Elevated BICD2 DNA methylation in blood of major depressive disorder patients and reduction of depressive-like behaviors in hippocampal Bicd2-knockdown mice.

Major depressive disorder (MDD) is a prevalent and devastating mental illness. To date, the diagnosis of MDD is largely dependent on clinical interviews and questionnaires and still lacks a reliable biomarker. DNA methylation has a stable and reversible nature and is likely associated with the course and therapeutic efficacy of complex diseases, which may play an important role in the etiology of a disease. Here, we identified and validated a DNA methylation biomarker for MDD from four independent cohorts of the Chinese Han population. First, we integrated the analysis of the DNA methylation microarray (n = 80) and RNA expression microarray data (n = 40) and identified BICD2 as the top-ranked gene. In the replication phase, we employed the Sequenom MassARRAY method to confirm the DNA hypermethylation change in a large sample size (n = 1,346) and used the methylation-sensitive restriction enzymes and a quantitative PCR approach (MSE-qPCR) and qPCR method to confirm the correlation between DNA hypermethylation and mRNA down-regulation of BICD2 (n = 60). The results were replicated in the peripheral blood of mice with depressive-like behaviors, while in the hippocampus of mice, Bicd2 showed DNA hypomethylation and mRNA/protein up-regulation. Hippocampal Bicd2 knockdown demonstrates antidepressant action in the chronic unpredictable mild stress (CUMS) mouse model of depression, which may be mediated by increased BDNF expression. Our study identified a potential DNA methylation biomarker and investigated its functional implications, which could be exploited to improve the diagnosis and treatment of MDD.

miR-AB, a miRNA-based shRNA viral toolkit for multicolor-barcoded multiplex RNAi at a single-cell level.

Uncovering the functions of genes in a complex biological process is fundamental for systems biology. However, currently there is no simple and reliable experimental tool available to conduct loss-of-function experiments for multiple genes in every possible combination in a single experiment, which is vital for parsing the interactive role of multiple genes in a given phenotype. In this study, we develop miR-AB, a new microRNA-based shRNA (shRNAmir) backbone for simplified, cost-effective, and error-proof production of shRNAmirs. After verification of its potent RNAi efficiency in vitro and in vivo, miR-AB was integrated into a viral toolkit containing multiple eukaryotic promoters to enable its application in diverse cell types. We further engineer eight fluorescent proteins emitting wavelengths across the entire visible spectrum into this toolkit and use it to set up a multicolor-barcoded multiplex RNAi assay where multiple genes are strongly and reliably silenced both individually and combinatorially at a single-cell level.

Minocycline Activates the Nucleus of the Solitary Tract-Associated Network to Alleviate Lipopolysaccharide-Induced Neuroinflammation.

Objective To examine the neuroanatomical substrates underlying the effects of minocycline in alleviating lipopolysaccharide (LPS)-induced neuroinflammation. Methods Forty C57BL/6 male mice were randomly and equally divided into eight groups. Over three conse-cutive days, saline was administered to four groups of mice and minocycline to the other four groups. Immediately after the administration of saline or minocycline on the third day, two groups of mice were additionally injected with saline and the other two groups were injected with LPS. Six or 24 hours after the last injection, mice were sacrificed and the brains were removed. Immunohistochemical staining across the whole brain was performed to detect microglia activation via Iba1 and neuronal activation via c-Fos. Morphology of microglia and the number of c-Fo-positive neurons were analyzed by Image-Pro Premier 3D. One-way ANOVA and Fisher's least-significant differences were employed for statistical analyses. Results Minocycline alleviated LPS-induced neuroinflammation as evidenced by reduced activation of microglia in multiple brain regions, including the shell part of the nucleus accumbens (Acbs), paraventricular nucleus (PVN) of the hypothalamus, central nucleus of the amygdala (CeA), locus coeruleus (LC), and nucleus tractus solitarius (NTS). Minocycline significantly increased the number of c-Fo-positive neurons in NTS and area postrema (AP) after LPS treatment. Furthermore, in NTS-associated brain areas, including LC, lateral parabrachial nucleus (LPB), periaqueductal gray (PAG), dorsal raphe nucleus (DR), amygdala, PVN, and bed nucleus of the stria terminali (BNST), minocycline also significantly increased the number of c-Fo-positive neurons after LPS administration. Conclusion Minocycline alleviates LPS-induced neuroinflammation in multiple brain regions, possibly due to increased activation of neurons in the NTS-associated network.

