebroventricular delivery of Dcf1 antibodies in the hippocampus reduced the area of senile plaques and reversed learning and memory deficits in APP-PS1-AD mice. CONCLUSION: Dcf1 causes Aß-plaque accumulation, inhibiting dcf1 expression could potentially offer therapeutic avenues.

Dcf1 deficiency induces hypomyelination by activating Wnt signaling.

Myelination is extremely important in achieving neural function. Hypomyelination causes a variety of neurological diseases. However, little is known about how hypomyelination occurs. Here we investigated the effect of dendritic cell factor 1(Dcf1) on myelination, using in vitro and in vivo models and found that Dcf1 is essential for normal myelination, motor coordination and balance. Lack of Dcf1 downregulated myelin-associated proteins, such as myelin basic protein (MBP), myelin associated glycoprotein (MAG), and 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in the hippocampus and corpus callosum of Dcf1-null mice, as a result, the myelin sheath of these mice became thinner. Transmission electron microscopy revealed hypomyelination in Dcf1-deficient mice. Motor coordination and balance tests confirmed impaired neurological function in Dcf1-null mice. Gain-of-function analysis via In utero electroporation showed that hypomyelination could be rescued by re-expression of Dcf1 in Dcf1-null mouse brain. Dcf1-null mice exhibited a phenotype similar to that of cuprizone-induced demyelinated mice, thereby supporting the finding of hypomyelination caused by Dcf1 knockout. Mechanistically, we further revealed that insufficient Dcf1 leads to hyperactivation of the Wnt/ß-catenin signaling pathway. Our work describes the role of Dcf1 in maintaining normal myelination, and this could help improve the current understanding of hypomyelination and its pathogenesis.

A Versatile Tiling Light Sheet Microscope for Imaging of Cleared Tissues.

We present a tiling light sheet microscope compatible with all tissue clearing methods for rapid multicolor 3D imaging of cleared tissues with micron-scale (4 × 4 × 10 µm3) to submicron-scale (0.3 × 0.3 × 1 µm3) spatial resolution. The resolving ability is improved to sub-100 nm (70 × 70 × 200 nm3) via tissue expansion. The microscope uses tiling light sheets to achieve higher spatial resolution and better optical sectioning ability than conventional light sheet microscopes. The illumination light is phase modulated to adjust the position and intensity profile of the light sheet based on the desired spatial resolution and imaging speed and to keep the microscope aligned. The ability of the microscope to align via phase modulation alone also ensures its accuracy for multicolor 3D imaging and makes the microscope reliable and easy to operate. Here we describe the working principle and design of the microscope. We demonstrate its utility by imaging various cleared tissues.

Tiling light sheet selective plane illumination microscopy using discontinuous light sheets.

Tiling light sheet selective plane illumination microscopy (TLS-SPIM) improves the 3D imaging ability of SPIM by using real-time optimized tiling light sheets. However, the imaging speed decreases and the raw image size increases due to the tiling process and additional camera exposures. The decreased imaging speed and the increased raw data could cause significant problems when TLS-SPIM is used to image large specimens at high spatial resolutions. Here, we present a novel method to solve the problem. Discontinuous light sheets created by scanning coaxial beam arrays synchronized with the detection camera rolling shutter are used in TLS-SPIM for 3D imaging. It improves the imaging efficiency of TLS-SPIM by reducing the number of tiles required per image plane without influencing the spatial resolution. We investigate the method via numerical simulations and experiments. We demonstrate the imaging ability of the TLS-SPIM using discontinuous light sheets and show the improved imaging efficiency by imaging optically cleared mouse brain.

Dcf1 Affects Memory and Anxiety by Regulating NMDA and AMPA Receptors.

The hippocampus is critical for memory and emotion and both N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl- 4-isoxazolepropionic acid (AMPA) receptors are known to contribute for those processes. However, the underlying molecular mechanisms remain poorly understood. We have previously found that mice undergo memory decline upon dcf1 deletion through ES gene knockout. In the present study, a nervous system-specific dcf1 knockout (NKO) mouse was constructed, which was found to present severely damaged neuronal morphology. The damaged neurons caused structural abnormalities in dendritic spines and decreased synaptic density. Decreases in hippocampal NMDA and AMPA receptors of NKO mice lead to abnormal long term potentiation (LTP) at DG, with significantly decreased performance in the water maze, elevated- plus maze, open field and light and dark test. Investigation into the underlying molecular mechanisms revealed that dendritic cell factor 1 (Dcf1) contributes for memory and emotion by regulating NMDA and AMPA receptors. Our results broaden the understanding of synaptic plasticity's role in cognitive function, thereby expanding its known list of functions.

ATP11B deficiency leads to impairment of hippocampal synaptic plasticity.

