The molecular neural mechanism underlying the acceleration of brain aging due to Dcf1 deficiency.

Owing to the continuous increase in human life expectancy, the management of aging-related diseases has become an urgent issue. The brain dominates the central nervous system; therefore, brain aging is a key area of aging-related research. We previously uncovered that dendritic cell factor 1 (Dcf1) maintains the stemness of neural stem cells and its expression in Drosophila can prolong lifespan, suggesting an association between Dcf1 and aging; however, the specific underlying neural mechanism remains unclear. In the present study, we show for the first time that hippocampal neurogenesis is decreased in aged Dcf1-/- mice, which leads to a decrease in the number of brain neurons and an increased number of senescent cells. Moreover, astrocytes proliferate abnormally and express elevated mRNA levels of aging-related factors, in addition to displaying increased activation of Akt and Foxo3a. Finally, behavioral tests confirm that aged Dcf1-/- mice exhibit a significant decline in cognitive abilities related to learning and memory. In conclusion, we reveal a novel mechanism underlying brain aging triggered by Dcf1 deficiency at the molecular, cellular, tissue, and behavioral levels, providing a new perspective for the exploration of brain aging.

TMEM59 ablation leads to loss of olfactory sensory neurons and impairs olfactory functions via interaction with inflammation.

The olfactory epithelium undergoes constant neurogenesis throughout life in mammals. Several factors including key signaling pathways and inflammatory microenvironment regulate the maintenance and regeneration of the olfactory epithelium. In this study, we identify TMEM59 (also known as DCF1) as a critical regulator to the epithelial maintenance and regeneration. Single-cell RNA-Seq data show downregulation of TMEM59 in multiple epithelial cell lineages with aging. Ablation of TMEM59 leads to apparent alteration at the transcriptional level, including genes associated with olfactory transduction and inflammatory/immune response. These differentially expressed genes are key components belonging to several signaling pathways, such as NF-κB, chemokine, etc. TMEM59 deletion impairs olfactory functions, attenuates proliferation, causes loss of both mature and immature olfactory sensory neurons, and promotes infiltration of inflammatory cells, macrophages, microglia cells and neutrophils into the olfactory epithelium and lamina propria. TMEM59 deletion deteriorates regeneration of the olfactory epithelium after injury, with significant reduction in the number of proliferative cells, immature and mature sensory neurons, accompanied by the increasing number of inflammatory cells and macrophages. Anti-inflammation by dexamethasone recovers neuronal generation and olfactory functions in the TMEM59-KO animals, suggesting the correlation between TMEM59 and inflammation in regulating the epithelial maintenance. Collectively, TMEM59 regulates olfactory functions, as well as neuronal generation in the olfactory epithelium via interaction with inflammation, suggesting a potential role in therapy against olfactory dysfunction associated with inflamm-aging.

Depression-like symptoms due to Dcf1 deficiency are alleviated by intestinal transplantation of Lactobacillus murine and Lactobacillus reuteri.

Depression, characterized by low mood, is a complex mental disorder that is a serious threat to human health. Depression is thought to be caused by a combination of genetic, environmental and psychological factors. However, the pathophysiology of depression remains unclear. In the present study, we found that Dcf1 knockout (KO) mice had depression-like symptoms and disruptive changes in gamma-aminobutyric acid (GABA) concentration and GABA receptor expression were found in the hippocampus of Dcf1 KO and WT mice. Furthermore, the gut microbiota composition of Dcf1 KO mice was significantly different from that of wildtype (WT) mice and Dcf1 KO mice showed lower Firmicutes and Lactobacillus content compared to WT mice. In addition, the depression-like behavior of Dcf1 KO mice was alleviated by the administration of microbiota. More surprisingly, after treatment with Lactobacillus murine and Lactobacillus reuteri, two Lactobacillus species with proportionally greater differences in content between the WT and KO groups, KO mice showed similar GABA content, as well as restored GABA-related receptor expression, as the WT group. Our data elucidated a possible mechanism of depression induction by gut microbiota in Dcf1 KO mice and provide a new avenue to explore the treatment of depression by gut microbiota.

