Biology subjects

Akula, S.

Publications and source records attributed to Akula, S..

10 recordsLinked to original sources

bioRxiv2026

TBX3 is required for the differentiation of a subset of retinal ganglion and cone photoreceptor cells

Retinogenesis is a key step driving the formation of the neurons that later coordinate angiogenesis in the retina. Understanding these complex interactions requires dissecting apart its cellular and molecular drivers. To tease apart the molecular links coupling retinogenesis to vascular development, we utilized conditional knockout mouse models to selectively delete the transcription factor T-box 3 (Tbx3) in 1) astrocytes, 2) postnatal retinal ganglion cells (RGCs), or 3) retinal progenitors. We demonstrate that the hypovascular retina phenotype observed upon Tbx3 loss most likely stems from a disruption of early retinogenesis. Embryonic analyses revealed that TBX3 is required during a critical retinogenic window coinciding with ATOH7 activity. While ATOH7+ progenitors are more abundant in Tbx3 mutants, the expression of key RGC and cone precursor factors (including TBX5, RXRgamma, ISL1/2, POU4F2, SHH, LHX4 and OTX2) is severely diminished, halting proper RGC and cone differentiation. Utilizing birthdating experiments and developmental immunostaining, we establish TBX3 as a novel upstream regulator of RGC and cone photoreceptor specification. Because the degeneration of RGCs is the definitive hallmark of diseases like glaucoma, identifying TBX3 as a driver of early RGC formation positions it as a promising new candidate biomarker for early-stage glaucoma diagnosis and potentially therapeutic targeting.

developmental biology↗

bioRxiv2024

Exploring a pico-well based scRNA-seq method (HIVE) for simplified processing of equine bronchoalveolar lavage cells

Single-cell RNA sequencing (scRNA-seq) is a valuable tool for investigating cellular heterogeneity in diseases such as equine asthma (EA). This study evaluates the HIVE scRNA-seq method, a pico-well-based technology, for processing bronchoalveolar lavage (BAL) cells from horses with EA. The HIVE method offers practical advantages, including compatibility with both field and clinical settings, as well as a gentle workflow suited for handling sensitive cells. Our results show that the major cell types in equine BAL were successfully identified; however, the proportions of T cells and macrophages deviated from cytological expectations, with macrophages being overrepresented and T cells underrepresented. Despite these limitations, the HIVE method confirmed previously identified T cell and macrophage subpopulations and defined other BAL cell subsets. However, compared to previous studies T helper subsets were less clearly defined. Additionally, consistent with previous scRNA-seq studies, the HIVE method detected fewer granulocytes and mast cells than anticipated in the total BAL samples. Nevertheless, applying the method to purified mast cells recovered an expected number of cells. A small set of eosinophils were also detected which have not been characterized in earlier studies. In summary these findings suggest that while the HIVE method shows promise for certain applications, further optimization is needed to improve the accuracy of cell type representation, particularly for granulocytes and mast cells, in BAL samples.

genomics↗

bioRxiv2022

Single cell transcriptomics delineates the immune-cell landscape in equine lower airways and reveals upregulation of the FKBP5 gene in horses with asthma

BackgroundEquine asthma (EA) is a heterogenous, complex disease with a significant negative impact on horse welfare and performance. EA and human asthma have fundamental similarities, making EA a useful large animal disease model. Bronchoalveolar lavage (BAL) fluid provides a snapshot sample of the immune cells occupying the alveolar space and is one of the most relevant sample types for studies of chronic inflammation in the lung. In this study, we sequenced single equine BAL-cells in order to study the immune cell landscape of the respiratory tract of horses diagnosed with mild-to-moderate EA and healthy controls. ResultsScRNA-seq analysis of ~63,000 cells from eleven horses diagnosed with mild-moderate asthma (mEA) and eight healthy controls were performed using the Drop-Seq technology. We identified five major immune cell populations in equine BAL; alveolar macrophages (AM), T cells, neutrophils, mast cells and dendritic cells, as well as subtypes thereof. The cellular subtypes demonstrated herein have previously not been characterized in horses. Differential gene expression analysis revealed upregulation of genes in mEA horses, including FKBP5 and CCL24, which have previously been associated with asthma in other species. The most significantly upregulated gene across the cell types in EA was FKBP5, a chaperone protein involved in regulating the assembly, activity, and sensitivity of the glucocorticoid receptor ConclusionHerein we demonstrate the first comprehensive scRNA-seq map of the immune-cell populations in BAL from horses with asthma and healthy individuals. The glucocorticoid receptor associated protein FKBP5 was identified as a potential biomarker for EA.

genomics↗

bioRxiv2022

The oncogenic fusion protein EWS-FLI1 promotes premature ageing of biomolecular condensates by catalyzing fibril formation.

