Journal of Neurochemistry最新文献

Neuroinflammation is involved in various neurodegenerative diseases, with glial cells playing crucial roles. It is known that neuroinflammation is initiated by microglia, which interact with astrocytes and neurons. However, the detailed molecular mechanisms underlying intercellular interactions during neuroinflammation are not fully understood. In this study, we developed a tri-culture system of neurons, astrocytes, and microglia derived from human induced pluripotent stem cells (iPSCs) to evaluate their relationships in neuroinflammation. Microglia cocultured with the astrocytes and neurons exhibited a morphology with branched processes compared to the monoculture system, suggesting a homeostatic state. By applying lipopolysaccharide (LPS) stimulation to induce inflammation, the microglial morphology shifted to an amoeboid shape, accompanied by an increase in the expression of pro-inflammatory cytokines. Additionally, nuclear translocation of NF-κB revealed that LPS specifically activates microglia through the TLR4 receptor, which subsequently releases TNF-α, leading to the activation of astrocytes. Furthermore, activated astrocytes were shown to enhance neuronal excitability. Using the tri-culture system, we elucidated a part of the cascade involving microglia, astrocytes, and neurons during neuroinflammation and demonstrated the amplification of inflammatory signals through cell communication. This culture system will be valuable for conducting detailed investigations into the interactions between glia and neurons, advancing research on neurodegenerative diseases associated with neuroinflammation.

Mutations in the SPAST gene, encoding the microtubule-severing protein Spastin, cause the most common type of hereditary spastic paraplegia (HSP): SPG4, a disorder primarily characterized by length-dependent axonal degeneration. Clinically, most SPG4 patients present with a pure phenotype marked by progressive spasticity in the lower extremities. It has also been reported that complex cases exhibit demyelination and cognitive deficits. Additionally, some SPAST variants have been determined in patients with multiple sclerosis (MS), indicating potential shared pathological mechanisms. Spastin is known to promote axonal regeneration by remodeling microtubules, whereas mutant Spastin disrupts microtubule dynamics and causes axonal transport defects in SPG4. However, whether Spastin dysfunction impairs regenerative processes such as myelination remains unknown. In this study, we investigated whether the SPG4-associated SPAST mutations affect axonal myelination. Using an in vitro cortical neuron-oligodendrocyte co-culture model, we found that pathogenic SPAST mutations result in a significant reduction in the myelination index. Furthermore, a cuprizone-induced demyelination mouse model revealed a decrease in Spastin protein levels in demyelinated white matter. Given Spastin's role in axonal regeneration, we hypothesized that Spastin may also protect against demyelination. Supporting this, wild-type Spastin expression protected neurons from demyelination in a cuprizone-induced cell culture demyelination model. Together, these results suggest a role for Spastin in axonal myelination, and its dysfunction may compromise myelin stability. Our findings highlight that impaired myelin stability may represent a secondary pathological feature of SPG4, contributing to disease complexity. This dual role of Spastin in axonal maintenance and myelin stability suggests its potential relevance for contributing to complex forms of SPG4.

Prenatal alcohol exposure (PAE) occurs in 10%-60% of pregnancies and can contribute to fetal alcohol spectrum disorders (FASD). FASD presents with diverse cognitive, behavioral, and motor impairments. Animal studies suggest PAE disrupts fetal iron homeostasis, but direct evidence in the human brain is lacking. Because iron is essential for neurodevelopmental processes including myelination, neurotransmitter synthesis, and energy metabolism, perturbations in iron deposition may represent a modifiable mechanism linking PAE to adverse outcomes. The aim of this study was to explore whether PAE is associated with brain iron levels at age 7 years, assessed using quantitative susceptibility mapping MRI. Children were recruited from the Asking Questions about Alcohol in Pregnancy (AQUA) prospective longitudinal cohort. Participants were categorized as unexposed (no PAE, n = 5), exposed in the first trimester only (PAE T1, n = 14), or exposed across the first to third trimester (PAE T1-3, n = 6). Quantitative susceptibility mapping (QSM), an MRI modality sensitive to iron, was used to estimate regional brain iron across 38 cortical and subcortical regions. Linear models assessed the effects of alcohol exposure and timing of exposure on brain iron, adjusting for age and sex. Compared with unexposed children, those with any PAE had significantly lower QSM in the substantia nigra (β = -18.93, p = 0.011). Stratified analyses revealed that substantia nigra QSM was lower even after first-trimester-restricted exposure (T1 β = -22.63 95% CI [-37.35, -7.91]; p = 0.004). Cortical analyses showed regionally variable alterations, with reductions in the superior parietal cortex (β = -1.33, p = 0.011), insula (β = -1.33, p = 0.051), and pars opercularis (β = -0.926, p = 0.078), and elevations in the postcentral gyrus (β = 2.82, p = 0.003) in those with any PAE compared with unexposed. PAE is associated with region-specific disruptions in brain iron, inferred by QSM, with early exposure particularly affecting the substantia nigra and extended exposure linked to broader cortical and subcortical changes. These exploratory findings provide the first evidence in humans that PAE alters brain iron homeostasis, highlighting iron metabolism as a potentially modifiable pathway contributing to the neurodevelopmental burden of FASD.

