Biology subjects

Hanna, J.

Publications and source records attributed to Hanna, J..

14 recordsLinked to original sources

bioRxiv2026

Regulation of the proteasome 20S core particle by the Parkinsonism-associated Proteins FBXO7 and PI31

FBXO7 and PI31 variants are linked to rare Parkinsonian syndromes, implicating their dysfunction in neurodegeneration. We define how both engage each other and regulate the proteasome 20S core particle (CP). In cells, each can associate independently with the proteasome, with multiple domains in FBXO7 contributing. Utilizing cryo-EM we visualized how FBXO7s C-terminal domain engages multiple subunits within the CP interior, blocking the {beta}5 peptidase activity. In contrast, we visualized PI31 engagement of all three catalytic sites within the 20S CP, revealing the previously unknown structural basis for {beta}1 inhibition. Furthermore, we establish how disease-associated variants impact both FBXO7 and PI31 function, including disruption of proteasome inhibition and SKP1-FBXO7-PI31 complex assembly. These results establish an unexpected function for FBXO7, providing a mechanistic basis for investigation of its role in proteasome regulation in Parkinsons disease.

biochemistry↗

bioRxiv2025

Computational Stabilization of the Human VH Germline Repertoire to Enable Conditional Multi-Specific Therapeutic Development

Protein-based biologic therapies, particularly antibody-like therapeutics, have emerged as a major modality to treat nearly all chronic and infectious diseases. Antagonists have dominated the first wave of antibody and antibody-like biologics, whereas agonism has generally been challenging due to systemic activation leading to issues with therapeutic index and problems with pleiotropic activity. Additionally, agonists that use native proteins such as cytokines can be challenging to produce at scale given these proteins evolved to act locally and are not designed for large scale manufacturing. To address these challenges, we generated a biologics platform comprised of stabilized human VH domains (VH-Select) which encompass the entire human germline repertoire with the goal of building multispecific biologics denoted Tentacles that use avidity-based binding to achieve conditional activity directed to specific cell types or tissues. Stabilizing disulfides and point mutations were identified computationally with Rosetta, evaluated in vitro, and combined into designs with 3-5 amino acid substitutions for each of the seven germline families (VH1-VH7). Computational design was also employed to reduce dimerization from both VL and homotypic VH-VH interactions. Optimization of specific sequences improved expression by greater than 600-fold and thermostability by more than 20{degrees}C. Each of the germline variants were screened for low HLA class II binding and incorporated into a library which demonstrated significant improvements in cellular protein production, thereby increasing sequence diversity for screening campaigns. These VH-Select scaffolds are useful for the discovery of novel binders used to build multispecific Tentacles designed for cis-interactions that achieve cell- and tissue-specific activation. As an example of the utility of the platform, we generated a set of Tentacles that use VH-Select binders to conditionally agonize IL2R{gamma}{beta} and 41BB on PD1+, LAG3+, or CD8+ T cells. These Tentacles demonstrate promising manufacturability, antibody-like exposure in vivo, and strong anti-tumor activity in a humanized tumor model. Overall, we believe the incorporation of VH-Select binders into multispecific Tentacles has the potential to create a host of conditionally active biologics to treat various chronic and acute diseases.

bioengineering↗

bioRxiv2025

Coil and flow diverting stents as drug delivery platforms for cerebral aneurysm treatment

Cerebral aneurysm occlusion with coils and flow diverting stents has become the first line treatment for both unruptured and ruptured cerebral aneurysms. As these technologies have advanced, there have been changes in device shape and surface coating to enhance aneurysm embolization while reducing stent thrombogenicity. Drug eluting stents have been used with great success in the targeted delivery of rapamycin, a mTOR complex 1 inhibitor to prevent restenosis in coronary and peripheral artery disease. However, few studies have investigated the use of coils and stents as delivery platforms for sustained drug release to cerebral aneurysm tissue. In this study, we used the bio-compatible and degradable polymers, gelatin and PLGA and a simple evaporative coating technique to investigate the release of rapamycin over time from coated platinum coils and Pipeline flow diverting stents. Rapamycin coated coils were incubated with human vascular endothelial cells in vitro to confirm therapeutic levels of rapamycin release. The rate of rapamycin release was similar in both gelatin and PLGA coated coils and was sustained for more than three weeks. Rapamycin was bioactive, at a therapeutic dose and inhibited mTOR complex 1 in human brain endothelial cells treated with a rapamycin coated coil. The relative degree of mTOR complex 1 inhibition was greater in PLGA compared to gelatin coated coils. Coating flow diverting stents with a rapamycin-PLGA coating demonstrated continuous rapamycin release over a 35 day period. Reducing the percent PLGA polymer concentration caused a robust and sustainable release of rapamycin. The PLGA coating was resilient enough to allow device recapturing without affecting rapamycin eluting rates or device deployment and expansion. This work provides a simple, feasible and tunable method to coat occlusion devices for preclinical studies investigating targeted drug delivery for improved parent vessel healing and aneurysm obliteration.

