Biology subjects

Welsbie, D. S.

Publications and source records attributed to Welsbie, D. S..

7 recordsLinked to original sources

bioRxiv2026

Identification a Compact Promoter using a New Promoter Selection Strategy and Engineering Hybrid Pol II/III Enable Efficient Genome Editing in Human Retinal Ganglion Cells

Promoters and vectors are critical components of gene therapy, enabling the delivery and expression of therapeutic genes to correct both loss- and gain-of-function mutations. Adeno-associated virus (AAV) vectors are the leading platform for in vivo gene delivery; however, the widely used Streptococcus pyogenes Cas9 (SpCas9, 4.1 kb) approaches the AAV packaging limit of 4.7 kb. This constraint often necessitates dual-vector systems, which reduce therapeutic efficiency, or the use of smaller nucleases such as SaCas9 (3.2 kb) and AacCas12b (3.4 kb), which have lower PAM site frequencies. To enhance promoter selection for gene therapy applications, we developed a strategy to identify compact, cell-preferred RNA polymerase II (Pol II) promoters. Analysis of approximately 300 compact Pol II promoters revealed that exogenous expression levels in one cell type correlate more strongly with those in other cell types than with endogenous expression, underscoring the importance of exogenous expression efficiency in promoter selection. Using this approach, we identified a compact Pol II promoter #2 (Pro2, 133 bp) that drives robust transgene expression in human retinal ganglion cells (RGCs). To enable single-AAV delivery of SpCas9, we analyzed three commonly used Pol III promoters (H1, 7SK and U6) and determined their minimal functional lengths using a CRISPR/Cas9 reporter assay. We further engineered three compact hybrid Pol II/III promoters which combined pro2 with minimal H1, 7SK and U6 (276, 294, and 323 bp) capable of co-expressing SpCas9 and gRNA, enabling efficient genome editing in both transfected HEK293 cells (approaching 100%) and human RGCs (up to 55.9%) from human stem cell-derived retinal ganglion cells (RGCs). Together, these findings establish a framework for developing single-AAV CRISPR-based gene therapy strategies. Authors contributionsPWZ and DJZ conceived the study, designed the experiments, performed data analysis and interpretation, and were the primary contributors to manuscript writing. STZ played a key role in data collection and correlation analysis. YYC, SL, LF, CJK, YD, CAB, JC, and DW contributed to the execution of essential experiments and subsequent data analysis. All authors have read and approved the final manuscript. Declaration of interestsThe authors declare no conflicts of interest.

molecular biology↗

bioRxiv2025

Cell-Intrinsic Vulnerability and Immune Activation Cooperate to Drive Degeneration in a Mitochondrial Complex I Deficiency Model of Optic Neuropathy

Mitochondrial dysfunction is a central hallmark of many optic neuropathies, yet the mechanisms linking intrinsic metabolic stress to retinal ganglion cell (RGC) degeneration remain unclear. To bridge this gap, we developed conditional transgenic models targeting the mitochondrial complex I subunit Ndufs4 in the retina. Broad deletion of Ndufs4 in the retina resulted in vision loss, progressive RGC degeneration, and pronounced immune activation before overt RGC death. Strikingly, depletion of myeloid cells significantly preserved RGCs, demonstrating that inflammation is not simply a downstream consequence but a participant in the degeneration process. To further distinguish between intrinsic and extrinsic mechanisms, we generated a mosaic model in which only subsets of retinal cells lacked Ndufs4. In this paradigm, the degeneration first appeared selectively in mutant regions, suggesting that mitochondrial impairment within RGCs is necessary to initiate vulnerability. At later stages, however, the degeneration extended beyond mutant territories, highly suggestive of a propagation through non-cell autonomous processes. Together, these findings support a model in which mitochondrial dysfunction creates the conditions for neuronal vulnerability, while immune responses govern the timing and extent of cell loss. This framework explains the consistent co-occurrence of metabolic deficits and neuroinflammation in optic neuropathies and highlights the importance of their interactions in disease progression. By clarifying the intersection of intrinsic and extrinsic mechanisms, this work advances our understanding of RGC degeneration and provides a conceptual basis for deciphering pathogenic processes across diverse optic neuropathies.