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.

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 deletion presents alterations in gut microbiota of mice similar to Parkinson's disease.

The gut-brain communication is increasingly being recognized as a profound effector on Parkinson's disease (PD). Gut microbiota changes have become the focus of attention. However, the mechanism leading to changes in the gut microbiota is not clear. In the present study, we found that knockout of Dcf1 (Dcf1-/-) caused changes in the gut microbiota in mice. Results indicated that the increased Proteobacteria (phylum-level) and decreased Prevotellaceae (family-level) in the microbiota composition of Dcf1-/- (KO) mice, which is consistent with the situation of PD patients. On species-level, Prevotellaceae_UCG-001 and Helicobacter_ganmani were significantly different between KO and WT mice, suggesting glycolipid metabolism disorders and inflammatory lesions in KO mice. In the behavior of Y-maze and Open field test, KO mice showed typical PD symptoms such as memory deficits, slowness of movement and anxiety. Further Nissl staining of brain tissue sections confirmed that the deletion of Dcf1 caused damage to amygdala neurons. These results provide a new mechanism for understanding gut microbiota changes, and provide a new basis for PD treatment from a new perspective of Gut-brain axis.

Elucidating the Relationship Between Diabetes Mellitus and Parkinson's Disease Using 18F-FP-(+)-DTBZ, a Positron-Emission Tomography Probe for Vesicular Monoamine Transporter 2.

Diabetes mellitus (DM) and Parkinson's disease (PD) have been and will continue to be two common chronic diseases globally that are difficult to diagnose during the prodromal phase. Current molecular genetics, cell biological, and epidemiological evidences have shown the correlation between PD and DM. PD shares the same pathogenesis pathways and pathological factors with DM. In addition, ß-cell reduction, which can cause hyperglycemia, is a striking feature of DM. Recent studies indicated that hyperglycemia is highly relevant to the pathologic changes in PD. However, further correlation between DM and PD remains to be investigated. Intriguingly, polycystic monoamine transporter 2 (VMAT2), which is co-expressed in dopaminergic neurons and ß cells, is responsible for taking up dopamine into the presynaptic vesicles and can specifically bind to the ß cells. Furthermore, we have summarized the specific molecular and diagnostic functions of VMAT2 for the two diseases reported in this review. Therefore, VMAT2 can be applied as a target probe for positron emission tomography (PET) imaging to detect ß-cell and dopamine level changes, which can contribute to the diagnosis of DM and PD during the prodromal phase. Targeting VMAT2 with the molecular probe 18F-FP-(+)-DTBZ can be an entry point for the ß cell mass (BCM) changes in DM at the molecular level, to clarify the potential relationship between DM and PD. VMAT2 has promising clinical significance in investigating the pathogenesis, early diagnosis, and treatment evaluation of the two diseases.

Dcf1 alleviates C99-mediated deficits in drosophila by reducing the cleavage of C99.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder. The generation of amyloid-ß from the amyloid precursor protein (APP) C-terminal fragment (C99) by γ-secretase cleavage is one of the main pathological mechanisms of AD. Dendritic cell factor 1 (Dcf1) is a membrane protein that was previously found to play a role in the development of AD. Bioinformatic analysis of AD patients indicated that Dcf1 may affect γ-secretase. In this study, we confirmed that Dcf1 attenuates the cleavage of C99 in vivo and in vitro. By using C99 transgenic AD drosophila, we found that Dcf1 reduces the cleavage of C99 by γ-secretase using Dcf1 overexpression. The climbing ability and lifespan of C99 drosophila were significantly increased, while learning and memory were also enhanced with Dcf1 expression. Increased levels of C99 protein in Dcf1-AD drosophila reveals inhibition of C99 cleavage by Dcf1 in vivo. Dcf1 inhibition of γ-secretase was further confirmed in vitro. These results provide a potential therapeutic target for the treatment of AD and also propose a new mechanism for understanding the occurrence of AD.

Kernel Differential Subgraph Analysis to Reveal the Key Period Affecting Glioblastoma.

