Levi B. Wood

Abstract Introduction Sleep disruption (SD) negatively impacts several aspects of cognitive function and increases the risk of developing dementia, including Alzheimer’s disease (AD). Synapses are particularly vulnerable to sleep loss and therefore represent a major target to prevent cognitive dysfunction. SD impairs cognition by altering synaptic molecular composition, ultrastructure, and function. Extensive evidence indicates that SD effects are especially detrimental when sleep loss is chronic. This study aims to investigate the impact of chronic SD on brain transcriptomic and cellular compartment-specific proteomic signatures to identify genes, proteins, and pathways that might confer AD risk. Methods Male wild-type mice (6.5-month-old) were randomly assigned to an undisrupted sleep control group and an SD group (N=10/group). For chronic SD, mice were placed into automated sleep fragmentation chambers that include a swipe bar, set to move every 30 sec for six hours per day, over six weeks. Next day, mice were euthanized and brains were quickly removed, bulk brain tissue was snap frozen and crude synaptosomes (P2 fraction) were prepared immediately by differential centrifugation. RNA was isolated from frozen brain tissue and then analyzed by RNAseq to identify differentially-enriched genes (DEGs). Brain homogenate and P2 fraction proteins were then analyzed by label-free quantitative mass spectrometry to identify differentially-enriched proteins (DEPs) and biological pathways by gene set variation analysis. Results Chronic SD mainly increased the gene expression of chaperones/heat shock proteins associated with cellular stress and the unfolded protein response. In contrast with a subtle transcriptional response, the effects of chronic SD on the brain proteome were much greater (518 DEPs in homogenate). Remarkably, chronic SD exerted unique proteomic effects on the synapse (556 DEPs in P2 fraction): Increased DEPs include cognitive resilience proteins, suggesting a compensatory mechanism, while reduced DEPs include mitochondrial proteins, potentially representing synaptic energy failure. The p38 MAPK pathway, implicated in the development and progression of AD, was uniquely increased in the synaptic compartment, supporting p38 inhibition as a neuroprotective strategy for improving synaptic pathology induced by chronic SD. Conclusion These results nominate synaptic-specific candidates for future mechanistic validation that might help clarify chronic SD effects linked to synapse dysfunction and AD risk. Support (if any) NIH R01AG071587

The tail suspension test (TST) is a well-known rodent behavioral test that assesses stress and depressive-like behavior. While several automatic tail suspension test scoring programs have emerged, many researchers still prefer a manual scoring method for accuracy and reliability. However, manual scoring can introduce significant errors. Thus, in this work, we present a novel graphical user interface that assists in the manual scoring process to minimize possibility for errors. The GUI, which we refer to as "TST Score Helper," minimizes errors through consolidation of the TST scoring procedure into a single cohesive program. Further, a rescore mode enhances rigor by enabling comparison of two different scorers' mobility status timelines and rereview of periods of disagreement. In a cohort of 64 male and 45 female mice subject to closed head injury or sham injury, we demonstrate the challenges with manual scoring and we characterize performance of the TST Score Helper program. The results show how this program can reduce sources of manual scoring error and improve the fidelity of results.

Mild traumatic brain injuries (mTBI) can substantially impact quality of life, and repetitive mTBIs (rmTBI) can amplify injury effects compared to a single injury. However, effective clinical treatments remain elusive, largely due to an incomplete understanding of the underlying injury mechanisms. Neuroinflammation has emerged as a key contributor to worse functional outcomes after mTBI/rmTBI. While microglia are traditionally viewed as primary mediators of post-injury inflammation, accumulating evidence suggests neurons play an immunomodulatory role in initiating the rmTBI inflammatory cascade through activation of intracellular proinflammatory pathways like p38 MAPK and secretion of cytokines that, in turn, stimulate microglial activation. Here, we tested whether inducible neuronal p38α knockout protects against functional, immune, and cerebrovascular consequences of a weight-drop closed head injury model of rmTBI. A battery of functional assays was conducted 4 weeks post-injury, and tissues were collected at both 4 hours and 4 weeks following final CHI. In males, neuronal p38α knockout protected against injury-induced depressive-like behavior, hyperactivity, synaptic loss, microglial reactivity, cytokine upregulation, and reduction in cerebral blood flow. In females, neuronal p38α knockout protected against risk-taking behavior and partially protected against cytokine upregulation but had limited effect on microglial reactivity and cerebral blood flow. Together, these findings identify neuronal p38α as a sex-dependent driver of rmTBI-associated neurological consequences, and they support neuronal p38α-immune signaling as a mechanistically relevant therapeutic target for future studies.

Mild traumatic brain injury (mTBI) is underdiagnosed and can lead to long-term symptoms in children. Currently, we lack diagnostic markers and effective therapies for pediatric mTBI. MicroRNAs (miRNAs) show promise for diagnosing and treating pediatric mTBIs due to their role in regulating key biological processes. Cyclosporine A (CsA) has also shown therapeutic effectiveness in modulating neuronal recovery and protection. We aimed at studying miRNA changes in the frontal lobe (FL) and hippocampus + amygdala (H&A) after a sagittal rapid non-impact head rotation (RNR) in 4 week old piglets (N = 50) at 1 day post-injury, 1 week post-injury, and following 1 day of 20 mg/kg/day Cyclosporine A (CsA) treatment compared to anesthesia-only shams. Interestingly, many miRNAs involved in disrupted neuronal and upregulated glial, epithelial, and endothelial functions were differentially expressed (DE-miRNAs) at 1 day post-injury and most returned to baseline by 1 week post-injury. However, miR-20a-3p, miR-10386, and miR-4331-3p were among the Top 10 DE-miRNAs at 1 day post-injury, and remained altered at 1 week. Upregulated neuroprotective miR-17-3p and miR-212 were also part of the Top 10 DE-miRNAs at 1 day post-injury, and their increases were correlated with decreases in axonal injury. Furthermore, to identify miRNAs that could serve as candidate diagnostic biomarkers of injury, we employed LASSO analysis and identified miRNAs miR-363, miR-15b, and miR-450c-3p as the best predictors of mTBI at 1 day post-injury. Lastly, WGCNA revealed the possible neuroprotective effects of CsA treatment in ameliorating neuronal, immune, stress, and vascular functions disrupted at 1 day post-injury. Overall, our data revealed key miRNAs that were differentially expressed at 1 day and 1 week post-injury, modulated by CsA treatment, and suggest that they may serve as diagnostic biomarkers of pediatric mTBI.

While previous work has shown a sustained protective effect of levodopa (L-DOPA) on retinal function in early-stage diabetic retinopathy (DR) in humans, its underlying biology is unknown. Using noninvasive measures in diabetic mice, we found L-DOPA protects retinal neurovascular function as measured by oscillatory potential timing and flicker-evoked retinal vasodilation, as well as visual behavior, for at least two weeks past treatment end. Assessing changes in retinal gene expression, differentially expressed genes were broadly comparable between diabetic mice experiencing washout of L-DOPA versus continued L-DOPA treatment, with gene co-expression network analysis identifying distinct modules across L-DOPA-treated diabetic mice associated with synaptic function and cytoskeletal organization that correlated with functional protection. Together, these findings demonstrate that L-DOPA restores and sustains retinal neurovascular function in early DR and links this protection to transcriptional programs supporting synapse activity and structural integrity.

