Biology subjects

Tsoulfas, P.

Publications and source records attributed to Tsoulfas, P..

10 recordsLinked to original sources

bioRxiv2026

AAV2-Retro-Mediated Gene Transfer Selectively Targets Outer Retinal Cells Following Intravitreal Injection

PurposeTo characterize the cellular tropism and temporal dynamics of adeno-associated virus 2 (AAV2)-retro-mediated gene delivery in the adult mouse retina following intravitreal injection. MethodsAdult C57BL/6J mice received single or sequential intravitreal injections of AAV2-retro carrying the mGreenLantern (mGL) reporter gene. Retinas were collected at 1-, 3-, and 14-days post-injection (dpi) and processed for immunofluorescence analysis. Transduced cell types were identified using cell-type markers, including cone arrestin, RBPMS, and AP-2. The number and distribution of mGL-positive cells were quantified on whole retinas or retinal cross-sections to assess transduction efficiency, specificity, and spatial coverage. ResultsReporter expression was detected in the outer retina at 1 dpi and increased markedly at 3 and 14 dpi. AAV2-retro demonstrated strong tropism for photoreceptors and retinal pigment epithelium (RPE), with robust labeling of both rods and cones. In contrast to the robust outer retinal expression, transduction in the inner nuclear layers was limited to a few retinal ganglion and amacrine cells, reflecting strong cell-type specificity. Reporter expression was distributed widely across the retina, exceeding the localized pattern typically observed following subretinal delivery with conventional AAV2 vectors. Sequential injections further increased reporter expression and spatial coverage compared with single injections. ConclusionsAAV2-retro enables efficient, outer retina-specific gene delivery following intravitreal administration. This approach overcomes the limitations of traditional intravitreal gene transfer and provides a minimally invasive alternative to subretinal injection. AAV2-retro- mediated transduction may facilitate preclinical studies of retinal degeneration and support the development of gene therapies aimed at preserving photoreceptors and RPE function.

neuroscience↗

bioRxiv2025

Retrograde viral delivery of hyper-IL-6 activates Stat3 in corticospinal tract neurons but causes severe tremors and weight loss in adult mice

Cytokines in the IL-6 family can improve axon regeneration after nervous system injury, acting in part by stimulating Stat3 phosphorylation and pro-regenerative signaling in injured neurons. Hyper-IL-6 (hIL-6) is an engineered ligand with enhanced signaling properties, shown previously to stimulate axon growth even more effectively than native IL-6 family members. Here we tested a method of hIL-6 delivery based on injection of retrograde AAV to the cervical spinal cord of adult mice. This was envisioned as a first step toward a regenerative treatment after spinal cord injury based on widespread stimulation of descending projection neurons. We found, however, that animals treated with AAV2-retro-hIL-6 developed severe tremors and weight loss within days of injection and reached criteria for humane euthanasia within five to eleven days, depending on viral dose. Examination of the cortex showed a pronounced increase in Stat3 phosphorylation in corticospinal tract (CST) neurons in hIL-6 animals versus control, confirming effective retrograde signaling. Stat3 phosphorylation and evidence of microglial activation were detected in tissue surrounding CST cell bodies and along descending CST axons, revealing extensive cytokine signaling and microglial response associated with secreted hIL-6. These results confirm the ability of hIL-6 expression to activate Stat3 signaling in CST neurons but uncover the potential for severe negative effects that must inform any further development of hIL-6 as a pro-regenerative therapeutic. SIGNIFICANCE STATEMENTThere is an urgent need to develop therapeutic strategies to improve repair after nervous system injury. In this study, we tested a new way to deliver Hyper-IL-6, an engineered cytokine with enhanced signaling properties, based on retrograde adeno-associated injection to the spinal cord and widespread uptake by descending axons. Expression of hIL-6 activated an expected pathway in descending neurons but also caused severe side effects and signs of widespread microglial activation in the cortex. These outcomes confirm potent signal activation by hIL-6 but reveal severe off-target effects that must be considered in any future application as a pro-regenerative treatment.

neuroscience↗

bioRxiv2025

Sox11 overexpression restores embryonic pro-growth transcription in mature corticospinal tract neurons

Neurons in the central nervous system (CNS) display a high capacity for axon growth during early development but lose this ability at a pivotal differentiation stage marked by synaptic maturation, circuit integration, and a profound shift in gene transcription. Once mature, most CNS neurons fail to reverse this transcriptional switch after axon injury, fundamentally constraining their intrinsic capacity for axon regeneration. Here, we show with single-nucleus RNA sequencing that forced expression of the transcription factor Sox11 in mature corticospinal tract (CST) neurons produces large-scale and stable changes in gene expression that are highly enriched for growth-relevant processes, and which strongly resemble those of pre-synaptic embryonic stages. Moreover, Sox11 is equally effective when delivered to chronically injured CST neurons. These data reveal the ability of Sox11 to reverse a critical step of neuronal maturation even in otherwise unperturbed neurons, clarifying the transcriptional underpinnings and highlighting the potential of Sox11 to act as a pro-regenerative stimulus.

