Protocol for evaluating mutualistic cooperative behavior in mice using a water-reward task assay.

Social cooperation is fundamentally important for group animals but rarely studied in mice because of their natural aggressiveness. Here, we present a new water-reward assay to investigate mutualistic cooperative behavior in mice. We describe the construction of the apparatus and provide details of the procedures and analysis for investigators to characterize and quantify the mutualistic cooperative behavior. This protocol has been validated in mice and can be used for investigating mechanisms of cooperation. For complete details on the use and execution of this protocol, please refer to Zhang et al. and Wang et al.1,2.

MicroRNA-451a is a candidate biomarker and therapeutic target for major depressive disorder.

Background: Increasing evidence supports the role of microRNAs (miRNAs) in major depressive disorder (MDD), but the pathophysiological mechanism remains elusive. Aims: To explore the mechanism of microRNA-451a (miR-451a) in the pathology and behaviours of depression. Methods: Abnormal miRNAs such as miR-451a reported previously in the serum of patients with MDD were screened and then confirmed in a mouse model of depression induced by chronic restraint stress (CRS). Eight-week-old male C57BL/6 mice had miR-451a overexpression in the medial prefrontal cortex (mPFC) via adeno-associated virus serotype 9 vectors encoding a pri-mmu-miR-451a-GFP fusion protein followed by behavioural and pathological analyses. Finally, molecular biological experiments were conducted to investigate the potential mechanism of miR-451a against depression. Results: The serum levels of miRNA-451a were significantly lower in patients with MDD, with a negative correlation with the Hamilton Depression Scale scores. Additionally, a negative association between serum miR-451a and behavioural despair or anhedonia was observed in CRS mice. Notably, miR-451a expression was significantly downregulated in the mPFC of CRS-susceptible mice. Overexpressing miR-451a in the mPFC reversed the loss of dendritic spines and the depression-like phenotype of CRS mice. Mechanistically, miR-451a could inhibit CRS-induced corticotropin-releasing factor receptor 1 expression via targeting transcription factor 2, subsequently protecting dendritic spine plasticity. Conclusions: Together, these results highlighted miR-451a as a candidate biomarker and therapeutic target for MDD.

Early growth response 2 in the mPFC regulates mouse social and cooperative behaviors.

Adolescent social neglect impairs social performance, but the underlying molecular mechanisms remain unclear. Here we report that isolation rearing of juvenile mice caused cooperation defects that were rescued by immediate social reintroduction. We also identified the transcription factor early growth response 2 (Egr2) in the medial prefrontal cortex (mPFC) as a major target of social isolation and resocialization. Isolation rearing increased corticosteroid production, which reduced the expression of Egr2 in the mPFC, including in oligodendrocytes. Overexpressing Egr2 ubiquitously in the mPFC, but not specifically in neurons nor in oligodendroglia, protected mice from the isolation rearing-induced cooperation defect. In addition to synapse integrity, Egr2 also regulated the development of oligodendroglia, specifically the transition from undifferentiated oligodendrocyte precursor cells to premyelinating oligodendrocytes. In conclusion, this study reveals the importance of mPFC Egr2 in the cooperative behavior that is modulated by social experience, and its unexpected role in oligodendrocyte development.

B lymphocytes ameliorate Alzheimer's disease-like neuropathology via interleukin-35.

Increasing evidence supports the involvement of the peripheral immune system in the pathogenesis of Alzheimer's disease (AD). In the present study, we found that B lymphocytes could mitigate beta-Amyloid (Aß) pathology and memory impairments in a transgenic AD mouse model. Specifically, in young 5 × FAD mice, we evidenced increased B cells in the frontal cortex and meningeal tissues; depletion of mature B cells aggravated these mice's Aß load and memory deficits. The increased B cells produced more interleukin-35 (IL-35) in the front cortex. We further found IL-35 neutralization exacerbated Aß pathology, while injecting IL-35 mitigated Aß load and cognitive dysfunction in 5 × FAD mice with or without mature B cell deficiency. Mechanistically, IL-35 inhibited neuronal BACE1 transcription through modulating the SOCS1/STAT1 pathway, and reduced Aß production accordingly. Reanalysis of the single-cell RNA sequencing data from blood samples of AD patients suggested an increased population of IL-35-producing B cells. Together, the present study revealed a novel effect of B lymphocyte-derived IL-35 on inhibiting Aß production in the frontal cortex, which may serve as a potential target for future AD treatment.