Repetitive mild traumatic brain injuries (rmTBI) sustained within a window of vulnerability can lead to long-term neurological impairments. Following mechanical impact, increasing evidence suggests that the brain undergoes an inflammatory cascade, leading to chronic neuroinflammation and persistent neurological deficits. While the p38 MAPK signaling pathway is implicated in severe TBI, its role in rmTBI is unclear. This study investigated whether small molecule inhibition of p38 MAPK with SB239063 reduces inflammatory response and functional deficits after repetitive mTBI in a mouse weight-drop model. In females, p38 MAPK inhibition reduced synaptic loss, cytokine upregulation, microglial reactivity, functional deficits, and transcript alterations while upregulating protective pathways. In males, p38 MAPK inhibition attenuated microglial changes and transcript alterations but had limited functional effects. Together, these findings suggest the role of p38 MAPK in driving injury consequences in a sex-dependent manner and highlight therapeutic potential for p38 MAPK inhibition after repetitive mTBI.

BACKGROUND: In Alzheimer's Disease (AD) microglia transition their phenotype from homeostatic to a potentially destructive disease associated phenotype (DAM). Prior research identified the ERK1/2 protein as being hyperactivated in microglia isolated from an AD mouse model. ERK1/2 is activated by the binding of upstream receptor tyrosine receptors (RTK) with their corresponding ligand(s). Further, DAM development is a progressive process, where cells transition from a homeostatic to DAM phenotype over time. METHOD: A single nucleus RNA sequencing dataset from a 5xFAD mouse model was obtained from the Gene Expression Omnibus database (GSE140511). 3 wildtype (WT) and 3 5xFAD mice samples were used for this work. The transcriptomes from these samples were processed with Seurat (v.5.0.3). The microglial population of cells was identified and loaded into NicheNetR (v2.1.0) to identify ligands suspected of driving the differential gene expression in the 5xFAD microglia. Next, a single nuclear trajectory was established using Monocle3 (v1.3.7), where the homeostatic microglia were labeled as the pseudotime origin. RESULT: Several RTK ligands were identified by NicheNet as having high likelihood of driving the DAM phenotype gene expression (Figure 1A). They include Csf1, Tgfb1, and Gas6, which are largely expressed by microglia. Notably, each of these ligands have been established as altering microglia behavior outside of AD. The Monocle3 trajectory of DAM development was then used to identify the stage in DAM development that the ligand may regulate. Csf1 and Tgfb1 changed as a function of pseudotime, shown in Figure 1B. Csf1 signaling began early in DAM development and plateaued later. Alternatively, Tgfb1 expression declined early in DAM development and further declined in the later stage. These trends align with what is known about each ligand in microglia, where Csf1 is proinflammatory and Tgfb1 is anti-inflammatory. Gas6 was generally expressed at the same level over time. This ligand is still suspected of regulating DAM, just not at a specific point of development. CONCLUSION: This analysis has identified three ligands for RTKs that are potential drivers of DAM and change expression temporally. Given their apparent role in DAM development, these ligands have therapeutic potential in AD through DAM modulation.

Vascular morphogenesis requires persistent endothelial cell motility that is responsive to diverse and dynamic mechanical stimuli. Here, we interrogated the mechanotransductive feedback dynamics that govern endothelial cell motility and vascular morphogenesis. We show that the transcriptional regulators, YAP and TAZ, are activated by mechanical cues to transcriptionally limit cytoskeletal and focal adhesion maturation, forming a conserved mechanotransductive feedback loop that mediates human endothelial cell motility in vitro and zebrafish intersegmental vessel (ISV) morphogenesis in vivo. This feedback loop closes in 4 hours, achieving cytoskeletal equilibrium in 8 hours. Feedback loop inhibition arrested endothelial cell migration in vitro and ISV morphogenesis in vivo. Inhibitor washout at 3 hrs, prior to feedback loop closure, restored vessel growth, but washout at 8 hours, longer than the feedback timescale, did not, establishing lower and upper bounds for feedback kinetics in vivo. Mechanistically, YAP and TAZ induced transcriptional suppression of RhoA signaling to maintain dynamic cytoskeletal equilibria. Together, these data establish the mechanoresponsive dynamics of a transcriptional feedback loop necessary for persistent endothelial cell migration and vascular morphogenesis.

Vascular morphogenesis requires persistent endothelial cell motility that is responsive to diverse and dynamic mechanical stimuli. Here, we interrogated the mechanotransductive feedback dynamics that govern endothelial cell motility and vascular morphogenesis. We show that the transcriptional regulators, YAP and TAZ, are activated by mechanical cues to transcriptionally limit cytoskeletal and focal adhesion maturation, forming a conserved mechanotransductive feedback loop that mediates human endothelial cell motility in vitro and zebrafish intersegmental vessel (ISV) morphogenesis in vivo. This feedback loop closes in 4 hours, achieving cytoskeletal equilibrium in 8 hours. Feedback loop inhibition arrested endothelial cell migration in vitro and ISV morphogenesis in vivo. Inhibitor washout at 3 hrs, prior to feedback loop closure, restored vessel growth, but washout at 8 hours, longer than the feedback timescale, did not, establishing lower and upper bounds for feedback kinetics in vivo. Mechanistically, YAP and TAZ induced transcriptional suppression of RhoA signaling to maintain dynamic cytoskeletal equilibria. Together, these data establish the mechanoresponsive dynamics of a transcriptional feedback loop necessary for persistent endothelial cell migration and vascular morphogenesis.

BACKGROUND/AIMS: Potassium channels regulate membrane potential, calcium flux, cellular activation and effector functions of adaptive and innate immune cells. The voltage-activated Kv1.3 channel is an important regulator of T cell-mediated autoimmunity and microglia-mediated neuroinflammation. Kv1.3 channels, via protein-protein interactions, are localized with key immune proteins and pathways, enabling functional coupling between K+ efflux and immune mechanisms. METHODS: To gain insights into proteins and pathways that interact with Kv1.3 channels, we applied a proximity-labeling proteomics approach to characterize protein interactors of the Kv1.3 channel in activated T-cells. Biotin ligase TurboID was fused to either N or C termini of Kv1.3, stably expressed in Jurkat T cells, and biotinylated proteins in proximity to Kv1.3 were enriched and quantified by mass spectrometry. RESULTS: We identified over 1,800 Kv1.3 interactors including known interactors (beta-integrins, Stat1), although the majority were novel. We found that the N-terminus of Kv1.3 preferentially interacts with protein synthesis and protein trafficking machinery, while the C-terminus interacts with immune signaling and cell junction proteins. T-cell Kv1.3 interactors we found consisted of 335 cell surface proteins, including T-cell receptor complex, mitochondrial, calcium and cytokine-mediated signaling pathway, and lymphocyte migration proteins. 178 Kv1.3 interactors in T-cells also represent genetic risk factors for T cell-mediated autoimmunity, including STIM1, which was further validated using co-immunoprecipitation. CONCLUSION: Our studies revealed novel proteins and molecular pathways that interact with Kv1.3 channels in adaptive (T-cell) and innate (microglia) immune cells, providing a foundation for understanding how Kv1.3 channels may regulate immune mechanisms in autoimmune.