Antibiotic-induced microbiome depletion in adult mice disrupts blood-brain barrier and facilitates brain infiltration of monocytes after bone-marrow transplantation.

The crosstalk between intestinal bacteria and the central nervous system, so called "the gut-brain axis", is critically important for maintaining brain homeostasis and function. This study aimed to investigate the integrity of the blood-brain barrier (BBB) and migration of bone marrow (BM)-derived cells to the brain parenchyma after intestinal microbiota depletion in adult mice. Gut microbiota dysbiosis was induced with 5 non-absorbable antibiotics in drinking water in mice that had received bone marrow transplantation (BMT) from green fluorescent protein (GFP) transgenic mice. Antibiotic-induced microbiome depletion reduced expression of tight-junction proteins of the brain blood vessels and increased BBB permeability. Fecal microbiota transplantation of antibiotics treated mice with pathogen-free gut microbiota decreased BBB permeability and up-regulated the expression of tight junction proteins. The BM-derived GFP+ cells were observed to infiltrate specific brain regions, including the nucleus accumbens (NAc), the septal nucleus (SPT) and the hippocampus (CA3). The infiltrated cells acquired a ramified microglia-like morphology and Iba1, a microglia marker, was expressed in all GFP+ cells, whereas they were negative for the astrocyte marker GFAP. Furthermore, treatment with CCR2 antagonist (RS102895) suppressed the recruitment of BM-derived monocytes to the brain. We report for the first time the migration of BM-derived monocytes to the brain regions involved in regulating emotional behaviors after depletion of intestinal microbiota in BMT background mice. However, mechanisms responsible for the migration and functions of the microglia-like infiltrated cells in the brain need further investigation. These findings indicate that monocyte recruitment to the brain in response to gut microbiota dysbiosis may represent a novel cellular mechanism that contributes to the development of brain disorders.

Non-neutralizing antibodies to SARS-Cov-2-related linear epitopes induce psychotic-like behavior in mice.

Objective: An increasing number of studies have reported that numerous patients with coronavirus disease 2019 (COVID-19) and vaccinated individuals have developed central nervous system (CNS) symptoms, and that most of the antibodies in their sera have no virus-neutralizing ability. We tested the hypothesis that non-neutralizing anti-S1-111 IgG induced by the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could negatively affect the CNS. Methods: After 14-day acclimation, the grouped ApoE-/- mice were immunized four times (day 0, day 7, day 14, day 28) with different spike-protein-derived peptides (coupled with KLH) or KLH via subcutaneous injection. Antibody level, state of glial cells, gene expression, prepulse inhibition, locomotor activity, and spatial working memory were assessed from day 21. Results: An increased level of anti-S1-111 IgG was measured in their sera and brain homogenate after the immunization. Crucially, anti-S1-111 IgG increased the density of microglia, activated microglia, and astrocytes in the hippocampus, and we observed a psychomotor-like behavioral phenotype with defective sensorimotor gating and impaired spontaneity among S1-111-immunized mice. Transcriptome profiling showed that up-regulated genes in S1-111-immunized mice were mainly associated with synaptic plasticity and mental disorders. Discussion: Our results show that the non-neutralizing antibody anti-S1-111 IgG induced by the spike protein caused a series of psychotic-like changes in model mice by activating glial cells and modulating synaptic plasticity. Preventing the production of anti-S1-111 IgG (or other non-neutralizing antibodies) may be a potential strategy to reduce CNS manifestations in COVID-19 patients and vaccinated individuals.

Enhanced contextual fear memory in central serotonin-deficient mice.