neuroscience↗

bioRxiv2025

Recovery of retinal terminal fields after traumatic brain injury: evidence of collateral sprouting and sexual dimorphism

The central nervous system is characterized by its limited regenerative potential, yet striking examples of functional recovery after injury in animal models and humans highlight its capacity for repair. Little is known about repair of pathways/circuits after traumatic brain injury (TBI), which results in disruption of connectivity. Here we utilize a mouse model of diffuse traumatic axonal injury (Impact-acceleration TBI) in order to explore, for the first time, the evolution of structural and functional changes in the terminal fields of the injured visual system. Retinal ganglion cell (RGC) axons and synapses were genetically labeled via AAV transduction, while anterograde and transsynaptic tracers were used to mark terminals and postsynaptic cells. Functional connectivity and visual integrity were assessed by monitoring c-Fos expression following light stimulation and pattern-reversal visual evoked potentials (pVEPs). Our findings demonstrate that, although TAI results in approximately a 50% loss of RGC axons and terminals, surviving RGCs undergo collateral sprouting, a form of compensatory branching of surviving axons, that restores terminal density to pre-injury levels. Transsynaptic tracing and c-Fos mapping confirmed the reestablishment of connectivity, which was also associated with significant improvements in visual function as measured by pVEPs. Interestingly, the recovery process exhibited sexual dimorphism, with female mice showing delayed or incomplete repair. Moreover, collateral sprouting proceeded normally in Sarm1 knockout mice, evidence of some independence from Wallerian degeneration. Our findings show that collateral sprouting may be an important mechanism of circuit repair in TAI and may represent a promising target for therapeutic interventions. SignificanceHomotypic collateral sprouting -the process by which uninjured axons from the same neuronal source extend new branches to reinnervate targets deprived of their original connections- is a fundamental yet understudied mechanism for CNS repair following injury. Unlike heterotypic sprouting, involving sprouting from unrelated pathways, homotypic sprouting offers potential to restore circuit architecture after partial lesions. Here, we employed a model of diffuse axonal injury in the mouse visual system to examine this mechanism. Our research demonstrates surviving retinal ganglion cell axons can re-establish terminal fields, achieving structural and functional connectivity. Importantly, we discovered significant sex differences: female mice showed delayed/incomplete recovery compared to males. These findings provide evidence of repair of brain circuits perturbed by TBI and the role of homotypic sprouting.

neuroscience↗

bioRxiv2024

Afadin Sorts Different Retinal Neuron Types into Accurate Cellular Layers

Neurons use cell-adhesion molecules (CAMs) to interact with other neurons and the extracellular environment: the combination of CAMs specifies migration patterns, neuronal morphologies, and synaptic connections across diverse neuron types. Yet little is known regarding the intracellular signaling cascade mediating the CAM recognitions at the cell surface across different neuron types. In this study, we investigated the neural developmental role of Afadin1-4, a cytosolic adapter protein that connects multiple CAM families to intracellular F-actin. We introduced the conditional Afadin mutant5 to an embryonic retinal Cre, Six3-Cre6-8. We reported that the mutants lead to the scrambled retinal neuron distribution, including Bipolar Cells (BCs), Amacrine Cells (ACs), and retinal ganglion cells (RGCs), across three cellular layers of the retina. This scrambled pattern was first reported here at neuron-type resolution. Importantly, the mutants do not display deficits for BCs, ACs, or RGCs in terms of neural fate specifications or survival. Additionally, the displayed RGC types still maintain synaptic partners with putative AC types, indicating that other molecular determinants instruct synaptic choices independent of Afadin. Lastly, there is a significant decline in visual function and mis-targeting of RGC axons to incorrect zones of the superior colliculus, one of the major retinorecipient areas. Collectively, our study uncovers a unique cellular role of Afadin in sorting retinal neuron types into proper cellular layers as the structural basis for orderly visual processing.