The progressive aggregation of amyloid beta (Aβ) monomers into oligomers is a critical factor in Alzheimer's disease (AD) pathogenesis. Although mutated forms of Aβ have been shown to display altered aggregation dynamics, the specific effects of deamidated Aβ on microglial function remain understudied. Our research group previously found that the deamidated variant Aβ-42-N27D modified Aβ aggregation, reduced neurotoxicity, and reduced microglial reactivity, but the impact of Aβ-42 side chain deamidation in general on such parameters remained unclear. Here, we expanded on our prior work by investigating how two site-specific Aβ-42 mutations (Q15E & N27D), where neutral amide side chains are replaced with negatively charged carboxylic acids, affect aggregation and microglial immune response using a mouse microglial cell line. Size exclusion chromatography revealed that Aβ-42-Q15E and Aβ-42-N27D exhibit distinct aggregation profiles compared to Aβ-42 wild type (WT). Multiplexed analysis of 8 cytokines secreted into the culture medium revealed that Aβ-42-Q15E and Aβ-42-N27D decrease the expression of inflammatory cytokines such as IL-6, IP-10, and MIP-1α relative to Aβ-42-WT. Immunocytochemistry revealed that Aβ-42-Q15E and Aβ-42-N27D decrease CD68 expression relative to Aβ-42-WT. These findings demonstrate that deamidation significantly alters Aβ-42 aggregation and microglial activation, suggesting structural modifications to Aβ-42 modulate inflammatory signaling in AD. This work provides a foundation for future studies on Aβ-42 post-translational modifications as potential therapeutic targets in AD.S.

Abstract The therapeutic efficacy of human mesenchymal stromal cells (hMSCs) is highly variable, limiting their clinical translation for musculoskeletal diseases and other regenerative medicine applications. There is a poor understanding of the critical quality attributes correlating to therapeutic efficacy of hMSCs. To address this challenge, we analyzed pre-clinical in vitro secretome profiles and in vivo therapeutic efficacy of hMSCs from multiple human donors. hMSCs from different donors showed significant differences between donors in therapeutic efficacy when assessed in a rat post-traumatic osteoarthritis (OA) model. A partial least squares feature learning model was trained to evaluate differences between more and less therapeutic donor hMSCs by examining cytokine secretion profiles, to predict donor-specific therapeutic outcomes. More therapeutic hMSCs exhibited increased secretion of GM-CSF, GRO, IL-4, and PDGF-AA, whereas less therapeutic donors had higher TNF-α, IL-6, and MCP-1 secretion. The cytokine profile was accompanied by evaluation of MAPK pathway, which revealed distinct differences in phospho-protein signaling between more and less therapeutic hMSC secretome profiles. Pharmacological inhibition of JNK signaling in more therapeutic donor cells decreased hMSC secretion of the key therapeutic associated cytokines and shifted hMSC secretome towards a less therapeutic profile. Prospective validation of cells from additional donors demonstrated significant correlations between predicted and observed pre-clinical in vivo efficacy to attenuate OA. This approach identifies critical quality attributes enabling consistent prediction of therapeutic potency, thereby addressing a major barrier to scalable and effective cell therapies. These findings advance precision cell-based therapies and offer a framework for standardized donor screening in clinical applications. Summary A feature learning model was developed, trained, and validated to identify critical quality attributes of MSCs that predict therapeutic potency.

Background: Understanding synapse-specific effects of neuroinflammation can provide mechanistic and therapeutically relevant insights across the spectrum of neurological diseases. Methods: model of systemic lipopolysaccharide (LPS) dosing, we examined the effects of neuroinflammation on whole neuron and synaptic compartments using a combination of MS, network analysis, confirmatory biochemical and ultrastructural assays and integrative approaches across our mouse-derived and existing human datasets. Results: Ultrastructural and biochemical analyses of P2 fractions verified enrichment in synaptic elements, including synaptic vesicles and mitochondria. MS of biotinylated proteins from Camk2a-specific bulk brain homogenates (whole neuron) and P2 fractions (synaptosome) showed enrichment of >1000 proteins, consistent with neuron-specific biotinylation, also confirmed by immunofluorescence microscopy. Camk2a-specific synaptic proteome revealed molecular signatures related to mitochondrial function, synaptic transmission, protein translation. LPS-treated mice displayed body weight loss and neuroinflammation, characterized by glial activation, increased pro-inflammatory cytokine levels and upregulated expression of Alzheimer's disease (AD)-related microglial genes. LPS-induced neuroinflammation exerted distinct effects on the synaptic proteome, including increased mitochondrial and reduced cytoskeletal-synaptic proteins, while suppressed synaptic MAPK signaling. Importantly, these changes were not observed at the whole neuron level, indicating unique vulnerability of the synapse to neuroinflammation. In line with synapse proteomic and signaling changes, LPS altered the ultrastructure of asymmetric synapses, suggesting dysregulation of excitatory neurotransmission. Co-expression network analysis of Camk2a neuronal proteins further resolved mitochondria- and synapse-specific protein modules, some of which were neuroinflammation-dependent. Neuroinflammation increased levels of a mitochondria-enriched module, and decreased levels of a pre-synaptic vesicle module, without impacting a post-synaptic membrane module. LPS-dependent mitochondrial and LPS-independent post-synaptic modules in mouse neurons mapped to post-mortem human AD brain proteomic modules which were decreased in cases with AD dementia and positively correlated to cognitive function, including pro-resilience markers for AD. Conclusion: Our findings using native-state proteomics of Camk2a neurons combined with synaptosome enrichment identify proteome-level mechanisms of early synaptic vulnerability to neuroinflammation relevant to AD.

Alzheimer's disease (AD) is a growing global challenge that imposes a tremendous burden on society and economies. Though recently approved anti-amyloid β (Aβ) immunotherapies show effectiveness in clearing amyloid and slowing cognitive decline, the removal of cerebral Aβ can also cause serious adverse events (SAEs). Therefore, decreasing the detrimental effects of Aβ in the brain without promoting SAEs is an unmet need in AD treatment. Here, we show that deamidation of Asparagine 27(N27) in Aβ1-42 can significantly reduce Aβ's neurotoxicity and decrease selective microglial pro-inflammatory cytokine production. We also show that deamidation of N27 produces a pronounced decrease in Aβ's aggregation propensity and decreases soluble oligomer formation, suggesting a potential mechanism for its mitigation of Aβ's detrimental cellular effects. Modulation of these Aβ properties by N27 deamidation represents a proof of concept for a potential strategy to alter the detrimental effects of Aβ that may not require its removal from the brain. Our findings on reducing Aβ's toxic properties by N27 deamidation may provide a basis for future therapeutic interventions.