bioengineering↗

bioRxiv2024

Glycolytic flux controls retinal progenitor cell differentiation via regulating Wnt signaling

Metabolic pathways are remodeled in response to energy and other homeostatic demands and are dynamically regulated during embryonic development, suggesting a role in guiding cellular differentiation. Here, we show that glycolytic flux is required and sufficient to bias multipotent retinal progenitor cells (RPCs) to acquire a rod photoreceptor fate in the murine retina. In RPC-specific Phosphatase and tensin homolog conditional knock-out (Pten-cKO) and RPC-specific conditional gain-of-function of dominant active PFKB3 (cytoPFKB3) mice, glycolytic gene expression and activity are elevated, correlating with precocious rod photoreceptor differentiation and outer segment maturation. Conversely, glycolytic inhibition in retinal explants suppresses RPC proliferation and photoreceptor differentiation, achieved either with 2-deoxy-d-glucose, a competitive inhibitor of glucose metabolism, by lowering media pH, which disables PKM2, a rate-limiting enzyme, or by inhibiting lactate/H+ symporters, which lowers intracellular pH. Mechanistically, we show that Wnt signaling, the top-upregulated pathway in Pten-cKO retinas, is a glycolysis-dependent pathway. Pharmacological and genetic perturbation of Wnt signaling by knocking-out Ctnnb1, encoding {beta}-catenin, phenocopies glycolytic inhibition, suppressing RPC proliferation, photoreceptor differentiation and outer segment maturation. Thus, developmental rewiring of glycolytic flux modulates Wnt signaling to drive rod photoreceptor differentiation and maturation, an instructive role that may be exploited therapeutically for cell replacement strategies. IMPACT STATEMENTTransgenic and pharmacological approaches reveal developmental elevations in glycolytic flux have an instructive role in promoting rod photoreceptor differentiation and maturation via activation of Wnt signaling.

developmental biology↗

bioRxiv2024

Visualizing chaperone-mediated multistep assembly of the human 20S proteasome

Dedicated assembly factors orchestrate stepwise production of many molecular machines, including the 28-subunit proteasome core particle (CP) that mediates protein degradation. Here, we report cryo-EM reconstructions of seven recombinant human subcomplexes that visualize all five chaperones and the three active site propeptides across a wide swath of the assembly pathway. Comparison of these chaperone-bound intermediates and a matching mature CP reveals molecular mechanisms determining the order of successive subunit additions, and how proteasome subcomplexes and assembly factors structurally adapt upon progressive subunit incorporation to stabilize intermediates, facilitate the formation of subsequent intermediates, and ultimately rearrange to coordinate proteolytic activation with gated access to active sites. The structural findings reported here explain many previous biochemical and genetic observations. This work establishes a methodologic approach for structural analysis of multiprotein complex assembly intermediates, illuminates specific functions of assembly factors, and reveals conceptual principles underlying human proteasome biogenesis.

biochemistry↗

bioRxiv2023

Decreasing alpha flow releases task-specific processing paths

Directing and maintaining attention toward relevant information and away from non-relevant information is a critical competence of higher-order nervous systems. Here, we used directed connectivity to investigate how the human brain prioritizes appropriate processing paths when participants are performing a behavioral task requiring attention in the visual or the auditory modality. We show that the efficient transfer of information relevant for the task is mediated by a region- and task-specific decrease of alpha band connectivity from parietal and, in case a motor response is required, motor cortex to the relevant sensory cortices. Further, alpha-band connectivity modulations reliably predict alpha power modulations in the task-related sensory cortices, particularly where the task-irrelevant cortex is inhibited via local alpha power increases. We suggest that the task- and region-specific modulation of alpha-band connectivity in the present study is a basic neuronal mechanism orchestrating the allocation of task-relevant neuronal resources related to and possibly underlying the previously reported sensory alpha power modulations in association with the allocation of competing attentional resources.

