Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with heterogeneous clinical presentations, including Neuropsychiatric SLE (NPSLE), which comprises a spectrum of central and peripheral nervous system manifestations attributable to immune-mediated neuronal and glial injury. Currently, diagnosing NPSLE is challenging due to the heterogeneous clinical manifestations and the lack of specific biomarkers. Breakthrough biomarkers are essential for improving diagnostic accuracy, prognostic assessment, and therapeutic monitoring in NPSLE. Serum biomarkers have been thoroughly examined, including inflammatory molecules such as cytokines, chemokines, and autoantibodies; however, these biomarkers are not brain-specific and have also been associated with other clinical domains of SLE. The present review focuses on neuronal and glial damage biomarkers in the context of NPSLE, highlighting their potential utility as diagnostic or prognostic biomarkers, while underscoring the need for further research in this area. Here, we discuss correlations between serum and cerebrospinal fluid (CSF) levels, supporting the use of serum as a minimally invasive surrogate for CNS assessment. Furthermore, findings on serum biomarkers of neurological damage were reviewed to explore their associations with clinical, demographic, and routine laboratory variables, which could provide insights into disease mechanisms. We identified potential biomarkers and highlighted important research gaps that may guide future investigations.

IntroductionThe ability to remember locations of objects and make spatial decisions is critical when navigating an environment. Such tasks, however, can be difficult for individuals with certain neurological conditions, such as attention deficit hyperactivity disorder (ADHD). The traveling salesperson problem (TSP), a naturalistic spatial foraging task, has been effectively used to examine these spatial navigational processes in rodents, as this optimization task requires subjects to identify the shortest route of travel between a certain number of targets in an open arena. Previous studies using the TSP task have found that rats with hippocampal or medial entorhinal cortex lesions are impaired on measures of spatial memory, but not spatial decision-making or route selection.MethodsThe current study examined the performance of male and female spontaneously hypertensive rats (SHR), the most widely used rodent model of ADHD, relative to their control model, Wistar Kyoto (WKY) rats, on the TSP task.ResultsOur behavioral findings suggest that both male and female SHRs have greater deficits in route selection compared to WKY rats, but they show intact performance on measures of spatial memory. We also examined microglia expression as a marker of neuroinflammation in the prefrontal cortex, hippocampus, and medial entorhinal cortex. SHRs had a greater percentage of hypertrophic microglia, indicating extended periods of inflammation or activation, in the infralimbic area of the prefrontal cortex and in the dentate gyrus. Within the dentate gyrus, the female SHRs showed an increased percentage of hypertrophic microglia compared to female WKY rats.DiscussionThis study expands on existing literature within ADHD-model male and female rats by exploring their ability to effectively route optimize using a naturalistic task.

The rise of single-cell transcriptomics and comprehensive reference atlases promised a unifying molecular framework to classify cell identity. Yet transcriptomic identities are often interpreted outside the environmental contexts in which they arise. Here, we analyzed primary cortical cultures, which lack native tissue architecture, to compare their transcriptional profiles to multiple in vivo mouse cortical reference datasets. We found that while core molecular signatures for major neuronal subclasses are largely preserved in vitro, the loss of in vivo structure triggers high transcriptional divergence associated with metabolic and physiological state. We also identified clusters that consistently show low confidence in the classification tool. These ambiguous populations express incomplete canonical marker profiles resulting from a lack of structural cues necessary for full maturation. These observations suggest that while transcriptomic reference frameworks capture major aspects of neuronal identity, their interpretation can become less certain when cells are profiled outside their native environment. Our findings highlight the importance of considering environmental context when interpreting transcriptome-based cell type annotations and provide a resource for understanding how neuronal transcriptional programs are reshaped in vitro.

[This retracts the article DOI: 10.3389/fncel.2015.00167.].