Glioblastoma (GBM) is a fast-growing type of malignant primary brain tumor. To explore the mechanisms in GBM, complex biological networks are used to reveal crucial changes among different biological states, which reflect on the development of living organisms. It is critical to discover the kernel differential subgraph (KDS) that leads to drastic changes. However, identifying the KDS is similar to the Steiner Tree problem that is an NP-hard problem. In this paper, we developed a criterion to explore the KDS (CKDS), which considered the connectivity and scale of KDS, the topological difference of nodes and function relevance between genes in the KDS. The CKDS algorithm was applied to simulated datasets and three single-cell RNA sequencing (scRNA-seq) datasets including GBM, fetal human cortical neurons (FHCN) and neural differentiation. Then we performed the network topology and functional enrichment analyses on the extracted KDSs. Compared with the state-of-art methods, the CKDS algorithm outperformed on simulated datasets to discover the KDSs. In the GBM and FHCN, seventeen genes (one biomarker, nine regulatory genes, one driver genes, six therapeutic targets) and KEGG pathways in KDSs were strongly supported by literature mining that they were highly interrelated with GBM. Moreover, focused on GBM, there were fifteen genes (including ten regulatory genes, three driver genes, one biomarkers, one therapeutic target) and KEGG pathways found in the KDS of neural differentiation process from activated neural stem cells (aNSC) to neural progenitor cells (NPC), while few genes and no pathway were found in the period from NPC to astrocytes (Ast). These experiments indicated that the process from aNSC to NPC is a key differentiation period affecting the development of GBM. Therefore, the CKDS algorithm provides a unique perspective in identifying cell-type-specific genes and KDSs.

Proteomics and molecular network analyses reveal that the interaction between the TAT-DCF1 peptide and TAF6 induces an antitumor effect in glioma cells.

Glioblastoma is the most lethal brain cancer in adults. Despite advances in surgical techniques, radiotherapy, and chemotherapy, their therapeutic effect is far from significant, since the detailed underlying pathological mechanism of this cancer is unclear. The establishment of molecular interaction networks has laid the foundation for the exploration of these mechanisms with a view to improving therapy for glioblastoma. In the present study, to further explore the cellular role of DCF1 (dendritic cell-derived factor 1), the proteins bound to TAT-DCF1 (transactivator of transcription-dendritic cell-derived factor 1) were identified, and biosystem analysis was employed. Functional enrichment analyses indicate that TAT-DCF1 induced important biological changes in U251 cells. Furthermore, the established molecular interaction networks indicated that TAT-DCF1 directly interacted with TAF6 in glioma cells and with UBC in HEK293T (human embryonic kidney 293T) cells. In addition, further biological experiments demonstrate that TAT-DCF1 induced the activation of the RPS27A/TOP2A/HMGB2/BCL-2 signaling pathway via interaction with TAF6 in U251 cells. Taken together, these findings suggest that the TAT-DCF1 peptide possesses great potential for the development of glioblastoma therapy through the interaction with TAF6-related pathways and provides further theoretic evidence for the mechanisms underlying the antitumor effects of TAT-DCF1.

DNF: A differential network flow method to identify rewiring drivers for gene regulatory networks.

Differential network analysis has become an important approach in identifying driver genes in development and disease. However, most studies capture only local features of the underlying gene-regulatory network topology. These approaches are vulnerable to noise and other changes which mask driver-gene activity. Therefore, methods are urgently needed which can separate the impact of true regulatory elements from stochastic changes and downstream effects. We propose the differential network flow (DNF) method to identify key regulators of progression in development or disease. Given the network representation of consecutive biological states, DNF quantifies the essentiality of each node by differences in the distribution of network flow, which are capable of capturing comprehensive topological differences from local to global feature domains. DNF achieves more accurate driver-gene identification than other state-of-the-art methods when applied to four human datasets from The Cancer Genome Atlas and three single-cell RNA-seq datasets of murine neural and hematopoietic differentiation. Furthermore, we predict key regulators of crosstalk between separate networks underlying both neuronal differentiation and the progression of neurodegenerative disease, among which APP is predicted as a driver gene of neural stem cell differentiation. Our method is a new approach for quantifying the essentiality of genes across networks of different biological states.