Transgenic mouse strains are essential tools in neuroscience, enabling targeted genetic manipulations to investigate brain function and neurological diseases. The NEX-Cre mouse line, which targets glutamatergic principal neurons in the neocortex and hippocampus by expressing Cre-recombinase under the NEX (NeuroD6) promoter, has been widely used for conditional gene manipulation. Contrary to previous reports suggesting no behavioral and histological abnormalities in NEX-Cre mice, our study reveals distinct behavioral and cellular phenotypes. Behavioral analyses indicate reduced anxiety-like behavior, altered reward-related behavior, and increased locomotor activity in NEX (Cre/Cre) mice. Additionally, Support Vector Machine (SVM) analysis uncovered subtle strain-specific and genotype-specific behavioral traits across all NEX-Cre genotypes relative to the commonly used C57BL/6J mouse strain. While overt behavioral abnormalities were most prominent in NEX (Cre/Cre) mice, SVM-based analysis revealed subtle genotype- and strain-specific behavioral signatures across NEX-Cre genotypes. This underlines the importance of using littermate controls rather than independently maintained or purchased C57BL/6J animals when interpreting genotype-related effects. Histological analyses of Golgi-Cox-stained brain slices revealed alterations in dendritic spine density across key brain regions, including the caudate putamen, hippocampal CA1, nucleus accumbens core region, lateral septum, and medial prefrontal cortex. These findings highlight significant inter- and intra-strain variability, emphasizing the importance of careful characterization of transgenic models and the need for appropriate control groups and experimental designs to ensure the reliability and validity of studies utilizing Cre-Driver lines.

Obesity and fatty diets are known to damage the structure and function of chemosensory systems. Consumption of a moderately high-fat diet (MHF) induces loss of olfactory sensory neurons (OSNs) and reduces the density of associated axonal projections to the olfactory bulb that are central in the coding of odor information. Previous work has demonstrated reduced alpha diversity, as well as signature changes in microbiome composition when mice are challenged with a MHF diet that precipitates diet-induced obesity. Herein, we tested the hypothesis that a dysbiotic gut microbiome is sufficient to induce olfactory damage. Male and female donor mice were randomly assigned to a control-fat (CF) or MHF diet for 5 months duration, followed by baseline measurements of body weight, body composition (EchoMRI), glucose tolerance, and metabolic phenotyping via indirect calorimetry. We next performed fecal microbiome transplantation (FMT) from these donors to CF-maintained recipient mice. After 8 weeks post FMT, we observed no difference in body weight, glucose clearance, body composition, or fat pad weights as a consequence of transfer from MHF-maintained donors. Following FMT, recipient male mice exhibited increased Erysipelotrichaceae abundance and decreased Lactobacillaceae abundance, similar to MHF-fed donors. Recipient brains were processed for tissue clearing using immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO) and then imaged using high resolution light sheet microscopy. The volume of olfactory glomeruli expressing Olfr160 odor receptors could be visualized using genetic reporters; the FMT from MHF-maintained donors failed to evoke structural changes to these defined olfactory synapses. We conclude that diet-induced obesity, associated adiposity, and metabolic dysfunctions drive functional loss and structural changes to the olfactory system, but that gut microbiome dysbiosis alone is not sufficient to yield olfactory circuitry deficits.