Abstract Rett syndrome (RTT) is a neurodevelopmental disorder caused by MECP2 mutations. Like other genetic neurodevelopmental disorders, it lacks molecular biomarkers to evaluate disease and therapeutic outcomes. We present a strategy to define biomarkers of MeCP2 dysfunction in brain with potential to delineate mechanisms and monitor therapeutic interventions. This strategy relies on a library of proteins responsive to Mecp2 gene dosage and correlated with molecular and clinical outcomes after AAV9-mediated MECP2 gene therapy in Mecp2 -KO mice. Gene rescue restored MeCP2 in brain, improved clinical phenotypes, and reverted transcriptome and proteome abnormalities. We identified 327 shared proteins among 1852 cortical and hippocampal proteins responsive to Mecp2 / MECP2 . Of these, 119 also displayed Mecp2/MECP2-dependent transcript changes. Both the Mecp2-responsive proteome and transcript–protein pairs were enriched in synaptic and metabolic pathways, including central carbon and NAD+ metabolism. We used this therapy-responsive protein library to guide selection of candidate cerebrospinal fluid (CSF) biomarkers in RTT. CSF composition from neurotypical and RTT groups was analyzed using ultrasensitive nucleic acid-based multiplexed ELISA. Twenty-eight proteins were altered in RTT, nine overlapping with Mecp2 dosage- and therapy-sensitive proteins. Multivariate regression linked several candidates to Mecp2 / MeCP2 abundance and phenotypic improvement in mice. This paradigm provides a rigorous molecular systems-level framework integrating genetics, preclinical gene therapy, and clinical metrics to define robust cross-species biomarkers and mechanisms in RTT, with potential applicability to other neurodevelopmental disorders. One Sentence Summary Genetic Identification of cross-species biomarkers and mechanisms in Rett Syndrome

Bone fracture repair initiates by periosteal expansion. The periosteum is a bilayered tissue composed of inner cambium and outer fibrous layers. Typically quiescent, periosteal progenitor cells proliferate upon fracture; however, the underlying transcriptional mechanisms remain unclear. Here, we show that deletion of the transcriptional regulators, yes-associated protein (YAP) and transcriptional coactivator with PDZ binding motif (TAZ), from Osterix-expressing cells, which reside in the cambium, impairs periosteal expansion. YAP activation increases chromatin accessibility, preferentially at TEA domain transcription factor (TEAD) binding sites, and regulates both cell-intrinsic and cell-extrinsic cellular functions. We identify bone morphogenetic protein 4 ( Bmp4 ) as a YAP-TEAD target gene expressed in the cambium. In YAP/TAZ knockout mice, BMP4 delivery increased periosteal expansion through matrix accumulation and fibrous layer cell proliferation. Conversely, in wild-type mice, BMP4 delivery increased osteogenic activity and angiogenesis. Together, these data identify YAP-mediated transcriptional programs that promote layer-specific periosteal expansion.

Chronic stress is a major risk factor for neuropsychiatric disorders, acting via increased neuroinflammation and disrupted synaptic plasticity. While non-invasive visual or audiovisual neurostimulation (AV flicker) at 40Hz has been shown to modulate brain immune signaling and improve cognitive performance in mouse models of Alzheimer's disease, its effects in the context of stress remain unknown. Here we show that AV flicker protects against stress-induced behavioral, microglial, astrocytic, and synaptic changes in a sex- and frequency-specific manner. Male and female mice underwent 28 days of chronic unpredictable stress with concomitant daily AV flicker exposure at 10Hz, 20Hz, or 40Hz. Stress-induced behaviors were most effectively mitigated by 10Hz AV flicker in males and 40Hz AV flicker in females. In the medial prefrontal cortex, AV flicker normalized the balance of mature and immature dendritic spines and counteracted stress-induced molecular changes in neurons, microglia, and astrocytes, including in key neuropsychiatric risk genes. These findings show that frequency optimized AV flicker induces resilience to chronic stress.

Current clinical trials are investigating gamma frequency sensory stimulation as a potential therapeutic strategy for Alzheimer's disease (AD); yet, we lack a comprehensive picture of the effects of this stimulation on multiple aspects of brain function. We previously showed that exposing mice to visual flickering stimulation increased mitogen activated protein kinase and nuclear factor kappa-light-chain-enhancer of activated B cells signaling in the visual cortex (VC) in a manner dependent on the duration and frequency of stimulation. Because these pathways control multiple neuronal and glial functions, here we aimed to define the transcriptional effects of different frequencies and durations of audiovisual flicker (AV flicker) stimulation on multiple brain functions. Within the VC, we found that all stimulation frequencies caused fast activation of a module of immune genes within 0.5 h and slower suppression of synaptic genes after 4 h. In the hippocampus, we found that a 20 Hz AV flicker activated a module of genes associated with mitochondrial function, metabolism, and synaptic translation, while 10 Hz rapidly suppressed a module of genes linked to neurotransmitter activity. Collectively, our data indicate that the frequency and duration of AV flicker stimulation control immune, neuronal, and metabolic genes in multiple regions of the brain affected by AD.

Background: Mild traumatic brain injury (mTBI) can cause long-term functional impairments, and repetitive mTBIs within a window of vulnerability can exacerbate these consequences compared to a single mTBI. However, current interventions for mTBI focus on alleviating symptoms, rather than targeting underlying mechanisms. Following the initial mechanical impact, increasing evidence suggests that the brain undergoes an inflammatory cascade consisting of pro-inflammatory intracellular signaling pathways and production of cytokines, ultimately leading to chronic neuroinflammation and persistent neurological deficits. Prior work in severe traumatic brain injury has shown that the p38 MAPK signaling pathway is a key regulator of microglial activation, proinflammatory cytokines, and synaptic dysfunction, but its role in the context of mTBI remains unclear. As such, this study aimed to determine if inhibition of p38 MAPK would attenuate the inflammatory response and longer-term functional deficits following a weight-drop mouse model of repetitive mTBI. Methods: C57BL/6J male and female mice were injected with a small molecule p38 MAPK inhibitor (SB239063) after each of 5 once-daily weight-drop closed head injuries (CHIs) or sham injuries. Functional outcome was assessed at 4-weeks post injury. Protein and transcriptional alterations associated with the immune response, synaptic function, microglial phenotype, and functional outcomes were assessed at both 4-hours and 4-weeks after the final CHI. Results: In females, acute inhibition of p38 MAPK attenuated i) cytokine expression and microglial reactivity at 4-hours post injury and ii) antidepressive-like behavior and synaptic loss at 4-weeks post injury. In males, p38 MAPK inhibition also attenuated microglial reactivity and up-regulation of specific cytokines, although changes in functional outcomes did not reach significance. Interestingly, bulk RNAseq analysis in both sexes showed that acute p38 MAPK inhibition both normalized the effects of injury and upregulated protective genes and pathways associated with recovery and maintenance of brain homeostasis. Together, these findings suggest a role for p38 MAPK in driving the acute and longer-term consequences post repetitive mTBI in a sex-dependent manner, and they suggest therapeutic potential of p38 MAPK inhibition. To our knowledge, this work is the first to investigate the effects of small molecule inhibitor SB239063 as a potential therapeutic treatment administrated following rmTBI.

Volumetric muscle loss (VML) injuries result in chronic fibrosis, inflammation, and persistent functional deficits. Fibro-adipogenic progenitor (FAP) cells are a heterogeneous, muscle-resident stromal cell population that play a crucial role in muscle regeneration, but also contribute to fibrosis in muscle disease. The role of FAPs in VML is not well established and may be critical target to ensure functional muscle regeneration after VML. We utilized a VML model in the mouse quadriceps to study the location, secretome, surface marker distribution, gene expression, and single-cell transcriptional profile of FAPs after VML. After VML, a subpopulation of FAPs highly expressed β1-integrin and were elevated in the post-VML muscle tissue; these FAPs had increased fibrotic gene expression and increased myofibroblast differentiation potential. Transforming growth factor-β1 (TGF-β1) and tissue inhibitor of matrix metalloproteinase 1 (TIMP1) were identified as secreted proteins from VML derived FAPs that produced both pro-fibrotic and anti-myogenic signaling. These data establish an aberrant FAP sub-population that are elevated in VML injury and provides novel targets for future scarless muscle regeneration in VML.