Locomotor Hyperactivity in the Early-Stage Alzheimer's Disease-like Pathology of APP/PS1 Mice: Associated with Impaired Polarization of Astrocyte Aquaporin 4.

Non-cognitive behavioral and psychological symptoms often occur in Alzheimer's disease (AD) patients and mouse models, although the exact neuropathological mechanism remains elusive. Here, we report hyperactivity with significant inter-individual variability in 4-month-old APP/PS1 mice. Pathological analysis revealed that intraneuronal accumulation of amyloid-ß (Aß), c-Fos expression in glutamatergic neurons and activation of astrocytes were more evident in the frontal motor cortex of hyperactive APP/PS1 mice, compared to those with normal activity. Moreover, the hyperactive phenotype was associated with mislocalization of perivascular aquaporin 4 (AQP4) and glymphatic transport impairment. Deletion of the AQP4 gene increased hyperactivity, intraneuronal Aß load and glutamatergic neuron activation, but did not influence working memory or anxiety-like behaviors of 4-month-old APP/PS1 mice. Together, these results demonstrate that AQP4 mislocalization or deficiency leads to increased intraneuronal Aß load and neuronal hyperactivity in the motor cortex, which in turn causes locomotor over-activity during the early pathophysiology of APP/PS1 mice. Therefore, improving AQP4 mediated glymphatic clearance may offer a new strategy for early intervention of hyperactivity in the prodromal phase of AD.

Miconazole Promotes Cooperative Ability of a Mouse Model of Alzheimer Disease.

BACKGROUND: Cooperative defect is 1 of the earliest manifestations of disease patients with Alzheimer disease (AD) exhibit, but the underlying mechanism remains unclear. METHODS: We evaluated the cooperative function of APP/PS1 transgenic AD model mice at ages 2, 5, and 8 months by using a cooperative drinking task. We examined neuropathologic changes in the medial prefrontal cortex (mPFC). Another experiment was designed to observe whether miconazole, which has a repairing effect on myelin sheath, could promote the cooperative ability of APP/PS1 mice in the early AD-like stage. We also investigated the protective effects of miconazole on cultured mouse cortical oligodendrocytes exposed to human amyloid ß peptide (Aß1-42). RESULTS: We observed an age-dependent impairment of cooperative water drinking behavior in APP/PS1 mice. The AD mice with cooperative dysfunction showed decreases in myelin sheath thickness, oligodendrocyte nuclear heterochromatin percentage, and myelin basic protein expression levels in the mPFC. The cooperative ability was significantly improved in APP/PS1 mice treated with miconazole. Miconazole treatment increased oligodendrocyte maturation and myelin sheath thickness without reducing Aß plaque deposition, reactive gliosis, and inflammatory factor levels in the mPFC. Miconazole also protected cultured oligodendrocytes from the toxicity of Aß1-42. CONCLUSIONS: These results demonstrate that mPFC hypomyelination is involved in the cooperative deficits of APP/PS1 mice. Improving myelination through miconazole therapy may offer a potential therapeutic approach for early intervention in AD.

Acquired immunity and Alzheimer's disease.