Stress is a major risk factor for neuropsychiatric disorders. However, how stress influences highly anxious populations and how pre-stress anxiety-related behavior variability mediates stress-elicited molecular responses remain elusive. Here, we investigated the effects of acute stress on the hippocampus in high anxiety-related behavior (HAB) male mice and explored how pre-stress high anxiety-related behavior shapes hippocampal and peripheral molecular signatures following acute stress. We first exposed HAB male mice to acute restraint stress (ARS) and investigated ARS effects on hippocampal proteome. We extensively characterized the pre-stress behavior of HAB mice, ranked them in high and low anxiety HAB subpopulations according to their pre-stress anxiety-related profiles and assessed whether divergent high anxiety-related behavior levels influence molecular stress responses. We found that ARS exerts imperceptible hippocampal proteome effects in HAB mice. However, when we compared high versus low anxiety HAB subpopulations following ARS, we observed profound stress-induced molecular changes in low anxiety ARS versus low anxiety control HAB mice, but not in high anxiety ARS versus high anxiety control HAB mice, predominantly impacting mitochondrial translation. When further exploring pre-stress anxiety variability effects in the presence and absence of stress, we observed that high versus low anxiety HAB subpopulations display divergent molecular profiles only after ARS, but not in its absence, leading to changes in RNA metabolism along with altered mitochondrial dynamics players. Taken together, our data showcase that individual behavioral variability largely shapes molecular stress responses in HAB male populations through modulating mitochondrial pathways.

Brain-derived neurotrophic factor (BDNF) plays a critical role in neuronal development and synaptic plasticity across various maturation stages. However, the extent to which BDNF modulates the neuronal transcriptome to mediate these effects, and the gene clusters most responsive at each culture stage, remain poorly understood. To address this, we investigated the time-dependent effects of BDNF on the transcriptomes of cultured cortical neurons at different culture durations. We found that the magnitude of the transcriptomic response to a 6-h BDNF treatment, relative to untreated controls, increased with longer culture duration. Furthermore, a BDNF-induced shift towards a more mature-like transcriptional state was observed specifically in neurons cultured for shorter durations, suggesting a response dependent on the length of time in culture. Specifically, matrix metalloproteinase 3 (MMP3) was robustly induced by BDNF. Single-nucleus RNA sequencing (snRNA-seq) revealed that this induction was primarily localized to Lhx6-positive inhibitory neurons. Additionally, BDNF regulated the expression of various ligand and receptor genes through a combination of cell type-specific and non-specific mechanisms. These findings provide a comprehensive view of BDNF-mediated transcriptional regulation over the course of cortical neuron culture.

Bipolar disorder (BD) is associated with mood dysregulation and neurobiological abnormalities such as oxidative stress, neuroinflammation, and neurodegeneration. While physical exercise shows promise in mental health, the mechanistic effects of strength training in BD remain poorly understood. This study aimed to investigate the impact of an 8-week strength training protocol on behavioral, oxidative, and cellular alterations in a validated rat model of BD induced by ouabain. Adult male Wistar rats were randomly assigned to sedentary or exercised groups. After the training period, animals underwent surgery for cannula implantation. Following recovery, they received either ouabain or artificial cerebrospinal fluid. Behavioral assessments were conducted during the manic- (Day 7) and depressive-like (Day 14) phases, and tissue samples were collected on Day 18 post-injection. Neurochemical assays and immunohistochemical analyses were performed in the cerebral cortex and hippocampus. Ouabain induced manic- and depressive-like behaviors, cognitive impairments along with oxidative imbalance, increased NF-κB activation, astrogliosis, and neuronal degeneration. Notably, prior strength training prevented these behavioral disturbances and significantly attenuated oxidative stress, neuroinflammation, and cell death. Physical exercise normalized antioxidant enzyme activities, reduced reactive species accumulation, prevented NF-κB activation, and decreased GFAP and Fluoro Jade-C labeling. Correlation analyses revealed significant associations among oxidative stress, inflammation, neurodegeneration, and cognitive impairment. These findings demonstrate, for the first time, that structured strength training exerts neuroprotective effects in a BD model by modulating redox homeostasis, inflammatory signaling, and neuronal integrity. Strength training emerges as a promising, low-cost, and mechanistically grounded adjunctive strategy in BD management.

