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Amyloid Precursor Protein (APP) has been reported to partially localize to mitochondria, and mitochondrial dysfunction is a key feature of Alzheimer's disease; however, the mechanisms linking APP to mitochondrial functions remain incompletely defined. In this study, we identified an interaction between APP and phosphoglycerate mutase family member 5 (PGAM5), a mitochondrial protein phosphatase. We confirmed their endogenous interaction in mouse brain tissue and determined that APP and PGAM5 are both present at mitochondria-ER contact sites (MERCS) and. Using in vitro binding assays, we demonstrate a direct interaction between the linker region of APP and a region of PGAM5 that includes the Kelch-like ECH-associated protein 1 (Keap-1) binding domain. PGAM5 is known to anchor a portion of Nuclear factor erythroid 2 p45-related factor 2 (Nrf2) through Keap1 at the outer mitochondrial membrane and regulates mitochondrial respiration and stress responses. We found that the Nrf2-regulated genes Hmox1 (Heme oxygenase-1) and Nqo1 (NADH:quinone oxidoreductase 1), which are involved in mitochondrial respiration, are downregulated in APP KO astrocytes. Accordingly, mitochondria isolated from the brains of APP knockout (KO) mice have impaired substrate-specific respiration and electron transport chain (ETC) function. Together, these findings suggest that APP supports mitochondrial respiration by binding to PGAM5 and modulating Keap1-Nrf2 signaling.

Prickles on blackberry and raspberry canes make pruning, harvesting, and handling more difficult and can increase labor costs for growers. The trait has been challenging to improve in these clonal crops because it is recessive and linked to undesirable agronomic traits. In blackberry and red raspberry, breeding programs have used recessive mutants at the S locus to generate prickleless cultivars for the last century. In this study, we identified independent loss-of-function mutations in a WUSCHEL-LIKE HOMEOBOX transcription factor, WOX1, as the genetic basis of the prickleless S locus in both blackberry and red raspberry. We mapped the S locus using integrated genome-wide association, bulked segregant analysis, and identity-by-descent analyses informed by breeding pedigrees. Additionally, we generated a genome sequence from Luther Burbank's prickleless blackberry variety Burbank Thornless that contained an additional allele of WOX1. To verify the gene's role, we used gene editing to knock out WOX1 in an elite prickled commercial blackberry line. All edited plants were prickleless and lacked glandular trichomes, confirming that WOX1 controls a joint developmental pathway. Other plant traits were unchanged, indicating WOX1 is a specific and safe target for improvement. Gene editing can enable breeders to remove prickles directly from elite varieties, reducing the need for extensive breeding cycles and delivering safer, easier-to-harvest cultivars to growers.

Using external cues to guide behavior is a core function that enables multiple aspects of cognition and attentional control, and deficits in this process are central to many theories of neuro-psychiatric, degenerative, and developmental disorders. Cue detection relies on the precise coordination of neural circuits, with the mediodorsal thalamus (MD) hypothesized to play a pivotal role in orchestrating the relay of cue-based associative information to the prefrontal cortex. The prefrontal cortex comprises multiple subregions, which are believed to differentially contribute to such associative cue-based behaviors. This regional specificity is likely seeded by projection-defined MD→PFC pathways, although the anatomical organization of these discrete channels and their dynamic roles in cue detection are still being defined. Here, we address this gap by combining anatomical circuit mapping of MD-PFC output pathways with in vivo calcium imaging during a cue-based reward conditioning task in mice. These experiments reveal that MD projections to distinct PFC subregions (prelimbic and anterior cingulate cortex) form topographically defined loops, that are characterized by unique patterns of activity across cue-reward learning. Using fiber photometry to monitor changes in calcium activity in axonal projections from the MD to the PFC, we show that during learning, MD projections to the prelimbic subregion are activated by cue presentation, and the dynamics of this activity remain stable across training days. In contrast, MD projections to the anterior cingulate exhibit a learning-dependent suppression of activity that predicts reward approach behavior in late training. Interestingly, the two pathways exhibit opposing activity patterns when the predictive validity of the cue is diminished by extinction training, suggesting distinct functional roles in detecting violations of learned contingencies. Together, these findings reveal previously unrecognized anatomical and functional distinctions within MD-PFC circuits and demonstrate that parallel thalamocortical pathways differentially support cue detection and behavioral flexibility. This work advances understanding of thalamocortical mechanisms underlying cue detection and may inform circuit-based approaches for treating cognitive dysfunction in psychiatric disorders.