neuroscience↗

bioRxiv2023

D-SPIN constructs gene regulatory network models from multiplexed scRNA-seq data revealing organizing principles of cellular perturbation response

Gene regulatory networks within cells modulate the expression of the genome in response to signals and changing environmental conditions. Reconstructions of gene regulatory networks can reveal the information processing and control principles used by cells to maintain homeostasis and execute cell-state transitions. Here, we introduce a computational framework, D-SPIN, that generates quantitative models of gene regulatory networks from single-cell mRNA-seq datasets collected across thousands of distinct perturbation conditions. D-SPIN constructs probabilistic models of regulatory interactions between genes or gene-expression programs to fit the cell state distributions under different perturbations. Using large Perturb-seq and drug-response datasets, we demonstrate that D-SPIN models reveal key regulators of cell fate decisions and the coordination of distant cellular pathways in response to gene knockdown perturbations. D-SPIN also dissects gene-level drug response mechanisms in heterogeneous cell populations, elucidating how combinations of immunomodulatory drugs acting on distinct regulators induce novel cell states through additive recruitment of gene expression programs. The D-SPIN model further predicts cell state distributions under drug dosage combinations beyond the training data. D-SPIN provides a computational framework for constructing interpretable models of gene regulatory networks to reveal principles of cellular information processing and physiological control.

bioinformatics↗

bioRxiv2022

Pten regulates endocytic trafficking of cell adhesion and signaling molecules to pattern the retina

The retina is an exquisitely patterned tissue, with neuronal somata positioned at regular intervals to completely sample the visual field. Cholinergic amacrine cells are spectacular exemplars of precision, distributing in two radial layers and tangentially, forming regular mosaics. Here, we investigated how the intracellular phosphatase Pten and the cell adhesion molecule Dscam cooperate to regulate amacrine cell patterning. Using double mutants to test epistasis, we found that Pten and Dscam function in parallel pathways to regulate amacrine cell positioning. Mechanistically, Pten regulates endocytic remodeling of cell adhesion molecules (Dscam, Megf10, Fat3), which are aberrantly redistributed in Pten conditional-knock-out (cKO) amacrine cells. Furthermore, extracellular vesicles derived from multivesicular endosomes have altered proteomes in PtencKO retinas. Consequently, Wnt signaling is elevated in PtencKO retinal amacrine cells, the pharmacological disruption of which phenocopies PtencKO patterning defects. Pten thus controls endocytic trafficking of critical cell adhesion/signaling molecules to control amacrine cell spacing. HIGHLIGHTSO_LIPten and Dscam act in parallel pathways to regulate amacrine cell spacing C_LIO_LIEndocytic remodeling of cell adhesion molecules is perturbed in PtencKO retinas C_LIO_LIExtracellular vesicle content is altered in PtencKO retinas C_LIO_LIPerturbation of Wnt signaling phenocopies defects in amacrine cell positioning C_LI eTOC BLURBPatterns in nature range from stereotyped distributions of colored patches on butterfly wings to precise neuronal spacing in the nervous system. Waddington proposed that built-in constraints canalize developmental patterns. Touahri et al. identified Pten-mediated endocytic trafficking of cell adhesion/signaling molecules as a novel constraint measure controlling retinal amacrine cell patterning.

neuroscience↗

bioRxiv2022

An accessible and versatile deep learning-based sleep stage classifier

Manual sleep analysis for research purposes and for the diagnosis of sleep disorders is labor-intensive and often produces unreliable results, which has motivated many attempts to design automatic sleep stage classifiers. With the recent introduction of large, publicly available hand-scored polysomnographic data, and concomitant advances in machine learning methods to solve complex classification problems with supervised learning, the problem has received new attention, and a number of new classifiers that provide excellent accuracy. Most of these however have non-trivial barriers to use. We introduce the Greifswald Sleep Stage Classifier (GSSC), which is free, open source, and can be relatively easily installed and used on any moderately powered computer. In addition, the GSSC has been trained to perform well on a large variety of electrode set-ups, allowing high performance sleep staging with portable systems. The GSSC can also be readily integrated into brain-computer interfaces for real-time inference. These innovations were achieved while simultaneously reaching a level of accuracy equal to, or exceeding, recent state of the art classifiers and human experts, making the GSSC an excellent choice for researchers in need of reliable, automatic sleep staging.