Central serotonin (5-HT) dysregulation contributes to the susceptibility for mental disorders, including depression, anxiety, and posttraumatic stress disorder, and learning and memory deficits. We report that the formation of hippocampus-dependent spatial memory is compromised, but the acquisition and retrieval of contextual fear memory are enhanced, in central 5-HT-deficient mice. Genetic deletion of serotonin in the brain was achieved by inactivating Lmx1b selectively in the raphe nuclei of the brainstem, resulting in a near-complete loss of 5-HT throughout the brain. These 5-HT-deficient mice exhibited no gross abnormality in brain structures and had normal locomotor activity. Spatial learning in the Morris water maze was unaffected, but the retrieval of spatial memory was impaired. In contrast, contextual fear learning and memory induced by foot-shock conditioning was markedly enhanced, but this enhancement could be prevented by intracerebroventricular administration of 5-HT. Foot shock impaired long-term potentiation and facilitated long-term depression in hippocampal slices in WT mice but had no effect in 5-HT-deficient mice. Furthermore, bath application of 5-HT in 5-HT-deficient mice restored foot shock-induced alterations of hippocampal synaptic plasticity. Thus, central 5-HT regulates hippocampus-dependent contextual fear memory, and 5-HT modulation of hippocampal synaptic plasticity may be the underlying mechanism. The enhanced fear memory in 5-HT-deficient mice supports the notion that 5-HT deficiency confers susceptibility to posttraumatic stress disorder in humans.

Hijacking Dorsal Raphe to Improve Metabolism and Depression-Like Behaviors via BDNF Gene Transfer in Mice.

Moods and metabolism modulate each other. High comorbidity of depression and metabolic disorders, such as diabetes and obesity, poses a great challenge to treat such conditions. Here we report the therapeutic efficacy of brain-derived neurotrophic factor (BDNF) by gene transfer in the dorsal raphe nucleus (DRN) in a chronic unpredictable mild stress model (CUMS) of depression and models of diabetes and obesity. In CUMS, BDNF-expressing mice displayed antidepressant- and anxiolytic-like behaviors, which are associated with augmented serotonergic activity. Both in the diet-induced obesity model (DIO) and in db/db mice, BDNF ameliorated obesity and diabetes, which may be mediated by enhanced sympathetic activity not involving DRN serotonin. Chronic activation of DRN neurons via chemogenetic tools produced similar effects as BDNF in DIO mice. These results established the DRN as a key nexus in regulating depression-like behaviors and metabolism, which can be exploited to combat comorbid depression and metabolic disorders via BDNF gene transfer.

Fat mass and obesity-associated protein regulates RNA methylation associated with depression-like behavior in mice.

Post-transcriptional modifications of RNA, such as RNA methylation, can epigenetically regulate behavior, for instance learning and memory. However, it is unclear whether RNA methylation plays a critical role in the pathophysiology of major depression disorder (MDD). Here, we report that expression of the fat mass and obesity associated gene (FTO), an RNA demethylase, is downregulated in the hippocampus of patients with MDD and mouse models of depression. Suppressing Fto expression in the mouse hippocampus results in depression-like behaviors in adult mice, whereas overexpression of FTO expression leads to rescue of the depression-like phenotype. Epitranscriptomic profiling of N6-methyladenosine (m6A) RNA methylation in the hippocampus of Fto knockdown (KD), Fto knockout (cKO), and FTO-overexpressing (OE) mice allows us to identify adrenoceptor beta 2 (Adrb2) mRNA as a target of FTO. ADRB2 stimulation rescues the depression-like behaviors in mice and spine loss induced by hippocampal Fto deficiency, possibly via the modulation of hippocampal SIRT1 expression by c-MYC. Our findings suggest that FTO is a regulator of a mechanism underlying depression-like behavior in mice.

Inverse changes in telomere length between the blood and brain in depressive-like mice.

BACKGROUND: Telomeres are nucleoprotein complexes located at the end of chromosomes. Previous studies have confirmed that telomere length is reduced in the peripheral blood of depression patients. However, studies regarding whether telomere length is altered in brain regions associated with depression are limited. It remains unclear whether the peripheral blood telomere length indicates telomere variation in the brain. METHODS: Using quantitative PCR, we measured telomere length in five brain regions (prefrontal cortex, amygdala, nucleus accumbens, paraventricular nucleus, and hippocampus) from depressive-like mice and in peripheral blood from depressive-like mice and major depressive disorder (MDD) patients. We also examined the expression of telomerase- and alternative lengthening of telomere (ALT)-related genes in the prefrontal cortex and amygdala of depressive-like mice. RESULTS: Telomeres were shortened in the peripheral blood of depressive-like mice and MDD patients, but were elongated in the prefrontal cortex and amygdala compared with healthy controls. We also observed that the expression of ALT-related genes increased in the prefrontal cortex and amygdala. LIMITATIONS: The amount of human sample was limited. The mechanism of telomere lengthening in the brain of depressive-like mice was not well explained. Mice and humans have inherently different telomere and telomere maintenance systems. CONCLUSION: These findings illustrate that the telomere length in the peripheral blood may not indicate the dynamics of telomere length in the brain. They offer a new perspective on variable telomere length in different brain regions affected in depression and provide a new basis for understanding the relationship between variable telomere length and MDD.