Mucolipidosis IV (MLIV) is an autosomal-recessive pediatric disease that leads to motor and cognitive deficits and loss of vision. It is caused by loss of function of the lysosomal channel transient receptor potential mucolipin-1, TRPML1, and is associated with an early brain phenotype consisting of glial reactivity, hypomyelination, and lysosomal abnormalities. Although the field is approaching the first translationally relevant therapy, we currently lack a molecular signature of disease that can be used to detect therapeutic efficacy. Here, we analyzed 7,322 proteins in the plasma proteome from 17 MLIV patients and 37 controls and compared protein profiles with clinical measures of disease severity (motor function, muscle tone, and age). We found a decrease in neuronal proteins and an increase in muscle proteins in MLIV, consistent with neuronal dysfunction and muscle pathology observed in patients. Reduced synaptic proteins (e.g., GABARAP) best correlated with disease severity. Comparing the MLIV plasma proteome to the brain proteome from the MLIV mouse model identified shared alterations in 45 proteins, including upregulated proteins related to lysosomal function (e.g., ACTN2, GLB1) and downregulated proteins related to myelination (e.g., TPPP3, CNTN2). These data indicate that peripheral blood plasma protein signatures mirror changes found in the MLIV brain.

Mild traumatic brain injury (mTBI) is a leading cause of morbidity in children with both short- and long-term neurological, cognitive, cerebrovascular, and emotional deficits. These deficits have been attributed to ongoing pathophysiological cascades that occur acutely and persist post-injury. Given our limited understanding of the transcriptional changes associated with these pathophysiological cascades, we studied formalin-fixed paraffin-embedded (FFPE) tissues from the frontal cortex (FC) and the hippocampus + amygdala (H&A) regions of swine (N = 40) after a sagittal rapid non-impact head rotation (RNR). We then sequenced RNA to define transcriptional changes at 1 day and 1 week after injury and investigated the protective influence of cyclosporine A (CsA) treatment. Differentially expressed genes (DEGs) were classified into five temporal patterns (Early, Transient, Persistent, Intensified, Delayed, or Late). DEGs were more abundant at 1 week than 1 day. Shared significant gene ontology annotations in both regions included terms associated with neuronal distress at 1 day and neurorecovery at 1 week. CsA (20 mg/kg/day) infused for 1 day (beginning at 6 h after injury) accelerated 466 DEGs in the FC and 2794 DEGs in the H&A, such that the CsA-treated transcriptional profile was associated with neurorecovery. Overall, our data reveal the effects of anatomic region and elapsed time on gene expression post-mTBI and motivate future studies of CsA treatment.

Abstract Manganese is an essential trace element required for various biological functions, but in excess is neurotoxic and leads to significant health concerns. The mechanisms underlying manganese neurotoxicity remain poorly understood. Neuropathological studies of affected brain regions reveal astrogliosis, neuronal loss, and neuroinflammation. Here, we present a novel manganese-dependent mechanism linking mitochondrial dysfunction to neuroinflammation. We found that manganese disruption of the mitochondrial transcriptome processing results in the accumulation of double stranded RNA (dsRNA). This dsRNA is released into the cytoplasm, where it activates the cytosolic sensor MDA5, triggering type I interferon responses and inflammatory cytokine production. This mechanism is evident in 100 day human cerebral organoids, where manganese-increased mitochondrial dsRNA and induced inflammatory responses in mature astrocytes. Similarly, we observed an increase in mitochondrial dsRNA content, the activation of an inflammatory transcriptome and the production of cytokines in female and male mouse brains carrying mutations in the Slc30a10 gene, a model for human hypermanganesemia with dystonia 1 disorder. These findings highlight a previously unrecognized role for mitochondrial dsRNA in manganese-induced neuroinflammation and provide insights into the molecular pathogenesis of manganism. We propose that this mitochondrial dsRNA-induced inflammatory pathway could be active in other neurological diseases caused by environmental or genetic factors. Significance Statement: Environmental exposures and genetic defects that perturb manganese homeostasis are an underappreciated cause of neurodegeneration and neuroinflammation. We describe a new paradigm for inducible neuroinflammation, where manganese disruption of mitochondrial transcriptome processing leads to the accumulation of mitochondrial double-stranded RNA (dsRNA), which activate antiviral responses in the cytoplasm driving type I interferon dependent inflammation. This manganese-dsRNA axis is induced in cell lines in vitro and a subpopulation of mature astrocytes in exposed human cerebral organoids. Brain cortex of mice deficient in the manganese efflux transporter Slc30a10, a genetic model of chronic manganese accumulation, show dsRNA accumulation, and up-regulation of type I interferon response and astrogliosis markers, supporting a role for this pathway in neurotoxicity and parkinsonism.

Regulation of the brain's neuroimmune system is central to development, normal function, and disease. Neuronal communication to microglia, the primary immune cells of the brain, is well known to involve purinergic signaling mediated via ATP secretion and the cytokine fractalkine. Recent evidence shows that neurons release multiple cytokines beyond fractalkine, yet these are less studied and poorly understood. In contrast to ATP, cytokines are a class of signaling molecule that are much larger, with longer signaling and farther diffusion. We posit that neuron-expressed cytokines are an essential mechanism of neuron-microglia communication that arises as part of both normal learning and memory and in response to tissue pathology. Thus, neurons are underappreciated immunomodulatory cells that express diverse immunomodulatory signals. While neuronally sourced cytokines have been understudied, new technical advances make this a timely topic. The goal of this review is to define what is known about the cytokines expressed from neurons, how they are regulated, and the effects of these cytokines on microglia. We delineate key knowledge gaps and needs for new tools to define and analyze neuronal roles in immunomodulation. Given that cytokines are central regulators of microglial function, a broad new body of work is required to illuminate functional links between these neuronally expressed cytokines and sustained and transient microglial function.

Bone fracture repair initiates by periosteal expansion. The periosteum is typically quiescent, but upon fracture, periosteal cells proliferate and contribute to bone fracture repair. The expansion of the periosteum is regulated by gene transcription; however, the molecular mechanisms behind periosteal expansion are unclear. Here, we show that Yes-Associated Protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) mediate periosteal expansion and periosteal cell proliferation. Bone fracture increases the number of YAP-expressing periosteal cells, and deletion of YAP and TAZ from Osterix (Osx) expressing cells impairs early periosteal expansion. Mechanistically, YAP regulates both 'cell-intrinsic' and 'cell-extrinsic' factors that allow for periosteal expansion. Specifically, we identified Bone Morphogenetic Protein 4 (BMP4) as a cell extrinsic factor regulated by YAP, that rescues the impairment of periosteal expansion upon YAP/TAZ deletion. Together, these data establish YAP mediated transcriptional mechanisms that induce periosteal expansion in the early stages of fracture repair and provide new putative targets for therapeutic interventions.