Contactin-associated protein-like 2 (CNTNAP2) is one of the largest and most evolutionarily conserved genes in the human genome that increasingly recognized as a pleiotropic and context-dependent regulator of human disorders. Genetic, immunological, and transcriptomic studies have implicated CNTNAP2 in a broad spectrum of neurological and psychiatric disorders, autoimmune encephalitis, and cancer. Early work focused primarily on the full-length isoform CNTNAP2-201, which encodes CASPR2 or CNTNAP2, and plays essential roles in neuronal development, axon-glia interactions, synaptic transmission, interneuron maturation, and maintenance of excitatory-inhibitory balance. Disruption of these functions contributes to impaired cortical connectivity and network dysfunction in neurodevelopmental disorders. Recent discoveries have substantially expanded this view by revealing isoform-specific and proteolytic fragment-dependent functions of CNTNAP2. Proteolytic processing of CNTNAP2 generates bioactive extracellular and intracellular fragments that regulate calcium homeostasis, gene expression, and neuronal network activity. In parallel, the short isoform CNTNAP2-203 has recently emerged as an oncogenic driver in oral squamous cell carcinoma, where its selective upregulation amplifies EGFR-E2F1 signaling and promotes tumor progression. This review synthesizes current knowledge of CNTNAP2 biology, highlighting isoform- and context-specific mechanisms and outlining key unanswered questions relevant to both neurological disease and cancer.

To establish a protocol for the simultaneous isolation and high-purity purification of primary astrocytes and microglia from neonatal rats cerebral cortex. Single-cell suspensions were prepared from cerebral cortices of postnatal day 2 (P2) rat pups. Fibroblasts were pre-removed using differential adhesion techniques. Mixed glial cultures were maintained with graded serum (from 10% to 5% to 2% FBS) to suppress fibroblast proliferation. On day 14, microglia were isolated by constant temperature shaking (200 rpm, 12 h, 37 °C), followed by manual agitation of remaining adherent cells to purify astrocytes. Cell purity was assessed by immunofluorescence (Iba1 and GFAP) and validated by multicolor flow cytometry (CD11b/CD45; ACSA-2) and ER-TR7 fibroblast exclusion staining. Cell viability was evaluated by trypan blue exclusion and CCK-8 assay. Microglial morphology was quantified by cell body area, circularity index, and primary process number. Day 14 was identified as the optimal separation time point. Immediately post-shaking, microglia purity (Iba1+) reached 98.6% ± 1.1%, and astrocyte purity (GFAP+) was 98.4% ± 1.7%. After subsequent purification culture, these values increased to 98.98% ± 1.21% and 98.81% ± 2.38%, respectively. Dual-label immunofluorescence confirmed minimal cross-contamination, with Iba1+/GFAP+ dual-positive cells constituting <1% in both populations. Multicolor flow cytometry corroborated these findings, yielding CD11b+ purity of 97.12% ± 1.58% for microglia (with 95.37% ± 1.84% classified as CD11b+/CD45^low homeostatic microglia) and ACSA-2+ purity of 94.65% ± 2.73% for astrocytes. No unequivocal ER-TR7+ fibroblasts were identified in either purified population. Microglial morphology progressively transitioned from amoeboid (Day 0: area 173.5 ± 32.8 μm2; circularity 0.847 ± 0.058; processes 0.8 ± 0.4) to ramified (Day 5: area 418.2 ± 68.3 μm2; circularity 0.438 ± 0.095; processes 4.3 ± 0.8 per cell). Cell viability remained above 92% following key procedural steps and recovered to over 95% post-purification; CCK-8 assay confirmed full metabolic recovery. This study establishes a combined method utilizing graded serum and constant temperature shaking for glial cell isolation, enabling simultaneous acquisition of both major glial cell types from a single animal. This cost-effective protocol provides a practical tool for functional studies of neuroglial cells.

Current ischemic stroke treatments largely focus on exogenous means of neural repair, with endogenous mechanisms being less understood. Here, we examine the cellular and molecular foundation of an endogenous neuroprotective mechanism using the in vitro stroke model oxygen-glucose deprivation (OGD). We demonstrate that after OGD, dying cortical neurons release ATP to activate microglia. There is a simultaneous increase in microglial release of B-NGF and IL-2, increased TrkA receptor expression on astrocytes, and a consequent downregulation in astrocyte P2Y1 receptors (P2Y1R), resulting in a decline in neuronal intracellular calcium levels and enhanced neuronal survival. This neuroprotective effect is mimicked when P2Y1R expression is directly knocked out in astrocytes or when exogenous microglial activators IL2 or NGF are added in place of microglia. Conversely, these neuroprotective effects are prevented by blockade of microglial activation or inhibition of TrkA or IL-2 receptors. Pharmacological buffering of intracellular Ca2+ with BAPTA-AM recapitulated the neuroprotective effect, whereas NMDA receptor blockade with Dizocilpine maleate did not, indicating that neuronal survival is mediated by reduced intracellular Ca2+ accumulation through an NMDA receptor-independent mechanism. Together, these results suggest the downregulation of P2Y1R in astrocytes by activated microglia is a critical endogenous neuroprotective mechanism after ischemic injury. By understanding these inherent non-cell autonomous mechanisms and their molecular mediators, it may be possible to improve intrinsic neuroprotection and recovery from stroke.