neuroscience↗

bioRxiv2022

Development of amidase-dependent pyrazinoic acid prodrugs with activity against pyrazinamide resistant Mycobacterium tuberculosis

Rapid emergence of drug resistance in Mycobacterium tuberculosis (Mtb) is one of the most significant healthcare challenges of our time. The cause of drug resistance is multifactorial, with the long course anti-tubercular therapy required to treat tuberculosis (TB) constituting a major contributing factor. Introduction of pyrazinamide (PZA) resulted in shortening of TB treatment from twelve to six months and consequently played a critical role in curbing drug resistance that developed over long course therapy. Nevertheless, because PZA is a prodrug activated by a nonessential amidase, PncA, resistance to PZA develops and frequently results in treatment failure. Here, we leveraged a whole cell drug screening approach to identify anti-tuberculars with unconventional mechanisms of action or activation that could be further developed into compounds effective at killing Mtb resistant to PZA. We discovered an amide containing prodrug, DG160, that was activated by the amidase, Rv2888c (AmiC). This amidase was capable of metabolizing a variety of amide containing compounds including a novel pyrazinoic acid-isoquinolin-1-amine prodrug, JSF-4302, which we developed as a potential PncA-independent replacement for PZA. As predicted, AmiC activation of JSF-4302 led to the generation of POA in Mtb including in a PZA resistant clinical isolate, thereby successfully delivering the active component of PZA while bypassing the need for activation by PncA. This work provides a framework for a new approach to drug development and prodrug activation in Mtb. SIGNIFICANCEPyrazinamide (PZA) is a vital component of Mycobacterium tuberculosis (Mtb) treatment since its inclusion shortened tuberculosis therapy by six months. However, PZA is a prodrug and resistance develops at a high frequency due to mutations in its activator PncA. Here, we present the discovery of amide-containing anti-tubercular prodrugs that are activated intracellularly by the Mtb amidase, AmiC. Taking advantage of this finding, we successfully designed and synthesized pyrazinoic acid (POA) prodrugs that were activated by AmiC and found that these compounds delivered intracellular POA to PZA- resistant Mtb isolates that contained a nonfunctional PncA. This new approach to prodrug development provides a method for delivering conjugated drugs into Mtb with the potential to overcome clinical drug resistance.

microbiology↗

bioRxiv2021

Towards optimization of oscillatory stimulation during sleep

BackgroundOscillatory rhythms during sleep such as slow oscillations (SO) and spindles, and most importantly their coupling, are thought to underlie processes of memory consolidation. External slow oscillatory transcranial direct current stimulation (so-tDCS) with a frequency of 0.75 Hz has been shown to improve this coupling and memory consolidation, however, effects varied quite markedly between individuals, studies, and species. Here, we aimed to determine how precisely the frequency of stimulation has to match the naturally occurring SO frequency in individuals to optimally improve SO-spindle coupling. Moreover, we systematically tested stimulation durations necessary to induce changes. MethodsWe addressed these questions by comparing so-tDCS with individualized frequency to standardized frequency of 0.75 Hz in a within-subject design with 28 older participants during napping while systematically varying stimulation train durations between 30 s, 2 min, and 5 min. ResultsStimulation trains as short as 30 s were sufficient to modulate the coupling between SOs and spindle activity. Contrary to our expectations, so-tDCS with standardized frequency indicated stronger aftereffects with regard to SO-spindle coupling compared to individualized frequency. Angle and variance of spindle maxima occurrence during the SO cycle were similarly modulated. ConclusionIn sum, short stimulation trains were sufficient to induce significant changes in sleep physiology allowing for more trains of stimulation, which provides methodological advantages and possibly even larger behavioral effects in future studies. With regard to individualized stimulation frequency, further options of optimization need to be investigated, such as closed-loop stimulation to calibrate stimulation frequency to the SO frequency at time of stimulation onset. Significance statementApplication of slow oscillatory transcranial direct current stimulation during sleep has been shown to enhance specific memory-relevant sleep parameters and memory performance after sleep, albeit with a high degree of variability. Here, we systematically explored two major stimulation parameters possibly accounting for this variability in humans: frequency and duration of stimulation. We found, contrary to our expectations, standardized frequency stimulation with 0.75 Hz being superior to individualized frequency stimulation in enhancing specific sleep parameters. Moreover short stimulation trains of 30 seconds were as effective as 5 min in modulating aftereffects. These are encouraging findings, implying methodological advantages as larger quantity of aftereffects data can be obtained within the same time window, which may also lead to enhanced behavioral stimulation effects.