neuroscience↗

bioRxiv2025

Chronic Cervical Midline Contusion in Rats Disrupts Aerobic, Muscular, and Cardiovascular Function

Cardiovascular dysfunction significantly contributes to morbidity and mortality following cervical spinal cord injury (SCI). Unfortunately, only a limited number of preclinical models have been developed for investigating cardiovascular dysfunction following cervical SCI. Furthermore, the broader consequences of cervical SCI on aerobic capacity and muscle endurance during physiological stress testing also remains understudied preclinically. Therefore, in this study we assessed potential deficits across multiple physiological systems in a rat model of cervical SCI using a battery of stress tests. Female Sprague-Dawley rats (n = 20) received either a C8 midline contusion (cSCI) or laminectomy alone as a control (LAM). Exercise stress testing was conducted to evaluate cardiorespiratory fitness and recovery using a metabolic treadmill, or forelimb fitness using the isometric pull task. Orthostatic stress testing and pharmacological stress testing were also performed to more directly challenge the cardiovascular system. Our findings demonstrate a decline in aerobic fitness in cSCI rats, as evidenced by dysregulated excess post-exercise oxygen consumption. cSCI rats also exhibited impaired muscle endurance compared to LAM. During orthostatic stress testing, 70% of cSCI rats experienced an approximately 25 mmHg decrease in systolic blood pressure and 20 mmHg decrease in diastolic blood pressure, in addition to modest but significant decreases in heart rate, myocardial contractility index, stroke volume index, and cardiac output index. During dobutamine infusion, cardiac output index and stroke volume index were significantly reduced following cSCI compared to LAM. Overall, these stress testing results suggest that preclinical cervical SCI in rats can lead to, and therefore model, clinically relevant impairments in cardiopulmonary exercise performance, muscle endurance, and cardiovascular function.

physiology↗

bioRxiv2024

Injury distance limits the transcriptional response to spinal injury

The ability of neurons to sense and respond to damage is fundamental to homeostasis and nervous system repair. For some cell types, notably dorsal root ganglia (DRG) and retinal ganglion cells (RGCs), extensive profiling has revealed a large transcriptional response to axon injury that determines survival and regenerative outcomes. In contrast, the injury response of most supraspinal cell types, whose limited regeneration constrains recovery from spinal injury, is mostly unknown. Here we employed single-nuclei sequencing in mice to profile the transcriptional responses of diverse supraspinal cell types to spinal injury. Surprisingly, thoracic spinal injury triggered only modest changes in gene expression across all populations, including corticospinal tract (CST) neurons. Moreover, CST neurons also responded minimally to cervical injury but much more strongly to intracortical axotomy, including upregulation of numerous regeneration and apoptosis-related transcripts shared with injured DRG and RGC neurons. Thus, the muted response of CST neuron to spinal injury is linked to the injurys distal location, rather than intrinsic cellular characteristics. More broadly, these findings indicate that a central challenge for enhancing regeneration after a spinal injury is the limited sensing of distant injuries and the subsequent modest baseline neuronal response.

neuroscience↗

bioRxiv2022

Single nuclei analyses reveal transcriptional profiles and marker genes for diverse supraspinal populations

The mammalian brain contains numerous neurons distributed across forebrain, midbrain, and hindbrain that project axons to the lower spinal cord and work in concert to control movement and achieve homeostasis. Extensive work has mapped the anatomical location of supraspinal cell types and continues to establish specific physiological functions. The patterns of gene expression that typify and distinguish these disparate populations, however, are mostly unknown. Here we combined retrograde labeling of supraspinal cell nuclei with fluorescence activated nuclei sorting and single nuclei RNA sequencing analyses to transcriptionally profile neurons that project axons from the mouse brain to lumbar spinal cord. We identified fourteen transcriptionally distinct cell types and used a combination of established and newly identified marker genes to assign an anatomical location to each. To validate the putative marker genes, we visualized selected transcripts and confirmed selective expression within lumbar-projecting neurons in discrete supraspinal regions. Finally, we illustrate the potential utility of these data by examining the expression of transcription factors that distinguish different supraspinal cell types and by surveying the expression of receptors for growth and guidance cues that may be present in the spinal cord. Collectively these data establish transcriptional differences between anatomically defined supraspinal populations, identify a new set of marker genes of use in future experiments, and provide insight into potential differences in cellular and physiological activity across the supraspinal connectome. SIGNIFICANCE STATEMENTThe brain communicates with the body through a wide variety of neuronal populations with distinct functions and differential sensitivity to damage and disease. We have employed single nuclei RNA sequencing technology to distinguish patterns of gene expression within a diverse set of neurons that project axons from the mouse brain to the lumbar spinal cord. The results reveal transcriptional differences between populations previously defined on the basis of anatomy, provide new marker genes to facilitate rapid identification of cell type in future work, and suggest distinct responsiveness of different supraspinal populations to external growth and guidance cues.