BACKGROUND: Neuroinflammation plays a critical role in Alzheimer's disease pathogenesis. Neurons are anatomically divided in subcellular compartments (axons, soma, and synapses), which may be distinctly impacted by neuroinflammation. This study aims to examine cellular compartment-specific proteomic signatures in excitatory neurons following a systemic neuroinflammatory stress. METHOD: We used our innovative CIBOP (cell type-specific in vivo biotinylation of proteins) approach to selectively label Camk2a excitatory neuron proteomes in vivo. Neuron-CIBOP transgenic mice and their littermate controls were treated with lipopolysaccharide (LPS, [500 µg/kg, i.p.]) during 4 consecutive days, which induces robust microglial activation and sickness behavior. After euthanasia, brains were quickly removed, and crude synaptosomal fractions (P2 fractions) were prepared by differential centrifugation. Neuron-specific biotinylated proteins were then enriched and analyzed by label-free quantitative mass spectrometry (MS) to identify differentially-enriched proteins (DEPs) and biological pathways (by gene set variation analysis/GSVA). Neuron-derived biotinylated key cellular signaling pathways (MAPK and Akt/mTOR) were directly measured by Luminex in homogenates and P2 fractions (Fig. 1A). RESULT: Electron micrographs validated the subcellular composition of the P2 fractions showing synaptosomes containing synaptic vesicles and mitochondria (Fig. 1B). MS studies confirmed that neuronal homogenates were enriched in microtubule and cytoskeleton-related proteins, while P2 fractions were enriched in mitochondria and synapse-related proteins (Fig. 1C). Interestingly, LPS induced unique compartment-specific proteomic effects, P2 fraction has 52 unique DEPs, while homogenate has 57 DEPs, and only 2 DEPs overlapped (Fig. 1D). Neuronal homogenate proteomes showed upregulation of detoxification and oxidoreductase activity, while a reduced neuron-synapse, somatodendritic compartment, and cytoskeleton organization. In P2 fraction proteomes, LPS upregulated mitochondrial envelope formation and metabolic activity, including purine containing compound metabolic process, but downregulated nucleoside triphosphate regulator activity. Increased aerobic respiration and mitochondria response overlapped among compartments (Fig. 1E). We also observed LPS-induced decrease in MAPK signaling specifically in the P2 fraction, not evident at the level of whole neurons, nor at the bulk brain tissue level (Fig. 1F). CONCLUSION: Our neuron and synaptosome-enriched proteomics approach revealed unique molecular and signaling effects of neuroinflammation that may preferentially impact the synapses of excitatory neurons.

BACKGROUND: Chronic stress promotes life-long risk for neuropsychiatric decline by increasing neuroinflammation and disrupting synaptic health and plasticity. Our lab and others have recently demonstrated that non-invasive gamma sensory stimulation (flicker) modulates immune signaling, restores microglial function, and improves cognitive performance in mouse models of Alzheimer's disease (AD). However, no research to date has studied the effects of flicker in the context of stress. Accordingly, our goal for this study was to determine if flicker mitigates neuropsychiatric-like behavioral deficits and rescues glial and synaptic pathology following chronic stress. METHOD: Daily audiovisual flicker intervention was introduced concomitantly with daily stress exposure (28 days) at multiple frequencies in male and female C57BL6 mice (n = 6/group per sex). The mice were then tested for anxiety-like behaviors and anhedonia using a range of behavioral tests. We quantified transcriptomic changes in cortical Thy1+ pyramidal neurons, microglia, and astrocytes to identify cell-type specific genes modulated by flicker intervention in chronically stressed mice. To determine how flicker mitigates stress-induced cellular changes in the prefrontal cortex, a region vulnerable to stress and AD pathology, we next quantified spine density changes in Thy1-GFP mice and measured glial morphology changes as a proxy for microglial and astrocyte reactivity using the semi-automated Imaris imaging software to determine synaptic health and inflammatory profile, respectively. RESULT: We found that stress-induced molecular changes in the prefrontal cortex are modulated in a sex-, and frequency-specific manner that coincides with behavioral resilience in stressed mice. Our findings indicates that audiovisual flicker protects against stress-induced behavioral deficits and mitigates stress-induced neuroimmune responses in a sex- and frequency-specific manner. CONCLUSION: Together, these findings show frequency optimized flicker intervention improves stress pathology and may prevent neuropsychiatric health decline in conditions with sex dimorphic symptoms and prevalence.

BACKGROUND: Mitogen activated protein kinase (MAPK) signaling is a critical regulator of microglial phenotype, including phagocytic function, cytokine expression, and motility, among others. Importantly, both canonical and non-canonical MAPK signaling is directly activated by RTKs, including Interestingly, CSF1R, is activated by two agonists, CSF1 and IL-34, which have been shown to activate the receptor in different ways that can lead to However, little is known about how the affect microglial MAPK signaling, and whether their effects are dependent on disease state/Aβ exposure. In this study, we hypothesized that IL-34 and CSF-1 elicit distinct patterns of MAPK signaling activation in microglia and MAPK activation would be dependent on whether the cells were exposed to Aβ. METHOD: We applied agonists of CSF1R (i.e., IL-34, CSF1) to the mouse microglial cell (MMC) line, either pre-treated with 500nM of Aβ1-42 or vehicle (1% NH4OH) for 48hr. Cells were serum starved (1hr), stimulated with each agonist for 5min, then lysed for Luminex quantification of a panel of 10 MAPK phosphoproteins. RESULT: In the absence of Aβ1-42, both CSF1R and IL-34 stimulated both canonical (ERK) and non-canonical (p38, JNK) MAPK signaling phospho-proteins (Wilcox test, p<0.05 vs control). While Aβ1-42 pre-treatment resulted in diminished activation in canonical MAPK signaling (i.e., MEK, ERK) following either CSF1 or IL-34 stimulation, CSF1 stimulation of non-canonical MAPK signaling through p38 and JNK was preserved with Aβ1-42 pre-treatment. These findings indicate that CSF1-induced signaling is robust regardless of Aβ1-42 exposure. Furthermore, different agonists of the same receptor distinctly activate non-canonical MAPK pathway signaling (p38). Collectively, this finding supports the overall hypothesis that CSF-1R-induced signaling is dependent on Aβ and Aβ pre-treatments shift the canonical ERK signaling to non-canonical p38 signaling. CONCLUSION: These findings show that different agonists of CSF1R can provoke distinct profiles of canonical and non-canonical MAPK pathway signaling in the presence of Aβ1-42. Understanding differences in agonist-specific microglial signaling in healthy and diseased microglia will be essential to designing microglial-targeted therapeutics for treatment of AD.

Abstract Background Chronic stress promotes life‐long risk for neuropsychiatric decline by increasing neuroinflammation and disrupting synaptic health and plasticity. Our lab and others have recently demonstrated that non‐invasive gamma sensory stimulation (flicker) modulates immune signaling, restores microglial function, and improves cognitive performance in mouse models of Alzheimer’s disease (AD). However, no research to date has studied the effects of flicker in the context of stress. Accordingly, our goal for this study was to determine if flicker mitigates neuropsychiatric‐like behavioral deficits and rescues glial and synaptic pathology following chronic stress. Method Daily audiovisual flicker intervention was introduced concomitantly with daily stress exposure (28 days) at multiple frequencies in male and female C57BL6 mice (n = 6/group per sex). The mice were then tested for anxiety‐like behaviors and anhedonia using a range of behavioral tests. We quantified transcriptomic changes in cortical Thy1+ pyramidal neurons, microglia, and astrocytes to identify cell‐type specific genes modulated by flicker intervention in chronically stressed mice. To determine how flicker mitigates stress‐induced cellular changes in the prefrontal cortex, a region vulnerable to stress and AD pathology, we next quantified spine density changes in Thy1‐GFP mice and measured glial morphology changes as a proxy for microglial and astrocyte reactivity using the semi‐automated Imaris imaging software to determine synaptic health and inflammatory profile, respectively. Result We found that stress‐induced molecular changes in the prefrontal cortex are modulated in a sex‐, and frequency‐specific manner that coincides with behavioral resilience in stressed mice. Our findings indicates that audiovisual flicker protects against stress‐induced behavioral deficits and mitigates stress‐induced neuroimmune responses in a sex‐ and frequency‐specific manner. Conclusion Together, these findings show frequency optimized flicker intervention improves stress pathology and may prevent neuropsychiatric health decline in conditions with sex dimorphic symptoms and prevalence.