Synaptic plasticity is known to regulate and support signal transduction between neurons, while synaptic dysfunction contributes to multiple neurological and other brain disorders; however, the specific mechanism underlying this process remains unclear. In the present study, abnormal neural and dendritic morphology was observed in the hippocampus following knockout of Atp11b both in vitro and in vivo. Moreover, ATP11B modified synaptic ultrastructure and promoted spine remodeling via the asymmetrical distribution of phosphatidylserine and enhancement of glutamate release, glutamate receptor expression, and intracellular Ca2+ concentration. Furthermore, experimental results also indicate that ATP11B regulated synaptic plasticity in hippocampal neurons through the MAPK14 signaling pathway. In conclusion, our data shed light on the possible mechanisms underlying the regulation of synaptic plasticity and lay the foundation for the exploration of proteins involved in signal transduction during this process.

Dendritic cell factor 1 inhibits proliferation and migration and induces apoptosis of neuroblastoma cells by inhibiting the ERK signaling pathway.

Neuroblastoma (NB) is the most common extracranial solid tumor that affects mainly children and has extremely high mortality and recurrence rates. A previous study revealed that dendritic cell factor 1 (DCF1), also called transmembrane protein 59, could activate apoptosis in glioma cells. In the present study, we applied immunofluorescence, western blot analysis, flow cytometry and cell tumorigenicity to investigate the DCF1 mechanisms involved in NB apoptosis. DCF1 was overexpressed in Neuro-2a and SK-N-SH cells through instantaneous transfection. The data revealed that overexpression of DCF1 could inhibit cell proliferation, migration, invasion and promote cell apoptosis in vitro, and suppress NB growth in vivo. The ERK1/2 signaling pathway, which promotes cell survival, was the target of DCF1 in neuroblastoma cells. All the results indicated that DCF1 could be a potential therapeutic target for the understanding and treatment of NB.

ARL3 subcellular localization and its suspected role in autophagy.

ADP-ribosylation factor-like3 (ARL3) is a member of the ADP-ribosylation factor family of GTP-binding proteins that plays important role in regulating Ciliary trafficking. It ubiquitously expressed in normal tissues and tumor cell lines. However, the location and function of ARL3 in organelles are rarely known. In this study, we explored ARL3 subcellular localization in an all-round way in HEK293T, Neuro-2A and U251 cells by density gradient centrifugation and immunofluorescence. The results showed that ARL3 is expressed in most of organelles, and an iodixonal step gradient was further confirmed that ARL3 is mainly localized to the mitochondria, endosomes, lysosomes, and proteasome. By molecular functional analysis, we observed that ARL3 promotes the aggregation of GFP-LC3, up-regulation of LC3-II/LC3-I and down-regulation of SQSMT1/BECN1, and knocking down of ARL3 inbibits autophagy, which suggested that ARL3 is necessary for autophagy. this study presents a comprehensive evaluation of the subcellular localization for ARL3 and provides important on understanding the functions of ARL3.

Degradation of alpha-synuclein by dendritic cell factor 1 delays neurodegeneration and extends lifespan in Drosophila.

Parkinson's disease (PD) is a common neurodegenerative disease associated with the progressive loss of dopaminergic neurons in the substantia nigra. Proteinaceous depositions of alpha-synuclein (α-syn) and its mutations, A30P and A53T, are one important characteristic of PD. However, little is known about their aggregation and degradation mechanisms. Dendritic cell factor 1 (DCF1) is a membrane protein that plays important roles in nerve development in mouse. In this study, we aimed to show that DCF1 overexpression in a PD Drosophila model significantly ameliorates impaired locomotor behavior in third instar larvae and normalizes neuromuscular junction growth. Furthermore, climbing ability also significantly increased in adult PD Drosophila. More importantly, the lifespan dramatically extended by an average of approximately 23%, and surprisingly, DCF1 could prevent α-syn-induced dopaminergic neuron loss by aggregating α-syn in the dorsomedial region of Drosophila. Mechanistically, we confirmed that DCF1 could degrade α-syn both in vivo and in vitro. Our findings revealed an important role of DCF1 in PD process and may provide new potential strategies for developing drugs to treat neurodegenerative diseases.

DCF1 subcellular localization and its function in mitochondria.

Dendritic cell factor 1 (DCF1) is a transmembrane protein that plays important roles in regulating neural stem cell differentiation and dendritic spine formation. Apart from its cytoplasmic functions, DCF1 plays a role in autophagy during the regulation of amyloid precursor proteins. However, the subcellular localization of DCF1 remains unknown. Therefore, in this study, DCF1 tagged with green fluorescent protein was transiently expression in HelaS3 and HEK293T cells. The results showed that DCF1 was widely expressed in different organelles, including the mitochondria, Golgi apparatus, endoplasmic reticulum, endosomes and lysosomes. An iodixanol step gradient further confirmed that DCF1 is localized to the mitochondria, endosomes, lysosomes, endoplasmic reticulum, and proteasome. Finally, functional analysis of the mitochondria revealed that DCF1 affected the expression and localization of MGST1. This study presents a comprehensive evaluation of the subcellular localization of DCF1, which provides important information on complex functions mediated by DCF1.