Hepatic progenitor cell activation is induced by the depletion of the gut microbiome in mice.

The homeostasis of the gut microbiome is crucial for human health and for liver function. However, it has not been established whether the gut microbiome influence hepatic progenitor cells (HPCs). HPCs are capable of self-renewal and differentiate into hepatocytes and cholangiocytes; however, HPCs are normally quiescent and are rare in adults. After sustained liver damage, a ductular reaction occurs, and the number of HPCs is substantially increased. Here, we administered five broad-spectrum antibiotics for 14 days to deplete the gut microbiomes of male C57BL/6 mice, and we measured the plasma aminotransferases and other biochemical indices. The expression levels of two HPC markers, SRY-related high mobility group-box gene 9 (Sox9) and cytokeratin (CK), were also measured. The plasma aminotransferase activities were not affected, but the triglyceride, lactate dehydrogenase, low-density lipoprotein, and high-density lipoprotein concentrations were significantly altered; this suggests that liver function is affected by the composition of the gut microbiome. The mRNA expression of Sox9 was significantly higher in the treated mice than it was in the control mice (p < 0.0001), and a substantial expression of Sox9 and CK was observed around the bile ducts. The mRNA expression levels of proinflammatory factors (interleukin [IL]-1ß, IL-6, tumor necrosis factor [TNF]-α, and TNF-like weak inducer of apoptosis [Tweak]) were also significantly higher in the antibiotic-treated mice than the levels in the control mice. These data imply that the depletion of the gut microbiome leads to liver damage, negatively impacts the hepatic metabolism and function, and activates HPCs. However, the underlying mechanisms remain to be determined.

BDNF Alleviates Neuroinflammation in the Hippocampus of Type 1 Diabetic Mice via Blocking the Aberrant HMGB1/RAGE/NF-κB Pathway.

Diabetes is a systemic disease that can cause brain damage such as synaptic impairments in the hippocampus, which is partly because of neuroinflammation induced by hyperglycemia. Brain-derived neurotrophic factor (BDNF) is essential in modulating neuroplasticity. Its role in anti-inflammation in diabetes is largely unknown. In the present study, we investigated the effects of BDNF overexpression on reducing neuroinflammation and the underlying mechanism in mice with type 1 diabetes induced by streptozotocin (STZ). Animals were stereotactically microinjected in the hippocampus with recombinant adeno-associated virus (AAV) expressing BDNF or EGFP. After virus infection, four groups of mice, the EGFP+STZ, BDNF+STZ, EGFP Control and BDNF Control groups, received STZ or vehicle treatment as indicated. Three weeks later brain tissues were collected. We found that BDNF overexpression in the hippocampus significantly rescued STZ-induced decreases in mRNA and protein expression of two synaptic plasticity markers, spinophilin and synaptophysin. More interestingly, BDNF inhibited hyperglycemia-induced microglial activation and reduced elevated levels of inflammatory factors (TNF-α, IL-6). BDNF blocked the increase in HMGB1 levels and specifically, in levels of one of the HMGB1 receptors, RAGE. Downstream of HMGB1/RAGE, the increase in the protein level of phosphorylated NF-κB was also reversed by BDNF in STZ-treated mice. These results show that BDNF overexpression reduces neuroinflammation in the hippocampus of type 1 diabetic mice and suggest that the HMGB1/RAGE/NF-κB signaling pathway may contribute to alleviation of neuroinflammation by BDNF in diabetic mice.

Inverse changes in L1 retrotransposons between blood and brain in major depressive disorder.

