Microglia critically shape neuronal circuit development and function, yet their region-specific properties and roles in distinct circuits of the human brain remain poorly understood. In this study, we generated region-specific brain organoids (cortical, striatal, and midbrain), each integrated with human microglia, to fill this critical gap. Single-cell RNA sequencing uncovered six distinct microglial subtypes exhibiting unique regional signatures, including a subtype highly enriched for the GABA _B receptor gene within striatal organoids. To investigate the contributions of microglia to neural circuitry, we created microglia-incorporated midbrain-striatal assembloids, modeling a core circuit node for many neuropsychiatric disorders, including autism. Using chemogenetics to activate this midbrain-striatal circuit, we observed increased calcium signaling in microglia involving GABA _B receptors. Leveraging this model, we examined microglial responses within neural circuits harboring an SCN2A nonsense (C959X) mutation associated with profound autism. Microglia displayed heightened calcium responses to SCN2A mutation–mediated neuronal hyperactivity and engaged in excessive synaptic pruning. These pathological effects were reversed not only by pharmacological inhibition of microglial GABA _B receptors but also by knockout of the GABBR1 gene in microglia. Collectively, our findings establish an advanced platform that can be used to dissect human neuroimmune interactions in subcortical regions and to evaluate previously undiscovered therapies, highlighting the important role of microglia in shaping critical circuitry related to neuropsychiatric disorders.

Neurodegeneration in Alzheimer’s disease (AD) is defined by pathology featuring amyloid-β (Aβ) deposition in the brain. Aβ monomers themselves are generally considered to be nontoxic, but misfold into β-sheets and aggregate to form neurotoxic oligomers. One suggested strategy to treat AD is to prevent the formation of toxic oligomers. The SG inhibitors are a class of pseudopeptides designed and optimized using molecular dynamics (MD) simulations for affinity to Aβ and experimentally validated for their ability to inhibit amyloid-amyloid binding using single molecule force spectroscopy (SMFS). In this work, we provide a review of our previous MD and SMFS studies of these inhibitors and present new cell viability studies that demonstrate their neuroprotective effects against Aβ(1–42) oligomers using mouse hippocampal-derived HT22 cells. Two of the tested SG inhibitors, predicted to bind Aβ in anti-parallel orientation, demonstrated neuroprotection against Aβ(1–42). A third inhibitor, predicted to bind parallel to Aβ, was not neuroprotective. Myristoylation of SG inhibitors, intended to enhance delivery across the blood-brain barrier (BBB), resulted in cytotoxicity. This is the first use of HT22 cells for the study of peptide aggregation inhibitors. Overall, this work will inform the future development of peptide aggregation inhibitors against Aβ toxicity.

The structure and biophysical properties of lipid biomembranes are important for normal function of plasma and organelle membranes, which is essential for proper functioning of living cells. In Alzheimer’s disease (AD) the structure of neuronal membranes becomes compromised by the toxic effect of amyloid- β (A β ) protein which accumulates at neuron synapses, resulting in membrane perforation and dysfunction, oxidative stress and cell death. Melatonin is an important pineal gland hormone that has been shown to be protective against A β toxicity in cellular and animal studies, but the molecular mechanism of this protection is not well understood. It has been shown that melatonin can interact with model lipid membranes and alter the membrane biophysical properties, such as membrane molecular order and dynamics. This effect of melatonin has been previously studied in simple model bilayers with one or two lipid components, we consider a more complex ternary lipid mixture as our membrane model. In this study, we used ² H-NMR to investigate the effect of melatonin on lipid phase behaviour of a three-component model lipid membranes composed of 1,2-dipalmitoyl- sn -glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC) and cholesterol. We used deuterium labelled palmitoyl- d ₃₁ in POPC- d ₃₁ and DPPC- d ₆₂ separately, to probe the changes in hydrocarbon chain order as a function of temperature and varying concentrations of melatonin. We found that melatonin concentration influences phase separation in these ternary mixtures somewhat differently depending on whether POPC- d ₃₁ or DPPC- d ₆₂ was used. At 5 mol% melatonin we observed phase separation in samples with POPC- d ₃₁ , but not with DPPC- d ₆₂ . However, at 10 mol% melatonin phase separation was observed in both samples with either POPC- d ₃₁ or DPPC- d ₆₂ . These results indicate that melatonin can have a strong effect on membrane structure and physical properties, which may provide some clues to understanding how melatonin protects against A β . SIGNIFICANCE: Melatonin has been shown to be protective against A β pathology in animal and cellular studies. Although the mechanism of this protection is not well-understood, melatonin’s membrane-active properties may be important in this regard. In this work solid-state deuterium nuclear magnetic resonance was used to study the effect of melatonin on the POPC/DPPC/cholesterol model membranes. Specifically, we showed that melatonin modifies lipid hydrocarbon chain order to promote phase separation. This knowledge helps to explain the role of melatonin in lipid domain formation and may provide a deeper understanding of the mechanism of melatonin neuroprotection in AD.

Objective: SCN2A pathogenic mutations, such as the recurrent heterozygous Nav1.2‐L1342P, are monogenic causes of epilepsy. In this human‐induced pluripotent stem cell–derived model system, we aim to investigate the molecular and cellular mechanisms underlying SCN2A ‐L1342P‐associated pathology. Methods: Using a human male induced pluripotent stem cell (iPSC) reference line (KOLF) carrying the Nav1.2‐L1342P mutation, we generated three‐dimensional (3D) cortical organoids for functional studies. Patch‐clamp, multi‐electrode array (MEA) recordings, immunocytochemistry, and RNA sequencing were used to characterize the disease phenotypes. Results: Nav1.2‐L1342P organoid neurons displayed increased intrinsic excitability and amplified excitatory post‐synaptic currents, which are consistent with an increase in excitatory synapse formation revealed by SYN1/PSD95 immunostaining. Moreover, elevated network firing activity, as demonstrated by MEA, indicates a pronounced network hyperexcitability. Transcriptomic profiling of organoids carrying the Nav1.2‐L1342P mutation further revealed significant alterations in synaptic, glutamatergic, developmental, and senescence/apoptotic pathways. Significance: Our findings demonstrate that the Nav1.2‐L1342P mutation drives a multifaceted disease phenotype, including network hyperexcitability and disruption of pathways related to neuronal and synaptic functions. These results advance our understanding of SCN2A ‐related developmental and epileptic encephalopathy (DEE), laying a foundation for personalized interventions.

This study uses contact-mode HS-AFM to probe Aβ interactions with neuronal lipid models, demonstrating oligomer-induced bilayer destabilization, especially in low-cholesterol membranes, emphasizing lipid composition and Aβ species dependent damage.