neuroscience↗

bioRxiv2021

AAV-PHP.S-mediated delivery of reporters to cranial ganglion sensory neurons

Because of their ease of use and low risk containment, Adeno-Associated Virus vectors are indispensable tools for much of neuroscience. Yet AAVs have been used relatively little to study the identities and connectivity of peripheral sensory neurons because methods to selectively target particular receptive fields or neuron types have been limited. The introduction of the AAV-PHP.S capsid with selective tropism for peripheral neurons (Chan et al., 2017) offered a solution, which we further elaborate here. We demonstrate using AAV-PHP.S with GFP or mScarlet reporters, that all cranial sensory ganglia, i.e. for cranial nerves V, VII, IX and X, are targeted. Pseudounipolar neurons of both somatic and visceral origin, but not satellite glia, are efficiently transduced rapidly and express the gene of interest within 1 week of injection. Fluorescent reporter proteins are transported into the central and peripheral axons of these sensory neurons, permitting visualization of terminals at high resolution, and/or in intact, cleared brain using light sheet microscopy. By combining a Cre-dependent reporter with the AAV-PHP.S capsid, we confirmed expression in a cell-type dependent manner for both anatomical and targeted functional analyses. The AAV-PHP.S capsid will be a powerful tool for mapping the receptive fields and circuits of molecular subtypes of many somatosensory, gustatory and visceral sensory neurons. SIGNIFICANCE STATEMENTAAV vectors have become an essential tool for visualizing, manipulating, and recoding from neurons of the central nervous system. However, the technology is not widely used for peripheral neurons because of several technical limitations. The AAV-PHP.S synthetic capsid, which targets peripheral neurons, was recently introduced (Chan et al., 2017). Here, we establish key parameters for using this virus, including which cells are transduced, the timing of expression in central and peripheral terminals, distant from neuronal somata, and the effectiveness of Cre-dependent constructs for cell type selective expression. This permits the use of AAV for constructing detailed anatomic and functional maps of the projections of molecular subtypes of peripheral sensory neurons.

neuroscience↗

bioRxiv2021

Brain-wide quantification of the supraspinal connectome

The supraspinal connectome is essential for normal behavior and homeostasis and consists of numerous sensory, motor, and autonomic projections from brain to spinal cord. Study of supraspinal control and its restoration after damage has focused mostly on a handful of major populations that carry motor commands, with only limited consideration of dozens more that provide autonomic or crucial motor modulation. We now provide an experimental platform and associated web-based resource to rapidly profile the entire supraspinal mesoconnectome in adult mice. Optimized viral labeling, 3D imaging, and registration to a mouse digital neuroanatomical atlas assigned tens of thousands of supraspinal neurons to more than 60 identified regions. We demonstrate the approachs ability to clarify essential points of topographic mapping between spinal levels, to measure population-specific sensitivity to spinal injury, and to resolve previously unexplained variability in functional recovery. This work will spur progress by broadening understanding and enabling analyses of essential but understudied supraspinal populations.

neuroscience↗

bioRxiv2020

Single cell analysis of the cellular heterogeneity and interactions in the injured mouse spinal cord

The wound healing process that occurs after spinal cord injury is critical for maintaining tissue homeostasis and limiting tissue damage, but eventually results in a scar-like environment that is not conducive to regeneration and repair. A better understanding of this dichotomy is critical to developing effective therapeutics that target the appropriate pathobiology, but a major challenge has been the large cellular heterogeneity that results in immensely complex cellular interactions. In this study, we used single cell RNA sequencing to assess virtually all cell types that comprise the mouse spinal cord injury site. In addition to discovering novel subpopulations, we used expression values of receptor-ligand pairs to identify signaling pathways that potentially drive specific cellular interactions during angiogenesis, gliosis, and fibrosis. Our dataset is a valuable resource that provides novel mechanistic insight into the pathobiology of not only spinal cord injury, but also other traumatic disorders of the CNS.

neuroscience↗

bioRxiv2018

Global connectivity and function of descending spinal input revealed by 3D microscopy and retrograde transduction

The brain communicates with the spinal cord through numerous axon tracts that arise from discrete nuclei, transmit distinct functions, and often collateralize to facilitate the coordination of descending commands. In efforts to restore supraspinal connectivity after injury or disease, this complexity presents a major challenge to interpreting functional outcomes, while the wide distribution of supraspinal nuclei complicates the delivery of therapeutics. Here we harness retrograde viral vectors to overcome these challenges. We demonstrate highly efficient retrograde transduction by AAV2-Retro in adult female mice, and employ tissue clearing to visualize descending tracts and create a mesoscopic projectome for the spinal cord. Moreover, chemogenetic silencing of supraspinal neurons with retrograde vectors results in complete and reversible forelimb paralysis, illustrating effective modulation of supraspinal function. Retrograde efficacy persists even after spinal injury, highlighting therapeutic potential. These data provide a global view of supraspinal connectivity and illustrate the potential of retrograde vectors to parse the functional contributions of supraspinal inputs.

neuroscience↗