Long interspersed nuclear element-1 (LINE-1 or L1) is a type of retrotransposons comprising 17% of the human and mouse genome, and has been found to be associated with several types of neurological disorders. Previous post-mortem brain studies reveal increased L1 copy number in the prefrontal cortex from schizophrenia patients. However, whether L1 retrotransposition occurs similarly in major depressive disorder (MDD) is unknown. Here, L1 copy number was measured by quantitative PCR analysis in peripheral blood of MDD patients (n = 105) and healthy controls (n = 105). The results showed that L1 copy number was increased in MDD patients possibly due to its hypomethylation. Furthermore, L1 copy number in peripheral blood and five brain regions (prefrontal cortex, hippocampus, amygdala, nucleus accumbens and paraventricular hypothalamic nucleus) was measured in the chronic unpredictable mild stress (CUMS) model of depression in mice. Intriguingly, increased L1 copy number in blood and the decreased L1 copy number in the prefrontal cortex were observed in stressed mice, while no change was found in other brain regions. Our results suggest that the changes of L1 may be associated with the pathophysiology of MDD, but the biological mechanism behind dysfunction of L1 retrotransposition in MDD remains to be further investigated.

An association study of the m6A genes with major depressive disorder in Chinese Han population.

BACKGROUND: Major depressive disorder (MDD) is a common, chronic and recurrent mental disease but the precise mechanism behind this disorder remains unknown. FTO is one of the N6-methyladenosine (m6A) modification genes and has recently been found to be associated with depression. N6-methyladenosine (m6A) is the most abundant internal modification on RNA, which is highly enriched within the brain. There are five genes involved in m6A modification including FTO, but whether these m6A modification genes could confer a risk of MDD is still unclear. This study aimed to investigate the genetic influence of the m6A modification genes on risk of MDD. METHODS: We genotyped 23 SNPs in 5 modification genes among 738 patients with MDD and 1098 controls. The UNPHASED program was applied to analyze the genotyping data for allelic and genotypic association with MDD. RESULTS: Of the 23 SNPs selected, rs12936694 from the m6A demethylase gene ALKBH5 showed allelic association (χ(2)=11.19, p=0.0008, OR=1.491, 95%CI 1.179-1.887) and genotypic association (χ(2)=12.26, df=2, p=0.0022) with MDD. LIMITATIONS: Replication and functional study are required to draw a firm conclusion. CONCLUSIONS: The ALKBH5 gene may play a role in conferring risk of MDD in the Chinese population.

Visualizing an emotional valence map in the limbic forebrain by TAI-FISH.

A fundamental problem in neuroscience is how emotional valences are represented in the brain. We know little about how appetitive and aversive systems interact and the extent to which information regarding these two opposite values segregate and converge. Here we used a new method, tyramide-amplified immunohistochemistry-fluorescence in situ hybridization, to simultaneously visualize the neural correlates of two stimuli of contrasting emotional valence across the limbic forebrain at single-cell resolution. We discovered characteristic patterns of interaction, segregated, convergent and intermingled, between the appetitive and aversive neural ensembles in mice. In nucleus accumbens, we identified a mosaic activation pattern by positive and negative emotional cues, and unraveled previously unappreciated functional heterogeneity in the D1- and D2-type medium-spiny neurons, which correspond to the Go and NoGo pathways. These results provide insights into the coding of emotional valence in the brain and act as a proof of principle of a powerful methodology for simultaneous functional mapping of two distinct behaviors.

Adult raphe-specific deletion of Lmx1b leads to central serotonin deficiency.

The transcription factor Lmx1b is essential for the differentiation and survival of central serotonergic (5-HTergic) neurons during embryonic development. However, the role of Lmx1b in adult 5-HTergic neurons is unknown. We used an inducible Cre-LoxP system to selectively inactivate Lmx1b expression in the raphe nuclei of adult mice. Pet1-CreER(T2) mice were generated and crossed with Lmx1b(flox/flox) mice to obtain Pet1-CreER(T2); Lmx1b(flox/flox) mice (which termed as Lmx1b iCKO). After administration of tamoxifen, the level of 5-HT in the brain of Lmx1b iCKO mice was reduced to 60% of that in control mice, and the expression of tryptophan hydroxylase 2 (Tph2), serotonin transporter (Sert) and vesicular monoamine transporter 2 (Vmat2) was greatly down-regulated. On the other hand, the expression of dopamine and norepinephrine as well as aromatic L-amino acid decarboxylase (Aadc) and Pet1 was unchanged. Our results reveal that Lmx1b is required for the biosynthesis of 5-HT in adult mouse brain, and it may be involved in maintaining normal functions of central 5-HTergic neurons by regulating the expression of Tph2, Sert and Vmat2.