Single-molecule microscopy has been widely used to study the structure and dynamics of RNA, but extension to larger systems such as long non-coding RNA (lncRNA) has proven challenging. Methods such as single-molecule kinetic analysis of RNA transient structure (SiM-KARTS), where the binding of a short, complementary oligonucleotide probe is used to determine accessibility of a specific region of the RNA, are promising. However, adapting SiM-KARTS to systems as complex as lncRNA requires careful optimization of experimental variables that have not been thoroughly explored. In this work, SiM-KARTS, thermal denaturation experiments, and circular dichroism spectroscopy were used to analyze the binding behaviors of probes with alternative backbone chemistries, specifically DNA with locked nucleic acid (LNA) residues incorporated and morpholinos. A segment of lncRNA that enabled control over the accessibility of the target sequence was used as a model. We show that optimizing probe backbone chemistry can allow for a more precise distinction between different structures of the target RNA, and for fine-tuning of probe binding stability without the structural impacts that other variables such as ionic concentration may have. Specifically, we demonstrate that LNA probes exhibit a high degree of structural sensitivity in both their binding and unbinding kinetics. We further show that when binding and unbinding rates are considered holistically, LNA probes allow traces arising from different target RNA structures to be individually classified with a high degree of accuracy. These results provide design principles for the application of SiM-KARTS to target RNAs of increased complexity such as lncRNA.

Spinal cord injury (SCI) disrupts corticospinal tract (CST) connectivity and impairs skilled voluntary movement. However, most human SCIs are anatomically incomplete, allowing spared CST pathways to engage in rehabilitation-mediated plasticity to promote functional recovery. How voluntary rehabilitation engages and reorganizes the supraspinal targets of the intact CST remains incompletely understood. Here, we combined unilateral pyramidotomy (uPyX) in male and female mice with continuous voluntary complex-wheel running to test whether fine motor-dependent rehabilitation drives supraspinal CST plasticity. uPyX mice rapidly resumed wheel running after a transient deficit. In contrast to lesion-only controls, rehabilitation significantly improved skilled forelimb performance on the horizontal ladder rung task. Immunohistochemical c-Fos labeling confirmed that complex-wheel running robustly activated the intact forelimb CST in motor cortex. Whole-brain CST projection mapping using intersectional viral vector tracing revealed targeted supraspinal reorganization localized to medullary motor nuclei. Three nuclei - the lateral paragigantocellular reticular nucleus (LPGi), gigantocellular reticular nucleus, alpha part (GiA), and ventral medullary reticular nucleus (MdV) - exhibited significant lesion- and/or rehabilitation-induced increases in CST innervation. Rehabilitation-driven CST sprouting correlated with regional c-Fos activation, indicating activity-dependent remodeling. Notably, CST projection density in the MdV, critical for skilled forelimb control, correlated with functional recovery. These findings identify a set of spinally-projecting medullary nuclei as key sites of rehabilitation-induced CST plasticity and highlight the MdV as a potential mediator of restored motor function. This work defines how voluntary rehabilitation reorganizes spared corticospinal pathways and provides targets for optimizing activity-based interventions after SCI.

