Denise M. O. Ramirez

Recovery after focal motor cortex stroke relies on structural remodeling of neuronal circuits in the perilesional cortex (PLC) and contralesional cortex (CLC). Although transcriptomic studies have identified distinct reactive glial states after stroke, how their spatial organization relates to processes of tissue degeneration and neuronal circuit remodeling remains poorly understood. We used a custom 347-gene Xenium spatial transcriptomics panel to profile approximately 650,000 cells across both hemispheres of male and female mice 7 days after photothrombotic stroke. We identified injury-associated myeloid populations concentrated within the infarct and reactive astrocytes enriched in the glial scar. Distinct reactive populations also extended into spared cortex, including an interferon-associated myeloid population and a reactive astrocyte population that was present in both PLC and CLC. Spatial mapping further revealed elevated neuronal growth-associated gene expression in both regions, particularly among layer 5 extratelencephalic projection neurons, which include corticospinal neurons. Intriguingly, reactive glial populations were enriched in regions exhibiting elevated neuronal growth-associated gene expression, including areas of the CLC containing axons and presynaptic terminals originating from the PLC. Together, these findings reveal a striking spatial association between reactive glial populations and neurons exhibiting elevated growth-associated gene expression and support glial-neuronal interactions as potential targets for promoting circuit remodeling and functional recovery after stroke.

Stroke induces a transient period of heightened plasticity during which functional recovery is most pronounced. Experimental models have identified repair-associated processes in both the ipsilesional and contralesional cortex, indicating that stroke recovery involves regions both remote and near the lesion. However, most transcriptional studies have focused on the infarct core and peri-lesional cortex (PLC), leaving it unclear whether comparable molecular responses occur in the contralesional cortex (CLC), a region that undergoes substantial remodeling in the absence of direct tissue injury, necrosis, or widespread cellular infiltration. In addition, potential sex-dependent differences in these responses remain incompletely defined, despite known influences of biological sex on post-stroke inflammation and vascular remodeling. To address these gaps, we performed bulk RNA-sequencing of the PLC and CLC at 7 days after photothrombotic stroke, a subacute time point associated with the initiation of repair, in male and female mice. Despite distinct positions relative to the lesion, both regions exhibited robust upregulation of inflammatory signaling, including cytokine-, astrocyte-, and myeloid-lineage-associated pathways. The CLC did not demonstrate a distinct region-specific transcriptional profile; instead, shared signatures between PLC and CLC included genes strongly associated with reactive microglial phenotypes. This shared neuroinflammatory response was largely conserved across sexes. Consistent with these findings, male and female mice exhibited comparable corticospinal tract axonal sprouting originating from the CLC at 6 weeks post-stroke. Together, these findings support a shared neuroinflammatory transcriptional response as a prominent early feature of cortical regions associated with post-stroke plasticity.

RNA therapeutics offer promising potential for treating heart disease; however, their clinical application has been limited by insufficient cardiac delivery. Here, we developed a screening platform using human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) to identify human cardiotropic lipid nanoparticles (LNPs). By screening a chemically diverse LNP library, we identified a lead LNP, 18:1 TAP10, with potent human cardiac transfection capability. Using the Ai14 LoxP-Stop-LoxP-tdTomato reporter mouse model, we assessed 18:1 TAP10 LNP delivery of Cre mRNA in vivo via four administration routes (intravenous, intrathoracic, intracoronary, and intramyocardial (IM)) and found that each administration path enabled both the spatial distribution and cellular tropism in the heart to varying degrees. While all routes achieved robust transfection across non-CM populations (up to 40% in endothelial cells), IM injection notably enabled substantial CM transfection (36% at injection site, 13% distally). To demonstrate therapeutic potential, 18:1 TAP10 LNPs were used to deliver adenine base editor (ABE) components to Duchenne muscular dystrophy (DMD) patient-derived hiPSC-CMs, resulting in 80% on-target gene correction and restoration of dystrophin expression. In a humanized DMD mouse model, we observed dystrophin-positive cardiomyocytes in the injected left ventricle following IM administration of LNP-ABE. This work establishes a human cell-based platform for discovering organ-tropic delivery vehicles and highlights LNPs as a potential modality for therapeutic mRNA delivery and gene editing in the heart.