neuroscience↗

bioRxiv2024

Optineurin-facilitated axonal mitochondria delivery promotes neuroprotection and axon regeneration

Optineurin (OPTN) mutations are linked to amyotrophic lateral sclerosis (ALS) and normal tension glaucoma (NTG), but a relevant animal model is lacking, and the molecular mechanisms underlying neurodegeneration are unknown. We found that OPTN C-terminus truncation (OPTN{Delta}C) causes late-onset neurodegeneration of retinal ganglion cells (RGCs), optic nerve (ON), and spinal cord motor neurons, preceded by a striking decrease of axonal mitochondria. Surprisingly, we discover that OPTN directly interacts with both microtubules and the mitochondrial transport complex TRAK1/KIF5B, stabilizing them for proper anterograde axonal mitochondrial transport, in a C- terminus dependent manner. Encouragingly, overexpressing OPTN/TRAK1/KIF5B reverses not only OPTN truncation-induced, but also ocular hypertension-induced neurodegeneration, and promotes striking ON regeneration. Therefore, in addition to generating new animal models for NTG and ALS, our results establish OPTN as a novel facilitator of the microtubule-dependent mitochondrial transport necessary for adequate axonal mitochondria delivery, and its loss as the likely molecular mechanism of neurodegeneration.

neuroscience↗

bioRxiv2019

Biomolecular Contrast Agents for Optical Coherence Tomography

Optical coherence tomography (OCT) has gained wide adoption in biological and medical imaging due to its exceptional tissue penetration, 3D imaging speed and rich contrast. However, OCT plays a relatively small role in molecular and cellular imaging due to the lack of suitable biomolecular contrast agents. In particular, while the green fluorescent protein has provided revolutionary capabilities to fluorescence microscopy by connecting it to cellular functions such as gene expression, no equivalent reporter gene is currently available for OCT. Here we introduce gas vesicles, a unique class of naturally evolved gas-filled protein nanostructures, as the first genetically encodable OCT contrast agents. The differential refractive index of their gas compartments relative to surrounding aqueous tissue and their nanoscale motion enables gas vesicles to be detected by static and dynamic OCT at picomolar concentrations. Furthermore, the OCT contrast of gas vesicles can be selectively erased in situ with ultrasound, allowing unambiguous assignment of their location. In addition, gas vesicle clustering modulates their temporal signal, enabling the design of dynamic biosensors. We demonstrate the use of gas vesicles as reporter genes in bacterial colonies and as purified contrast agents in vivo in the mouse retina. Our results expand the utility of OCT as a unique photonic modality to image a wider variety of cellular and molecular processes.

bioengineering↗

bioRxiv2018

Thyroid hormone signaling specifies cone subtypes in human retinal organoids

The mechanisms underlying the specification of diverse neuronal subtypes within the human nervous system are largely unknown. The blue (shortwavelength/S), green (medium-wavelength/M) and red (long-wavelength/L) cone photoreceptors of the human retina enable high-acuity daytime vision and trichromatic color perception. Cone subtypes are specified in a poorly understood two-step process, with a first decision between S and L/M fates, followed by a decision between L and M fates. To determine the mechanism controlling S vs. L/M fates, we studied the differentiation of human retinal organoids. We found that human organoids and retinas have similar distributions, gene expression profiles, and morphologies of cone subtypes. We found that S cones are specified first, followed by L/M cones, and that thyroid hormone signaling is necessary and sufficient for this temporal switch. Temporally dynamic expression of thyroid hormone degrading and activating proteins supports a model in which the retina itself controls thyroid hormone levels, ensuring low signaling early to specify S cones and high signaling late to produce L/M cones. This work establishes organoids as a model for determining the mechanisms of cell fate specification during human development.\n\nOne sentence summaryCone specification in human organoids

developmental biology↗