Significance statement: Effective rehabilitation after spinal cord injury (SCI) must harness the plasticity of spared motor pathways, yet the supraspinal circuits that support rehabilitation-mediated recovery remain unknown. Using a model that preserves voluntary motor engagement, we show that continuous fine motor-dependent rehabilitation activates intact corticospinal neurons and drives highly specific remodeling of their supraspinal terminals. Rehabilitation selectively strengthens CST inputs to motor regions of the medulla, particularly the ventral medullary reticular nucleus (MdV), and CST plasticity within this region predicts enhanced behavioral recovery. These findings highlight the MdV as a central locus by which rehabilitation re-establishes descending control of the impaired limb, providing mechanistic insight to guide targeted, circuit-based rehabilitation therapies for incomplete SCI.

Size is a fundamental property of cells that influences many aspects of their physiology. This is because cell size sets the scale for all subcellular components and drives changes in the composition of the proteome. Given that large and small cells differ in their biochemical composition, we hypothesize that they should also differ in how they respond to signals and make decisions. Here, we investigated how cell size affects susceptibility to cell death. We found that large cells are more resistant to ferroptosis caused by system x_c ^- inhibition. Ferroptosis is a type of cell death characterized by the iron-dependent accumulation of toxic lipid peroxides. This process is opposed by cysteine-dependent lipid peroxide detoxification mechanisms. We found that larger cells exhibit higher concentrations of the cysteine-containing metabolite glutathione and lower concentrations of membrane lipid peroxides. Mechanistically, this can be explained by the fact that larger cells had lower concentrations of an enzyme that enriches cellular membranes with peroxidation-prone polyunsaturated fatty acids, ACSL4, and increased concentrations of the iron-chelating protein ferritin, the glutathione-producing enzymes glutamate-cysteine ligase and glutathione synthetase, and the lysosomal protease cathepsin B, which can catabolize cysteine-rich extracellular proteins to produce additional cystine for fueling the synthesis of glutathione. Taken together, our results highlight the significant impact of cell size on cellular function and survival, revealing a size-dependent vulnerability to ferroptosis that could influence therapeutic strategies based on this cell death pathway.

The hippocampal formation is a highly curved and topographically complex forebrain structure. This complex geometry presents persistent challenges for analyzing subregional, laminar, and connectivity patterns. Here, we present a computational workflow that generates curvilinear-coordinate flatmaps from Common Coordinate Framework (CCF) registered hippocampal and retrohippocampal regions by solving the Laplacian equation to derive geodesic streamlines. This transformation unfolds the hippocampus into a planar slab, bounded by the meningeal and ventricular surfaces, with the depth defined along the radial axis. We apply this transform to image volumes, single neuron reconstructions, and point data, including spatial transcriptomic and rabies tracing datasets, revealing topographic variations in the dorsoventral and radial axes that are obscured in the CCF coordinate space. As proof of principle, we use flatmaps to show connectivity loss in a mouse model of Alzheimer's disease and track postnatal development of microglial distribution in the hippocampus. This work provides an efficient and accessible resource for visualizing hippocampal organization across development and disease, offering new opportunities to interrogate the structure and function of this important brain region.

Cancer cachexia is characterized by unintentional weight loss and wasting away of fat and muscle tissues. Anorexia, or reduced food intake, is often implicated as a contributor to the negative energy balance in this condition. However, to what extent anorexia alone accounts for body weight loss and wasting of different tissues, and whether anorexia is responsible for other cachectic phenotypes such as physical performance impairment remains insufficiently characterized in preclinical models and patients. In this study, we demonstrate the critical need to address these questions in cancer cachexia research. Using the colon carcinoma 26 (C26) model of cancer cachexia as an example, we systematically determined how much each of the key phenotypes of cancer cachexia is driven by anorexia. Anorexia was the predominant driver for body weight loss, adipose tissue wasting, and muscle wasting, strikingly suggesting the lack of other mechanisms for causing these phenotypes in this model. In contrast, anorexia had no impact on physical performance, pointing to the existence of anorexia-independent mechanisms in causing fatigue. Thus, for a given preclinical model or patient group, anorexia can be the main cause for certain cachectic phenotypes and play no role in causing other cachectic phenotypes. Discriminating between anorexia-mediated and independent effects is essential for guiding research focus and ultimately unraveling the causal pathways of cancer cachexia.