Lipopolysaccharides (LPSs) from Gram-negative bacteria are widely used to model neuroinflammation in vitro and in vivo. However, this paradigm assumes that all LPS chemotypes are uniformly pro-inflammatory, despite significant structural diversity between enterobacterial pathogens and gut-resident commensals. Whether microglia can discriminate among these chemotypes remains largely unexplored. We performed a comparative analysis of canonical Escherichia coli LPS and commensal-derived Phocaeicola vulgatus LPS in murine (BV2) and human (HMC3) microglial cells. Pro-inflammatory mediators were quantified by ELISA, and TLR4-downstream signaling was assessed by western blotting. Conditioned media (CM) from LPS-treated BV2 and HMC3 cells was applied to PC12 neuronal cells to evaluate cell viability and differentiation by immunofluorescence. In BV2 microglial cells, P. vulgatus LPS did not induce nitric oxide (NO) production or iNOS expression. In both BV2 and HMC3 cells, it failed to trigger pro-inflammatory cytokine release or TLR4 pathway activation. CM from E. coli-treated microglia disrupted MAP2 expression in PC12 neurons, whereas media from P. vulgatus-treated microglia did not. Overall, our data argue that "LPS-induced neuroinflammation" is not a universal phenomenon, but a chemistry-dependent outcome shaped by specific LPS structures. This study therefore highlights the need to consider LPS structural diversity in neuroinflammation models, particularly in the context of gut-brain communication.

The axon initial segment (AIS) is essential for initiating action potentials and maintaining neuronal polarity, yet the developmental roles of its core molecular components-Neurofascin 186 (NF186) and Ankyrin G (AnkG)-remain incompletely defined in cerebellar Purkinje cells. Here, we generated Purkinje cell-specific NF186 and AnkG single- and double-knockout mice to investigate how these adhesion and scaffolding proteins cooperatively regulate AIS formation, ion channel localization, synaptic targeting, and neuronal survival. We found that genetic ablation of either Nfasc NF186 (NFKO) or Ankyrin3 (AnkGKO) disrupted assembly and maintenance of the AIS cytoskeleton, and that this defect was exacerbated by combined loss of both proteins during postnatal development. Other AIS-enriched proteins, including βIV Spectrin (βIVSpec), voltage-gated sodium (Nav), and potassium (Kv1.2) channels, failed to properly localize to the AIS and progressively disintegrated between postnatal days 10 and 30. Notably, Kv1.2 clustering at the pinceau synapse was disrupted, and basket cell axons showed misaligned terminal organization, indicating defective inhibitory synapse innervation. By 2 months of age, degeneration of Purkinje cells was evident, accompanied by cerebellar dysfunction. Notably, AnkG ablation caused a progressive postnatal loss of NF186 at the AIS, whereas NF ablation resulted in much slower loss of AnkG at the AIS in Purkinje cells and closely phenocopied the severe AIS destabilization observed in NF/AnkG double-knockout mice. In addition, our RNA-seq analysis revealed that Purkinje cell-specific loss of NF186 predominantly activated immune-inflammatory pathways; AnkG loss significantly disrupted neuronal developmental and metabolic processes; and the dual loss of NF186/AnkG produced transcriptional changes that were distinct from, and in part intermediate to, those observed in NF186 and AnkG single knockout. Collectively, our results show that NF186 and AnkG have complementary, non-redundant roles in establishing and maintaining the Purkinje cell AIS, and that their loss disrupts synaptic organization at the AIS. These findings advance our understanding of AIS development in cerebellar neurons and have implications for diseases involving AIS dysfunction, including cerebellar ataxia and demyelinating neuropathies.