Deep-UV microscopy enables high-resolution, label-free molecular imaging by leveraging biomolecular absorption properties in the UV spectrum. Recent advances in UV-imaging hardware have renewed interest in this technique for quantitative live cell imaging applications. However, UV-induced photodamage remains a concern for longitudinal dynamic imaging studies. Here, we quantify UV phototoxicity with several cell types at notable UV wavelengths. We find that the fluence required for cell death via UV phototoxicity with continuous UV exposure varies with cell type and wavelength from ∼0.5µJ/µm 2 to 2µJ/µm 2 , but is independent of typical illumination power/radiant flux of UV microscopy (e.g., 0.1-20 nW/µm 2 ). We also show results from fractionation studies that reveal cell repair following UV exposure, which increases the tolerance to UV radiation by a factor of 2 or more, depending on the fractionation paradigm. Results further show that UV tolerance exceeds ANSI guidelines for maximum permissible exposure. Finally, we calculate imaging limits for a typical application of UV microscopy, such as hematology analysis. Together, this work provides UV fluence thresholds that can serve as guidelines for nondestructive, longitudinal, and dynamic deep-UV microscopy experiments.

Increased risk of developing glaucoma has recently been associated with early age of menopause. Here, we examined how age and surgically-induced menopause via ovariectomy (OVX) impacted gene expression in gene pathways previously linked to glaucoma, such as extracellular matrix (ECM) remodeling and TGF-β signaling. Using bulk RNA sequencing, we analyzed changes in young (3-4 months) and middle-aged (9-10 months) Long-Evans rats. We focused on posterior pole tissues (sclera and optic nerve head) but also examined the retina to compare observed changes across different tissue regions. Our results demonstrated that aging and OVX significantly alter gene expression in the sclera and optic nerve head. Generally, OVX triggered the enrichment of immune-related processes. However, OVX in young rats also led to significant enrichment of ECM and TGF-β gene sets. At the same time, these effects were diminished in middle-aged rats, indicating an age dependency of the effects of OVX on matrix-related pathways. Notably, the transcriptional factor Fos was downregulated in the posterior eye and retina in aged and OVX animals. Fos is a major regulator of cell proliferation and survival, and its dysregulation may play an important role in aging and menopause for women. These findings underscore the important role of menopause timing in modulating molecular pathways associated with glaucoma, which is consistent with clinical studies showing that early menopause may heighten the risk of developing this condition. This study also highlights the importance of considering women's health factors, such as menopause, in understanding and managing glaucoma risk.

BACKGROUND: Mesenchymal stromal cell derived extracellular vesicles (MSC-EVs) are a promising therapeutic for neuroinflammation. MSC-EVs can interact with microglia, the resident immune cells of the brain, to exert their immunomodulatory effects. In response to inflammatory cues, such as cytokines, microglia undergo phenotypic changes indicative of their function e.g. morphology and secretion. However, these changes in response to MSC-EVs are not well understood. Additionally, no disease-relevant screening tools to assess MSC-EV bioactivity exist, which has further impeded clinical translation. Here, we developed a quantitative, high throughput morphological profiling approach to assess the response of microglia to neuroinflammation- relevant signals and whether this morphological response can be used to indicate the bioactivity of MSC-EVs. RESULTS: Using an immortalized human microglia cell-line, we observed increased size (perimeter, major axis length) and complexity (form factor) upon stimulation with interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). Upon treatment with MSC-EVs, the overall morphological score (determined using principal component analysis) shifted towards the unstimulated morphology, indicating that MSC-EVs are bioactive and modulate microglia. The morphological effects of MSC-EVs in TNF-α /IFN-γ stimulated cells were concomitant with reduced secretion of 14 chemokines/cytokines (e.g. CXCL6, CXCL9) and increased secretion of 12 chemokines/cytokines (e.g. CXCL8, CXCL10). Proteomic analysis of cell lysates revealed significant increases in 192 proteins (e.g. HIBADH, MEAK7, LAMC1) and decreases in 257 proteins (e.g. PTEN, TOM1, MFF) with MSC-EV treatment. Of note, many of these proteins are involved in regulation of cell morphology and migration. Gene Set Variation Analysis revealed upregulation of pathways associated with immune response, such as regulation of cytokine production, immune cell infiltration (e.g. T cells, NK cells) and morphological changes (e.g. Semaphorin, RHO/Rac signaling). Additionally, changes in microglia mitochondrial morphology were measured suggesting that MSC-EV modulate mitochondrial metabolism. CONCLUSION: This study comprehensively demonstrates the effects of MSC-EVs on human microglial morphology, cytokine secretion, cellular proteome, and mitochondrial content. Our high-throughput, rapid, low-cost morphometric approach enables screening of MSC-EV batches and manufacturing conditions to enhance EV function and mitigate EV functional heterogeneity in a disease relevant manner. This approach is highly generalizable and can be further adapted and refined based on selection of the disease-relevant signal, target cell, and therapeutic product.

Current treatments for congenital and acquired craniofacial (CF) bone abnormalities are limited and costly. Conventional methods involve surgical correction, short-term stabilization, and long-term bone grafting, which may include problematic allografts and limited autografts. While bone morphogenetic protein 2 (BMP2) has been used for bone regeneration, it can cause bone overgrowth and life-threatening inflammation. Bone marrow-derived mesenchymal stem cell therapies, though promising, are not Food and Drug Administration approved and are resource intensive. Thus, there is a need for effective, affordable, and less side-effect-prone bone regenerative therapies. Previous research demonstrated that JAGGED1 induces osteoblast commitment in murine cranial neural crest cells through a NOTCH-dependent non-canonical pathway involving JAK2–STAT5. We hypothesize that delivery of JAGGED1 and induction of its downstream NOTCH non-canonical signaling in pediatric human osteoblasts constitutes an effective bone regenerative treatment. Delivering pediatric human bone-derived osteoblast-like cells to an in vivo murine bone loss model of a critically sized cranial defect, we identified that JAGGED1 promotes human pediatric osteoblast commitment and bone formation through p70 S6K phosphorylation. This approach highlights the potential of JAGGED1 and its downstream activators as innovative treatments for pediatric CF bone loss.