Necrotizing granulomas, the pathological hallmark of active tuberculosis, are characterized by the accumulation of lipid droplet-laden macrophage foam cells that contribute to tissue destruction, bacterial persistence, and transmission. Despite their central role in tuberculosis pathogenesis, the molecular mechanisms driving foam cell formation remain poorly defined. Here, we show that the mycobacterial lipoglycan mannose-capped lipoarabinomannan (ManLAM) induces macrophage lipid droplet accumulation through coordinated engagement of Toll-like receptor 2 and Dectin-2. Distinct structural moieties within ManLAM are selectively required for recognition by each receptor. Engagement of both receptors induces lipid metabolic reprogramming and enhances NF-κB-mediated inflammatory signaling, yet lipid accumulation proceeds through an mTORC1-PPARγ-dependent pathway that is largely independent of NF-κB activation. ManLAM-induced lipid metabolic changes closely mirror those elicited during Mycobacterium tuberculosis infection, both in neutral lipid composition and in their dependence on the mTORC1-PPARγ axis. These findings identify ManLAM as a major mycobacterial input into foam cell-associated lipid metabolism and establish ligand-level coordination of innate receptor engagement as a mechanism linking mycobacterial recognition to macrophage lipid metabolic reprogramming.

Significance statement: Characterizing how lipid-laden foam cells form is central to understanding tuberculosis pathogenesis because foam cells define the necrotizing lesions that drive lung damage and transmission. We show that a single mycobacterial component uses distinct structural features to engage two innate immune receptors on macrophages and induce lipid metabolic remodeling and foam cell formation. This finding establishes the principle that microbial ligand architecture can encode engagement of multiple receptors to shape host responses. These insights provide a mechanistic framework for tuberculosis pathogenesis and identify host pathways that may represent targets for host-directed intervention.

Pathogenic variants in leucine-rich repeat kinase 2 (LRRK2), vacuolar protein sorting 35 (VPS35 ), and RAB32 cause dominantly inherited parkinsonism, indistinguishable from idiopathic late-onset Parkinson's disease (PD). All three causes constitutively activate LRRK2 kinase activity to augment immune responses, enhancing immunity to fight pathogens, but similar mechanisms in the brain increase the vulnerability of dopaminergic neurons to degeneration. Although VPS35 p.D620N possess the highest constitutive increase in LRRK2 kinase activity among known variants in LRRK2 or RAB32, its effects on the immune system remain poorly understood. LRRK2 and Rab32 are highly expressed in myeloid cells including microglia; thus we examined the transcriptomic and functional consequences of Vps35 p.D620N in knock-in mice (VKI). Microglia were isolated from brains of six-month-old VKI mice and were analyzed via single-cell RNA sequencing. Differential gene expression highlighted pathways involved in antimicrobial humoral immune response, lysosomal stress sensing, and phagocytosis. Notably, genes of S100 family proteins, along with lipocalin 2 ( Lcn2 ), were significantly upregulated, and those measures were complimented by immunohistochemistry and quantitative PCR. In contrast, pathways involved in synaptic transmission, neuronal development, and homeostatic immune signaling were downregulated. Peripheral stimulation with lipopolysaccharide amplified microglial activation and phagocytic markers in wildtype mice, and VKI mice also display enhanced morphological activation and increased synaptic engulfment. Collectively, Vps35 p.D620N drives a chronic pro-inflammatory microglial phenotype characterized by heightened innate immune signaling, lysosomal stress, and enhanced phagocytic activity. VKI microglia are sensitized to peripheral immune challenges and may promote synaptic remodeling and neurodegenerative vulnerability in PD. These results provide mechanistic insight into how retromer dysfunction and LRRK2 kinase hyperactivity intersect with microglial biology to influence PD pathogenesis.