Alzheimer's disease (AD) is an age-related neurodegenerative disease characterized by progressive cognitive defects. The role of the central immune system dominated by microglia in the progression of AD has been extensively investigated. However, little is known about the peripheral immune system in AD pathogenesis. Recently, with the discovery of the meningeal lymphatic vessels and glymphatic system, the roles of the acquired immunity in the maintenance of central homeostasis and neurodegenerative diseases have attracted an increasing attention. The T cells not only regulate the function of neurons, astrocytes, microglia, oligodendrocytes and brain microvascular endothelial cells, but also participate in the clearance of ß-amyloid (Aß) plaques. Apart from producing antibodies to bind Aß peptides, the B cells affect Aß-related cascades via a variety of antibody-independent mechanisms. This review systemically summarizes the recent progress in understanding pathophysiological roles of the T cells and B cells in AD.

miR-124 regulates early isolation-induced social abnormalities via inhibiting myelinogenesis in the medial prefrontal cortex.

Patients with autism spectrum disorder (ASD) typically experience substantial social isolation, which may cause secondary adverse effects on their brain development. miR-124 is the most abundant miRNA in the human brain, acting as a pivotal molecule regulating neuronal fate determination. Alterations of miR-124 maturation or expression are observed in various neurodevelopmental, neuropsychiatric, and neurodegenerative disorders. In the present study, we analyzed a panel of brain-enriched microRNAs in serums from 2 to 6 year old boys diagnosed with ASD. The hsa-miR-124 level was found significantly elevated in ASD boys than in age and sex-matched healthy controls. In an isolation-reared weanling mouse model, we evidenced elevated mmu-miR-124 level in the serum and the medial prefrontal cortex (mPFC). These mice displayed significant sociability deficits, as well as myelin abnormality in the mPFC, which was partially rescued by expressing the miR-124 sponge in the bilateral mPFC, ubiquitously or specifically in oligodendroglia. In cultured mouse oligodendrocyte precursor cells, introducing a synthetic mmu-miR-124 inhibited the differentiation process through suppressing expression of nuclear receptor subfamily 4 group A member 1 (Nr4a1). Overexpressing Nr4a1 in the bilateral mPFC also corrected the social behavioral deficits and myelin impairments in the isolation-reared mice. This study revealed an unanticipated role of the miR-124/Nr4a1 signaling in regulating early social experience-dependent mPFC myelination, which may serve as a potential therapy target for social neglect or social isolation-related neuropsychiatric disorders.

Drainage of senescent astrocytes from brain via meningeal lymphatic routes.

Recent progress on the central lymphatic system has greatly increased our understanding of how the brain maintains its own waste homeostasis. Here, we showed that perivascular spaces and meningeal lymphatic vessels form a functional route for clearance of senescent astrocytes from the aging brain. Blocking meningeal lymphatic drainage by ligation of the deep cervical lymph nodes impaired clearance of senescent astrocytes from brain parenchyma, subsequently increasing neuroinflammation in aged mice. By contrast, enhancing meningeal lymphatic vessel diameter by a recombinant adeno-associated virus encoding mouse vascular endothelial growth factor-C (VEGF-C) improved clearance of senescent astrocytes and mitigated neuroinflammation. Mechanistically, VEGF-C was highly expressed in senescent astrocytes, contributing themselves to migrate across lymphatic vessels along C-C motif chemokine ligand 21 (CCL21) gradient by interacting with VEGF receptor 3. Moreover, intra-cisternal injection of antibody against CCL21 hampered senescent astrocytes into the lymphatic vessels and exacerbated short memory defects of aged mice. Together, these findings reveal a new perspective for the meningeal lymphatics in the removal of senescent astrocytes, thus offering a valuable target for therapeutic intervention.

Aquaporin 4 deficiency eliminates the beneficial effects of voluntary exercise in a mouse model of Alzheimer's disease.