Tissue-nonspecific alkaline phosphatase (TNAP) has emerged as a crucial regulator of neuronal circuit formation and maintenance; however, the complexities of its sex- and cell type-specific roles within microglia remain largely unexplored. To address this critical knowledge gap, this study examined how TNAP deficiency differentially affects microglial morphology, function, and signaling in both male and female mice, and investigated its broader implications for neurodevelopment and disease susceptibility. Using Alpl^+/+ (wild-type) and Alpl^-/- (TNAP knockout) mice, we conducted behavioral assessments at postnatal Days 13-14 to evaluate early neurobehavioral outcomes. Microglia were subsequently isolated for molecular, metabolic, and morphological analyses. TNAP-deficient mice of both sexes exhibited profound physiological deficits, including stunted growth and significant sensorimotor impairments, confirming effective TNAP knockout and indicating that systemic TNAP loss affects multiple cell types beyond microglia. At the cellular level, TNAP loss induced notable morphological changes in microglia, characterized by enlarged cell soma and shortened processes, hallmarks of microglial activation. Molecular profiling revealed upregulation of neuroinflammatory and phagocytic markers, implicating TNAP as a modulator of the innate immune response. Furthermore, metabolic analyses uncovered a dramatic shift in tryptophan-kynurenine metabolism, with increased quinolinic acid production signifying a transition to a neurotoxic, pro-inflammatory state. Additionally, TNAP-deficient microglia displayed extensive dysregulation in purinergic signaling pathways, exemplified by increased expression of key purinergic receptors, and acquired a senescent phenotype evidenced by elevated canonical senescence gene expression. Given the influence of TNAP deficiency on multiple cell populations, some observed microglial phenotypes may result from altered intercellular signaling or indirect effects. To delineate cell-autonomous effects, siRNA-mediated TNAP knockdown was performed in primary microglia isolated from wild-type (WT) mice. TNAP depletion modulated inflammatory responses, suggesting an intrinsic role for TNAP in microglial regulation; however, these effects may not fully recapitulate the extent of deficiency observed in vivo. Overall, TNAP emerges as a key modulator of microglial structure and function, with its dysfunction potentially increasing susceptibility to neurodevelopmental and neurodegenerative disorders. This highlights the potential of TNAP as a therapeutic target for central nervous system health and disease.

Liquid chromatography with electrochemical detection (LC-ED) after batch alumina extraction has been the mainstay for assaying levels of catecholamines and related 3,4-dihydroxy compounds (catechols) as part of the clinical laboratory workup of patients with neurogenic orthostatic hypotension, especially in the setting of the autonomic synucleinopathies Parkinson disease with orthostatic hypotension (PD + OH), pure autonomic failure (PAF), and multiple system atrophy (MSA). Liquid chromatography with tandem mass spectrometry (LC-MS/MS) is faster and measures catechols and non-catechol metabolites simultaneously but has not yet been validated sufficiently against LC-ED or used to assess catechol vs. non-catechol neurochemical abnormalities in autonomic synucleinopathies. We measured plasma catechols by LC-MS/MS and LC-ED in patients with PAF, PD + OH, or MSA and healthy controls. Cardiac sympathetic neuroimaging by ¹⁸F-dopamine positron emission tomography (PET) was used to indicate myocardial norepinephrine (NE) content in the same subjects. Across 41 participants (12 PAF, 9 PD + OH, 10 MSA, 10 controls) individual values for plasma 3,4-dihydroxyphenylglycol (DHPG), NE, and 3,4-dihydroxyphenylalanine (DOPA) by LC-MS/MS correlated positively with values by LC-ED (r = 0.97, 0.98, and 1.00, p < 0.0001 each). The PAF group had low mean NE, DHPG, normetanephrine, 3-methoxy-4-hydroxyphenylglycol, epinephrine, and metanephrine compared to the PD + OH group, while cardiac PET did not separate the 2 groups. We therefore conclude that LC-MS/MS validly assays plasma catechols. Several catechol and non-catechol biomarkers of generalized catecholamine deficiency separate PAF from PD + OH but not PD + OH from MSA, while ¹⁸F-dopamine PET separates PAF and PD + OH from MSA but not PAF from PD + OH. Combining LC-MS/MS with cardiac sympathetic neuroimaging efficiently differentiates among these conditions.