Stroke induces a transient period of heightened plasticity during which functional recovery is most pronounced. Work in rodent models of stroke has identified key mechanisms in both the ipsilesional and contralesional cortex that contribute to functional and structural post-stroke plasticity. To date, most gene expression studies have focused on the infarct core and the immediately surrounding tissue, peri-lesional cortex (PLC). We sought to understand whether the contralesional cortex (CLC), a region that shows robust structural and molecular remodeling despite its distance from the lesion, mounts a distinct transcriptional response to stroke. Direct comparisons of molecular pathways governing these regions, particularly across sexes, are limited. To address this gap, we performed bulk RNA sequencing of the PLC and CLC at 7 days post-stroke, a critical time point for initiating repair, in male and female mice. Our results indicate that despite distinct positions from the lesion, both regions share a robust upregulation of inflammatory signaling, with Gene Ontology enrichment indicating activation of cytokine signaling, leukocyte activation, and gliogenesis pathways. Further analysis of this shared gene expression signature revealed reactive microglia signaling as the dominant pathway. Surprisingly, the CLC did not show a distinct transcriptional response. These findings were consistent across males and females, which also showed similar CLC-derived corticospinal tract axonal sprouting at 6 weeks post-stroke. Together, these findings support a shared microglia-centered neuroinflammatory transcriptional response in the PLC and CLC and suggest that microglial reactivity is a key early process for post-stroke cortical plasticity in both male and female mice.

The corticospinal tract (CST) is essential for forelimb-specific fine motor skills. In rodents, it undergoes extensive structural remodeling across development, injury, and disease states, with major implications for motor function. A vast body of literature, spanning numerous injury models, frequently assesses these projections. Despite this, a cohesive imaging modality for rapid, quantitative assessment of the bilateral cervical spinal cord projectome is lacking. To address this, we developed SpinalTRAQ (Spinal cord Tomographic Registration and Automated Quantification), a novel mouse cervical spinal cord volumetric reference atlas and machine learning-based analytical pipeline. Using serial two-photon tomography, SpinalTRAQ enables unbiased, region-specific quantification of fluorescently labeled CST presynaptic terminals. In healthy male mice, the CST exhibits a distinct bilateral synaptic projectome, with the densest innervation in laminae 5 and 7 on the contralateral side and lamina 7 on the ipsilateral side. We additionally observed sparse synaptic input in lamina 9, specifically axial motor neuron pools, which we found was targeted to spinal motoneurons. Following focal motor cortical stroke, the injured CST axons are depleted, and contralesional CST projections are significantly increased after 4 and 6 weeks. By 6 weeks post-stroke, ipsilateral CST synapses were increased by fivefold, with the greatest increases seen in homotopic laminae and all motor neuron pools. SpinalTRAQ offers detailed, level- and lamina-specific quantification of the bilateral cervical spinal cord synaptic projectome, revealing previously unrecognized CST connectivity and plasticity after injury.

/Summary: Descending corticospinal tract (CST) connections to the neurons of the cervical spinal cord are vital for performance of forelimb-specific fine motor skills. In rodents, CST axons are almost entirely crossed at the level of the medullary decussation. While specific contralateral axon projections have been well-characterized using anatomic and molecular approaches, the field currently lacks a cohesive imaging modality allowing rapid quantitative assessment of the entire, bilateral cervical cord projectome at the level of individual laminae and cervical levels. This is potentially important as the CST is known to undergo marked structural remodeling in development, injury, and disease. We developed SpinalTRAQ ( S pinal cord T omographic R egistration and A utomated Q uantification), a novel volumetric cervical spinal cord atlas and machine learning-driven microscopy acquisition and analysis pipeline that uses serial two-photon tomography-images to generate unbiased, region-specific quantification of the fluorescent pixels of anterograde AAV-labeled CST pre-synaptic terminals. In adult mice, the CST synaptic projectome densely innervates the contralateral hemicord, particularly in laminae 5 and 7, with sparse, monosynaptic input to motoneurons in lamina 9. Motor pools supplying axial musculature in the upper cervical cord are bilaterally innervated. The remainder of the ipsilateral cord has sparse labeling in a distinct distribution compared to the contralateral side. Following a focal stroke of the motor cortex, there is a complete loss of descending corticospinal axons from the injured side. Consistent with prior reports of axon collateralization, the CST spinal projectome increases at four weeks post-stroke and continues to elevate by six weeks post stroke. At six weeks post-stroke, we observed striking synapse formation in the denervated hemicord from the uninjured CST in a homotopic distribution. Additionally, CST synaptic reinnervation increases in the denervated lamina 9 in nearly all motoneuron pools, exhibiting novel patterns of connectivity. Detailed level- and lamina-specific quantification of the bilateral cervical spinal cord synaptic projectome reveals previously undescribed patterns of CST connectivity in health and injury-related plasticity.

Neurodegenerative tauopathies are hypothesized to propagate via brain networks. This is uncertain because we have lacked precise network resolution of pathology. We therefore developed whole-brain staining methods with anti-p-tau nanobodies and imaged in 3D PS19 tauopathy mice, which have pan-neuronal expression of full-length human tau containing the P301S mutation. We analyzed patterns of p-tau deposition across established brain networks at multiple ages, testing the relationship between structural connectivity and patterns of progressive pathology. We identified core regions with early tau deposition, and used network propagation modeling to determine the link between tau pathology and connectivity strength. We discovered a bias towards retrograde network-based propagation of tau. This novel approach establishes a fundamental role for brain networks in tau propagation, with implications for human disease. One-Sentence Summary: Novel whole brain imaging of p-tau deposition reveals retrograde-dominant network propagation in a tauopathy mouse model.