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.

Dendritic cell factor 1 deletion leads to developmental defects in mushroom-shaped dendritic spines.

Dendritic spines are divided into four subtypes, namely, Mushroom, Stubby, Thin, and Branched. The mushroom-shaped spines are related to learning and memory. Previous studies have shown that the dendritic cell factor 1 (Dcf1, a transmembrane protein) affects the memory process and regulates the development of dendritic spines by inhibiting the expression of lipocalin 2 (Lcn2, a member of the family containing over 20 small secreted proteins). However, the exact subtype of dendritic spines that are specifically affected by Dcf1 remains unknown. Here, we identified that deletion of Dcf1 leads to developmental defects in mushroom-shaped spines. We provide evidence for memory defects caused by Dcf1-knockout in mice. We discovered and report for the first time that Dcf1 affects the development of mushroom-shaped spines by inhibiting the expression of Lcn2. Further, we demonstrated that environmental enrichment can effectively stimulate Dcf1-knockout mice and rescue development defects in mushroom-shaped spines caused by Dcf1 deletion. Our results provide a novel direction for further studies on dendritic spine development and mechanisms associated with learning and memory.

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.

Zebrafish olfactory receptors ORAs differentially detect bile acids and bile salts.

The fish olfactory receptor ORA family is orthologous to the mammalian vomeronasal receptors type 1. It consists of six highly conserved chemosensory receptors expected to be essential for survival and communication. We deorphanized the zebrafish ORA family in a heterologous cell system. The six receptors responded specifically to lithocholic acid (LCA) and closely related C24 5ß-bile acids/salts. LCA attracted zebrafish as strongly as food in behavioral tests, whereas the less potent cholanic acid elicited weaker attraction, consistent with the in vitro results. The ORA-ligand recognition patterns were probed with site-directed mutagenesis guided by in silico modeling. We revealed the receptors' structure-function relationship underlying their specificity and selectivity for these compounds. Bile acids/salts are putative fish semiochemicals or pheromones sensed by the olfactory system with high specificity. This work identified their receptors and provided the basis for probing the roles of ORAs and bile acids/salts in fish chemosensation.

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.

Dcf1 Deficiency Attenuates the Role of Activated Microglia During Neuroinflammation.

Microglia serve as the principal immune cells and play crucial roles in the central nervous system, responding to neuroinflammation via migration and the execution of phagocytosis. Dendritic cell-derived factor 1 (Dcf1) is known to play an important role in neural stem cell differentiation, glioma apoptosis, dendritic spine formation, and Alzheimer's disease (AD), nevertheless, the involvement of the Dcf1 gene in the brain immune response has not yet been reported. In the present paper, the RNA-sequencing and function enrichment analysis suggested that the majority of the down-regulated genes in Dcf1-/- (Dcf1-KO) mice are immune-related. In vivo experiments showed that Dcf1 deletion produced profound effects on microglial function, increased the expression of microglial activation markers, such as ionized calcium binding adaptor molecule 1 (Iba1), Cluster of Differentiation 68 (CD68) and translocator protein (TSPO), as well as certain proinflammatory cytokines (Cxcl1, Ccl7, and IL17D), but decreased the migratory and phagocytic abilities of microglial cells, and reduced the expression levels of some other proinflammatory cytokines (Cox-2, IL-1ß, IL-6, TNF-α, and Csf1) in the mouse hippocampus. Furthermore, in vitro experiments revealed that in the absence of lipopolysaccharide (LPS), the majority of microglia were ramified and existed in a resting state, with only approximately 10% of cells exhibiting an amoeboid-like morphology, indicative of an activated state. LPS treatment dramatically increased the ratio of activated to resting cells, and Dcf1 downregulation further increased this ratio. These data indicated that Dcf1 deletion mediates neuroinflammation and induces dysfunction of activated microglia, preventing migration and the execution of phagocytosis. These findings support further investigation into the biological mechanisms underlying microglia-related neuroinflammatory diseases, and the role of Dcf1 in the immune response.