neuroscience↗

bioRxiv2021

Direct observation of adaptive tracking on ecological timescales in Drosophila

Direct observation of evolution in response to natural environmental change can resolve fundamental questions about adaptation including its pace, temporal dynamics, and underlying phenotypic and genomic architecture. We tracked evolution of fitness-associated phenotypes and allele frequencies genome-wide in ten replicate field populations of Drosophila melanogaster over ten generations from summer to late fall. Adaptation was evident over each sampling interval (1-4 generations) with exceptionally rapid phenotypic adaptation and large allele frequency shifts at many independent loci. The direction and basis of the adaptive response shifted repeatedly over time, consistent with the action of strong and rapidly fluctuating selection. Overall, we find clear phenotypic and genomic evidence of adaptive tracking occurring contemporaneously with environmental change, demonstrating the temporally dynamic nature of adaptation. One sentence summaryRapid environmental change drives continuous phenotypic and polygenic adaptation, demonstrating the temporal dynamism of adaptation.

evolutionary biology↗

bioRxiv2021

Plagl1 is part of the mammalian retinal injury response and a critical regulator of Muller glial cell quiescence

Retinal damage triggers reactive gliosis in Muller glia across vertebrate species, but only in regenerative animals, such as teleost fish, do Muller glia initiate repair; proliferating and undergoing neurogenesis to replace lost cells. By mining scRNA-seq and bulk RNA-seq datasets, we found that Plagl1, a maternally imprinted gene, is dynamically regulated in reactive Muller glia post-insult, with transcript levels transiently increasing before stably declining. To study Plagl1 retinal function, we examined Plagl1+/-pat null mutants postnatally, revealing defects in retinal architecture, visual signal processing and a reactive gliotic phenotype. Plagl1+/-pat Muller glia proliferate ectopically and give rise to inner retinal neurons and photoreceptors. Transcriptomic and ATAC-seq profiles revealed similarities between Plagl1+/-pat retinas and neurodegenerative and injury models, including an upregulation of pro-gliogenic and pro-proliferative pathways, such as Notch, not observed in wild-type retinas Plagl1 is thus an essential component of the transcriptional regulatory networks that retain mammalian Muller glia in quiescence.

neuroscience↗

bioRxiv2021

Non-canonical Hedgehog signaling through L-type voltage gated Ca2+ channels controls CD8+ T cell killing

Cytotoxic CD8+ T lymphocytes (CTLs) are critical to the immune response against intracellular pathogens and cancer and act by eliminating infected and malignant cells through targeted secretion of cytotoxic granules. Hedgehog (Hh) signaling has been shown to be critical for CTL killing. Interestingly, Hh signaling in CD8+ T cells is not induced by extracellular Hh ligands but is initiated upon T cell receptor (TCR) engagement. How the TCR induces the Hh pathway independently of extracellular Hh ligands is unknown. Here we show that the Hh transcription factor Gli1 is essential for efficient CTL function and is induced downstream of the TCR by an extracellular Ca2+ influx selectively controlled by L-type voltage gated Ca2+ channels localized at the plasma membrane. We demonstrate that this novel mode of Hh signaling induction is independent of the canonical Hh pathway and represents the primary mechanism of Gli1 induction in naive CD8+ T cells, while CTLs can also activate Gli1 via MAP Kinase signaling. Importantly, we show that this L-type voltage gated Ca2+ channel-controlled Gli1 induction is functionally required for CTL killing in mice and humans. Gli inhibitors are currently in clinical trials against various cancers and our observations indicate that they likely inhibit the anti-tumor response. Significance statementCytotoxic CD8+ T cells (CTLs) kill infected and malignant cells by targeted secretion of cytotoxic granules. Hedgehog signaling is critical for effective CTL killing and is activated by the T cell receptor (TCR) independently of exogenous Hedgehog ligands. This study shows that Hedgehog transcription factor Gli1 is required for CTL killing and identifies L-type voltage gated Ca2+ channels (Cav1) as essential regulators of CTL killing in mouse and human, by virtue of their ability to activate Gli1 downstream of the TCR. This Cav1-Gli1 axis operates independently of canonical Hedgehog signaling. Our work suggests that caution is required when using Gli inhibitors, currently in trials as anti-cancer therapeutics, since they may dampen the anti-tumor response.

immunology↗