Dcf1 Triggers Dendritic Spine Formation and Facilitates Memory Acquisition.

Dendritic spines, a special kind of structure in nerve cells, play a key role in performing cellular function. Structural abnormalities of the dendritic spine may contribute to synaptic dysfunction and have been implicated in memory formation. However, the molecular mechanisms that trigger dendritic spine loss remain unclear. Here, we show that the absence of dendritic cell factor 1 (Dcf1) appeared dendritic spines dysplasia, which in turn leads to the damage of learning and memory; in contrast, enhancing Dcf1 expression rescues dendritic spines morphology and function, indicating a pivotal role of Dcf1 in cellular function. Electrophysiological test indicates that there is a significant reduction in the frequency of miniature excitatory postsynaptic currents in Dcf1 -/- knockout (KO) mice. Subsequent to optogenetic ignition, we observed a weaker neuronal activation in Dcf1 KO mice, explaining the neural circuit cause. On molecular mechanism, we demonstrated an unprecedented discovery that Dcf1 triggers the dendritic spine and synaptic function through the recruitment of Lcn2 and activation of PSD95-NMDAR signaling. Removing this brake leads to memory damage. Our results highlight an unexpected regulatory mechanism of dendritic spine development and formation.

A biosystems approach to identify the molecular signaling mechanisms of TMEM30A during tumor migration.

Understanding the molecular mechanisms underlying cell migration, which plays an important role in tumor growth and progression, is critical for the development of novel tumor therapeutics. Overexpression of transmembrane protein 30A (TMEM30A) has been shown to initiate tumor cell migration, however, the molecular mechanisms through which this takes place have not yet been reported. Thus, we propose the integration of computational and experimental approaches by first predicting potential signaling networks regulated by TMEM30A using a) computational biology methods, b) our previous mass spectrometry results of the TMEM30A complex in mouse tissue, and c) a number of migration-related genes manually collected from the literature, and subsequently performing molecular biology experiments including the in vitro scratch assay and real-time quantitative polymerase chain reaction (qPCR) to validate the reliability of the predicted network. The results verify that the genes identified in the computational signaling network are indeed regulated by TMEM30A during cell migration, indicating the effectiveness of our proposed method and shedding light on the regulatory mechanisms underlying tumor migration, which facilitates the understanding of the molecular basis of tumor invasion.

LK6/Mnk2a is a new kinase of alpha synuclein phosphorylation mediating neurodegeneration.

Parkinson's disease (PD) is a movement disorder due to the loss of dopaminergic (DA) neurons in the substantia nigra. Alpha-synuclein phosphorylation and α-synuclein inclusion (Lewy body) become a main contributor, but little is known about their formation mechanism. Here we used protein expression profiling of PD to construct a model of their signalling network from drsophila to human and nominate major nodes that regulate PD development. We found in this network that LK6, a serine/threonine protein kinase, plays a key role in promoting α-synuclein Ser129 phosphorylation by identification of LK6 knockout and overexpression. In vivo test was further confirmed that LK6 indeed enhances α-synuclein phosphorylation, accelerates the death of dopaminergic neurons, reduces the climbing ability and shortens the the life span of drosophila. Further, MAP kinase-interacting kinase 2a (Mnk2a), a human homolog of LK6, also been shown to make α-synuclein phosphorylation and leads to α-synuclein inclusion formation. On the mechanism, the phosphorylation mediated by LK6 and Mnk2a is controlled through ERK signal pathway by phorbolmyristate acetate (PMA) avtivation and PD98059 inhibition. Our findings establish pivotal role of Lk6 and Mnk2a in unprecedented signalling networks, may lead to new therapies preventing α-synuclein inclusion formation and neurodegeneration.

Overexpression of DCF1 inhibits glioma through destruction of mitochondria and activation of apoptosis pathway.

Gliomas are the most common brain tumors affecting the central nervous system and are associated with a high mortality rate. DCF1 is a membrane protein that was previously found to play a role in neural stem cell differentiation. In the present study, we found that overexpression of dcf1 significantly inhibited cell proliferation, migration, and invasion and dramatically promoted apoptosis in the glioblastoma U251 cell line. DCF1 deletion mutations in the functional region showed that the complete structure of DCF1 was necessary for apoptosis. Furthermore, significantly lower tumorigenicity was observed in athymic nude mice by transplanting U251 cells overexpressing dcf1. To decode the apoptosis induced by dcf1, mitochondrial structure and membrane potential in glioma cells were investigated and the results indicated obvious mitochondrial swelling, destruction of cristae, and a significant decline in membrane potential. Mechanismly, caspase-3 signaling was activated. Finally, endogenous dcf1 silence in U251 cells was investigated. Results showed a highly methylation at -1339 and -1322 position at dcf1 promoter sequence, revealing the causal relationship between dcf1 gene and tumorigencicity. The present study identified a previously unknown cancer apoptosis mechanism involving dcf1 overexpression and provided a novel approach to potentially treat glioma patients.