Small extracellular vesicles (sEVs, “exosomes”) in milk have attracted considerable attention for use in delivering therapeutics to diseased tissues because of the following qualities. The production of milk sEVs is scalable, e.g., more than 10²¹ sEVs may be obtained annually from a single cow. Milk EVs protect their cargo against degradation in the gastrointestinal tract and during industrial processing. Milk sEVs and their cargo are absorbed following oral administration and they cross barriers such as intestinal mucosa, placenta and the blood–brain barrier in humans, pigs, and mice. Milk sEVs do no alter variables of liver and kidney function, or hematology, and do not elicit immune responses in humans, rats, and mice. Protocols are available for loading milk sEVs with therapeutic cargo, and a cell line is available for assessing effects of milk sEV modifications on drug delivery. Future research will need to assess and optimize sEV shelf-life and storage and effects of milk sEV modifications on the delivery of therapeutic cargo to diseased tissues.

Human milk contains large amounts of small extracellular vesicles (sEVs) and their microRNA cargos, whereas infant formulas contain only trace amounts of sEVs and microRNAs. We assessed the transport of sEVs across the blood-brain barrier (BBB) and sEV accumulation in distinct regions of the brain in brain endothelial cells and suckling mice. We further assessed sEV-dependent gene expression profiles and effects on the dendritic complexity of hippocampal granule cells and phenotypes of EV depletion in neonate, juvenile and adult mice. The transfer of sEVs across the BBB was assessed by using fluorophore-labeled bovine sEVs in brain endothelial bEnd.3 monolayers and dual chamber systems, and in wild-type newborn pups fostered to sEV and cargo tracking (ECT) dams that express sEVs labeled with a CD63-eGFP fusion protein for subsequent analysis by serial two-photon tomography and staining with anti-eGFP antibodies. Effects of EVs on gene expression and dendritic architecture of granule cells was analyzed in hippocampi from juvenile mice fed sEV and RNA-depleted (ERD) and sEV and RNA-sufficient (ERS) diets by using RNA-sequencing analysis and Golgi-Cox staining followed by integrated neuronal tracing and morphological analysis of neuronal dendrites, respectively. Spatial learning and severity of kainic acid-induced seizures were assessed in mice fed ERD and ERS diets. bEnd.3 cells internalized sEVs by using a saturable transport mechanism and secreted miR-34a across the basal membrane. sEVs penetrated the entire brain in fostering experiments; major regions of accumulation included the hippocampus, cortex and cerebellum. Two hundred ninety-five genes were differentially expressed in hippocampi from mice fed ERD and ERS diets; high-confidence gene networks included pathways implicated in axon guidance and calcium signaling. Juvenile pups fed the ERD diet had reduced dendritic complexity of dentate granule cells in the hippocampus, scored nine-fold lower in the Barnes maze test of spatial learning and memory, and the severity of seizures was 5-fold higher following kainic acid administration in adult mice fed the ERD diet compared to mice fed the ERS diet. We conclude that sEVs cross the BBB and contribute toward optimal neuronal development, spatial learning and memory, and resistance to kainic acid-induced seizures in mice.

Background: Morphea is an autoimmune, sclerosing skin disorder. Despite the recent emphasis on immune dysregulation in morphea, the role of autoantibodies in morphea pathogenesis or utility as biomarkers are poorly defined. Methods: Autoantigen microarray was used to profile autoantibodies from the serum of participants from the Morphea in Adults and Children (MAC) cohort. Clinical and demographic features of morphea patients with myelin basic protein (MBP) autoantibodies were compared to those without. MBP immunohistochemistry staining was subsequently performed in morphea skin to assess for perineural inflammation in areas of staining. Immunofluorescence staining on mouse brain tissue was also performed using patient sera and mouse anti-myelin basic protein antibody to confirm the presence of MBP antibodies in patient sera. Results: Myelin basic protein autoantibodies were found in greater frequency in morphea (n = 50, 71.4%) compared to systemic sclerosis (n = 2, 6.7%) and healthy controls (n = 7, 20%). Patients with MBP antibodies reported pain at higher frequencies. Morphea skin biopsies, highlighted by immunohistochemistry, demonstrated increased perineural inflammation in areas of MBP expression. Immunofluorescence staining revealed an increased fluorescence signal in myelinated areas of mouse brain tissue (i.e. axons) when incubated with sera from MBP antibody-positive morphea patients compared to sera from MBP antibody-negative morphea patients. Epitope mapping revealed target epitopes for MBP autoantibodies in morphea are distinct from those reported in MS, and included fragments 11–30, 41–60, 51–70, and 91–110. Conclusions: A molecular classification of morphea based on distinct autoantibody biosignatures may be used to differentially classify morphea. We have identified anti-MBP as a potential antibody associated with morphea due to its increased expression in morphea compared to healthy controls and systemic sclerosis patients.