Regular exercise has been shown to reduce the risk of Alzheimer's disease (AD). Our previous study showed that the protein aquaporin 4 (AQP4), which is specifically expressed on the paravascular processes of astrocytes, is necessary for glymphatic clearance of extracellular amyloid beta (Aß) from the brain, which can delay the progression of Alzheimer's disease. However, it is not known whether AQP4-regulated glymphatic clearance of extracellular Aß is involved in beneficial effects of exercise in AD patients. Our results showed that after 2 months of voluntary wheel exercise, APP/PS1 mice that were 3 months old at the start of the intervention exhibited a decrease in Aß burden, glial activation, perivascular AQP4 mislocalization, impaired glymphatic transport, synapse protein loss, and learning and memory defects compared with mice not subjected to the exercise intervention. In contrast, APP/PS1 mice that were 7 months old at the start of the intervention exhibited impaired AQP4 polarity and reduced glymphatic clearance of extracellular Aß, and the above-mentioned impairments were not alleviated after the 2-month exercise intervention. Compared with age-matched APP/PS1 mice, AQP4 knockout APP/PS1 mice had more serious defects in glymphatic function, Aß plaque deposition, and cognitive impairment, which could not be alleviated after the exercise intervention. These findings suggest that AQP4-dependent glymphatic transport is the neurobiological basis for the beneficial effects of voluntary exercises that protect against the onset of AD.

Microglia prevent beta-amyloid plaque formation in the early stage of an Alzheimer's disease mouse model with suppression of glymphatic clearance.

BACKGROUND: Soluble beta-amyloid (Aß) can be cleared from the brain through various mechanisms including enzymatic degradation, glial cell phagocytosis, transport across the blood-brain barrier, and glymphatic clearance. However, the relative contribution of each clearance system and their compensatory effects in delaying the pathological process of Alzheimer's disease (AD) are currently unknown. METHODS: Fluorescent trace, immunofluorescence, and Western blot analyses were performed to compare glymphatic clearance ability and Aß accumulation among 3-month-old APP695/PS1-dE9 transgenic (APP/PS1) mice, wild-type mice, aquaporin 4 knock out (AQP4-/-) mice, and AQP4-/-/APP/PS1 mice. The consequence of selectively eliminating microglial cells, or downregulating apolipoprotein E (apoE) expression, on Aß burden, was also investigated in the frontal cortex of AQP4-/-/APP/PS1 mice and APP/PS1 mice. RESULTS: AQP4 deletion in APP/PS1 mice significantly exaggerated glymphatic clearance dysfunction, and intraneuronal accumulation of Aß and apoE, although it did not lead to Aß plaque deposition. Notably, microglia, but not astrocytes, increased activation and phagocytosis of Aß in the cerebral cortex of AQP4-/-/APP/PS1 mice, compared with APP/PS1 mice. Selectively eliminating microglia in the frontal cortex via local injection of clodronate liposomes resulted in deposition of Aß plaques in AQP4-/-/APP/PS1 mice, but not APP/PS1 mice. Moreover, knockdown of apoE reduced intraneuronal Aß levels in both APP/PS1 mice and AQP4-/-/APP/PS1 mice, indicating an inhibitory effect of apoE on Aß clearance. CONCLUSION: The above results suggest that the glymphatic system mediated Aß and apoE clearance and microglia mediated Aß degradation synergistically prevent Aß plague formation in the early stages of the AD mouse model. Protecting one or both of them might be beneficial to delaying the onset of AD.

Persistent Malfunction of Glymphatic and Meningeal Lymphatic Drainage in a Mouse Model of Subarachnoid Hemorrhage.

Subarachnoid hemorrhage (SAH) is a devastating cerebrovascular event that often is followed by permanent brain impairments. It is necessary to explore the pathogenesis of secondary pathological damages in order to find effective interventions for improving the prognosis of SAH. Blockage of brain lymphatic drainage has been shown to worsen cerebral ischemia and edema after acute SAH. However, whether or not there is persistent dysfunction of cerebral lymphatic drainage following SAH remains unclear. In this study, autologous blood was injected into the cisterna magna of mice to establish SAH model. One week after surgery, SAH mice showed decreases in fluorescent tracer drainage to the deep cervical lymph nodes (dcLNs) and influx into the brain parenchyma after injection into the cisterna magna. Moreover, SAH impaired polarization of astrocyte aquaporin-4 (AQP4) that is a functional marker of glymphatic clearance and resulted in accumulations of Tau proteins as well as CD3+, CD4+, and CD8+ cells in the brain. In addition, pathological changes, including microvascular spasm, activation of glial cells, neuroinflammation, and neuronal apoptosis were observed in the hippocampus of SAH mice. Present results demonstrate persistent malfunction of glymphatic and meningeal lymphatic drainage and related neuropathological damages after SAH. Targeting improvement of brain lymphatic clearance potentially serves as a new strategy for the treatment of SAH.