Abstract Rare inherited diseases caused by mutations in the copper transporters SLC31A1 (CTR1) or ATP7A induce copper deficiency in the brain, causing seizures and neurodegeneration in infancy through poorly understood mechanisms. Here, we used multiple model systems to characterize the molecular mechanisms by which neuronal cells respond to copper deficiency. Targeted deletion of CTR1 in neuroblastoma cells produced copper deficiency that was associated with a metabolic shift favoring glycolysis over oxidative phosphorylation. Proteomic and transcriptomic analysis of CTR1 KO cells revealed simultaneous upregulation of mTORC1 and S6K signaling and reduced PERK signaling. Patterns of gene and protein expression and pharmacogenomics show increased activation of the mTORC1-S6K pathway as a pro-survival mechanism, ultimately resulting in increased protein synthesis. Spatial transcriptomic profiling of Atp7a flx/Y :: Vil1 Cre/+ mice identified upregulated protein synthesis machinery and mTORC1-S6K pathway genes in copper-deficient Purkinje neurons in the cerebellum. Genetic epistasis experiments in Drosophila demonstrated that copper deficiency dendritic phenotypes in class IV neurons are partially rescued by increased S6k expression or 4E-BP1 (Thor) RNAi, while epidermis phenotypes are exacerbated by Akt, S6k, or raptor RNAi. Overall, we demonstrate that increased mTORC1-S6K pathway activation and protein synthesis is an adaptive mechanism by which neuronal cells respond to copper deficiency. Significance Copper deficiency is present in rare conditions such as Menkes disease and CTR1 deficiency and in more common diseases like Alzheimer’s. The mechanisms of resilience and ultimate susceptibility to copper deficiency and associated pathology in the brain remain unknown. We demonstrate that in a human cell line, Drosophila , and the mouse cerebellum, copper-deficient neuronal cells exhibit increased protein synthesis through mTORC1 activation and decreased PERK (EIF2AK3) activity. Upregulation of protein synthesis facilitates resilience of neuronal cells to copper deficiency, including partial restoration of dendritic arborization. Our findings offer a new framework for understanding copper deficiency-related pathology in neurological disorders.

Abstract Mucolipidosis IV (MLIV) is an autosomal-recessive pediatric disease that leads to motor and cognitive deficits and loss of vision. It is caused by the loss of function of the lysosomal channel transient receptor potential mucolipin-1, TRPML1, and is associated with an early brain phenotype consisting of glial reactivity, hypomyelination, lysosomal abnormalities, and increased cytokine expression. Although the field is approaching the first translationally relevant therapy, we currently lack a molecular signature of disease that can be used to detect therapeutic efficacy. In the current study, we analyzed 7,322 proteins in the plasma proteome and compare protein profiles with clinical measures of disease severity (motor function, muscle tone, and age). To do so, we used aptamer-based protein profiling on plasma isolated from 18 MLIV patients and 37 aged-matched controls from a biorepository. We identified a total of 1,961 differentially expressed proteins between MLIV and control subjects, with functions spanning many major hallmarks of MLIV. Our analysis revealed a decrease in the abundance of neuronal proteins and an increase in muscle proteins, consistent with the neuronal dysfunction and muscle pathology observed in patients. In particular, lower levels of synaptic proteins (e.g., GABARAP) best correlated with disease severity. Next, we compared the plasma proteome of patients to the brain proteome from the mouse model of MLIV and identified shared alterations in 45 proteins. The up-regulated overlapping proteins were largely related to lysosomal function (e.g., ACTN2, GLB1), while the down-regulated proteins were largely related to myelination (e.g. TPPP3, CNTN2). Both signatures are consistent with our understanding of key disease hallmarks: impaired myelination and modified lysosomal function. Collectively, these data indicate that peripheral blood plasma protein signatures mirror changes found in the MLIV brain and suggest candidate markers relevant to MLIV pathology to be validated in future studies.

Treatments for congenital and acquired craniofacial (CF) bone abnormalities are limited and expensive. Current reconstructive methods include surgical correction of injuries, short-term bone stabilization, and long-term use of bone grafting solutions, including implantation of (i) allografts which are prone to implant failure or infection, (ii) autografts which are limited in supply. Current bone regenerative approaches have consistently relied on BMP2 application with or without addition of stem cells. BMP2 treatment can lead to severe bony overgrowth or uncontrolled inflammation, which can accelerate further bone loss. Bone marrow-derived mesenchymal stem cell-based treatments, which do not have the side effects of BMP2, are not currently FDA approved, and are time and resource intensive. There is a critical need for novel bone regenerative therapies to treat CF bone loss that have minimal side effects, are easily available, and are affordable. In this study we investigated novel bone regenerative therapies downstream of JAGGED1 (JAG1).We previously demonstrated that JAG1 induces murine cranial neural crest (CNC) cells towards osteoblast commitment via a NOTCH non-canonical pathway involving JAK2-STAT5 () and that JAG1 delivery with CNC cells elicits bone regeneration in vivo. In this study, we hypothesize that delivery of JAG1 and induction of its downstream NOTCH non-canonical signaling in pediatric human osteoblasts constitute an effective bone regenerative treatment in an in vivo murine bone loss model of a critically-sized cranial defect. Using this CF defect model in vivo, we delivered JAG1 with pediatric human bone-derived osteoblast-like (HBO) cells to demonstrate the osteo-inductive properties of JAG1 in human cells and in vitro we utilized the HBO cells to identify the downstream non-canonical JAG1 signaling intermediates as effective bone regenerative treatments. In vitro, we identified an important mechanism by which JAG1 induces pediatric osteoblast commitment and bone formation involving the phosphorylation of p70 S6K. This discovery enables potential new treatment avenues involving the delivery of tethered JAG1 and the downstream activators of p70 S6K as powerful bone regenerative therapies in pediatric CF bone loss.

Abstract Treatments for congenital and acquired craniofacial (CF) bone abnormalities are limited and expensive. Current reconstructive methods include surgical correction of injuries, short-term bone stabilization, and long-term use of bone grafting solutions, including implantation of (i) allografts which are prone to implant failure or infection, (ii) autografts which are limited in supply. Current bone regenerative approaches have consistently relied on BMP2 application with or without addition of stem cells. BMP2 treatment can lead to severe bony overgrowth or uncontrolled inflammation, which can accelerate further bone loss. Bone marrow-derived mesenchymal stem cell-based treatments, which do not have the side effects of BMP2, are not currently FDA approved, and are time and resource intensive. There is a critical need for novel bone regenerative therapies to treat CF bone loss that have minimal side effects, are easily available, and are affordable. In this study we investigated novel bone regenerative therapies downstream of JAGGED1 (JAG1). We previously demonstrated that JAG1 induces murine cranial neural crest (CNC) cells towards osteoblast commitment via a NOTCH non-canonical pathway involving JAK2-STAT5 (1) and that JAG1 delivery with CNC cells elicits bone regeneration in vivo. In this study, we hypothesize that delivery of JAG1 and induction of its downstream NOTCH non-canonical signaling in pediatric human osteoblasts constitute an effective bone regenerative treatment in an in vivo murine bone loss model of a critically-sized cranial defect. Using this CF defect model in vivo, we delivered JAG1 with pediatric human bone-derived osteoblast-like (HBO) cells to demonstrate the osteo-inductive properties of JAG1 in human cells and in vitro we utilized the HBO cells to identify the downstream non-canonical JAG1 signaling intermediates as effective bone regenerative treatments. In vitro, we identified an important mechanism by which JAG1 induces pediatric osteoblast commitment and bone formation involving the phosphorylation of p70 S6K. This discovery enables potential new treatment avenues involving the delivery of tethered JAG1 and the downstream activators of p70 S6K as powerful bone regenerative therapies in pediatric CF bone loss.