Blocking meningeal lymphatic drainage aggravates Parkinson's disease-like pathology in mice overexpressing mutated α-synuclein.

BACKGROUND: Abnormal aggregation of brain α-synuclein is a central step in the pathogenesis of Parkinson's disease (PD), thus, it is reliable to promote the clearance of α-synuclein to prevent and treat PD. Recent studies have revealed an essential role of glymphatic system and meningeal lymphatic vessels in the clearance of brain macromolecules, however, their pathophysiological aspects remain elusive. METHOD: Meningeal lymphatic drainage of 18-week-old A53T mice was blocked via ligating the deep cervical lymph nodes. Six weeks later, glymphatic functions and PD-like phenotypes were systemically analyzed. RESULTS: Glymphatic influx of cerebrospinal fluid tracer was reduced in A53T mice, accompanied with perivascular aggregation of α-synuclein and impaired polarization of aquaporin 4 expression in substantia nigra. Cervical lymphatic ligation aggravated glymphatic dysfunction of A53T mice, causing more severe accumulation of α-synuclein, glial activation, inflammation, dopaminergic neuronal loss and motor deficits. CONCLUSION: The results suggest that brain lymphatic clearance dysfunction may be an aggravating factor in PD pathology.

Aquaporin-4-dependent glymphatic solute transport in the rodent brain.

The glymphatic system is a brain-wide clearance pathway; its impairment contributes to the accumulation of amyloid-ß. Influx of cerebrospinal fluid (CSF) depends upon the expression and perivascular localization of the astroglial water channel aquaporin-4 (AQP4). Prompted by a recent failure to find an effect of Aqp4 knock-out (KO) on CSF and interstitial fluid (ISF) tracer transport, five groups re-examined the importance of AQP4 in glymphatic transport. We concur that CSF influx is higher in wild-type mice than in four different Aqp4 KO lines and in one line that lacks perivascular AQP4 (Snta1 KO). Meta-analysis of all studies demonstrated a significant decrease in tracer transport in KO mice and rats compared to controls. Meta-regression indicated that anesthesia, age, and tracer delivery explain the opposing results. We also report that intrastriatal injections suppress glymphatic function. This validates the role of AQP4 and shows that glymphatic studies must avoid the use of invasive procedures.

Deletion of aquaporin-4 aggravates brain pathology after blocking of the meningeal lymphatic drainage.

The glymphatic pathway and meningeal lymphatic vessels are involved in clearance of metabolic macromolecules from the brain. However, the functional interaction between the two systems in the maintenance of brain homeostasis remains unclear. Here we reported that deletion of aquaporin-4 (AQP4), a functional regulator of glymphatic clearance, aggravated brain pathology of 3 month-old mice after blocking of the meningeal lymphatic drainage for 2 weeks via ligation of the deep cervical lymphatic nodes (LdcLNs). LdcLNs increased total and phosphorylated Tau protein levels in the hippocampus of both genotype mice, but increased hippocampal amyloid beta 1-40 and 1-42 levels only in AQP4 null mice, with up-regulation of beta-site amyloid precursor protein-cleaving enzyme 1 and down-regulation of insulin degrading enzyme. Consistently, LdcLNs caused microglial reactivity and activation of nod-like receptor protein-3 inflammasomes in the AQP4 null hippocampus. These mice also showed hippocampal neuronal apoptosis and declines in exploring and cognitive abilities. Deletion of AQP4, but not LdcLNs, increased brain water content. Together, these findings have revealed respective and interactive roles of the glymphatic system and the dural lymphatic system in maintaining amyloid beta, Tau proteins and water homeostasis in the brain, helping to understand the pathogenesis of neurological diseases associated with mis-accumulation of brain macromolecules.