Ewing sarcoma (EwS) is an aggressive pediatric cancer of bone and soft tissue. A chromosomal translocation that joins the low-complexity domain of EWS (EWSLCD) with the DNA-binding domain of FLI1 (FLI1DBD) creates EWS-FLI1, a fusion oncoprotein essential for EwS development and accounts for 85% of all EwS cases. EWS-FLI1 acts as an aberrant transcription factor and interferes with the normal functions of nucleic acid-binding proteins via multivalent interactions and biomolecular condensation. The FLI1DBD was found to directly interact with the EWSLCD causing enhanced phase separation and induced hardening of EWSLCD condensates. Three related ETS DBDs (ERG, ETV1 and PU.1) also induced EWSLCD condensate hardening. DNA binding blocked the interaction with the EWSLCD, and NMR spectroscopy confirmed that ETS DBDs interact with EWSLCD via the DNA-binding interface. Our results provide a physical basis for the dominant-negative effect EWS-FLI1 exerts on EWS and highlight the need for further investigations of the FLI1DBD-EWSLCD interaction in vivo.

biophysics↗

bioRxiv2022

Tbx3 is required for mammalian retinal angiogenesis.

PurposeIn Retinopathy of Prematurity (ROP), infants often have incomplete vascularization, affecting the temporal region. A factor expressed in this region during retinal development is the T-box factor, Tbx3, which has not been studied in the mammalian eye. The purpose of this study was to determine if Tbx3 is required during eye formation for retinal angiogenesis. MethodsConditional removal of Tbx3 from both retinal progenitors and astrocytes was done using the optic cup-Cre recombinase driver, BAC-Dkk3-CRE and analyzed using standard immunohistochemical techniques. ResultsWith Tbx3 loss, the retinas were hypovascular, as seen in patients with ROP and Familial Exudative Vitreoretinopathy (FEVR). Retinal vasculature failed to form the stereotypic tri-layered plexus in the dorsal-temporal region. Astrocyte precursors were reduced in number and failed to form a lattice at the dorsal-temporal edge. We next examined retinal ganglion cells, as they have been shown to play a critical role in retinal angiogenesis. We found that melanopsin expression and Islet1/2-positive retinal ganglion cells were reduced in the dorsal half of the retina. In previous studies, loss of melanopsin has been linked to hyaloid artery persistence, which we also observed in the Tbx3 cKO retina, as well as in infants with ROP or FEVR. ConclusionsTogether, these results show that TBX3 is required for normal mammalian eye formation for the first time. This potentially provides a new genetic model for retinal hypovascular diseases.

neuroscience↗

bioRxiv2022

Glial dysregulation in human brain in Fragile X-related disorders

AbstractWhile large trinucleotide repeat expansions at the FMR1 locus cause Fragile X Syndrome (FXS), smaller "premutations" are associated with the late-onset condition Fragile X-associated tremor/ataxia syndrome (FXTAS), which shows very different clinical and pathological features, with no clear molecular explanation for these marked differences. One prevailing theory posits that the premutation uniquely causes neurotoxic increases in FMR1 mRNA (i.e., 4-8-fold increases), but evidence to support this hypothesis is largely derived from analysis of peripheral blood. We applied single- nucleus RNA-sequencing to post-mortem frontal cortex and cerebellum from 9 individuals with Fragile X mutations as well as age and sex matched controls (n=6) to assess cell-type specific molecular neuropathology. We found robust reduction of FMR1 mRNA in FXS as expected, with modest but significant upregulation ([~]1.3 fold) of FMR1 in glial clusters associated with premutation expansions. In premutation cases we identified alterations in glia number in cortex and cerebellum. Differential expression analysis demonstrated altered cortical oligodendrocyte development, while gene ontology analysis revealed alterations in neuroregulatory roles of glia, such as glial modulation of neurotransmission and synaptic structure. We identified significant enrichment of known FMR1 protein target genes in differentially expressed gene lists in FXS as well as the premutation, suggesting FMR1 protein target pathways may represent a shared source of dysfunction in both conditions despite opposite FMR1 mRNA changes. These findings challenge existing dogma regarding FXTAS and implicate glial dysregulation as a critical facet of premutation pathophysiology, representing novel therapeutic targets directly derived from the human condition.

neuroscience↗

bioRxiv2021

Evaluation of the Efficacy of Omega-3 Fatty Acids Formulations in the Lipopolysaccharide-Induced Acute Lung Inflammation Rat Model

Many current treatment options for lung inflammation and thrombosis come with unwanted side effects. The natural omega-3 fatty acids (O3FA) are generally anti-inflammatory and antithrombotic. The O3FA are always administered orally and occasionally by intravenous (IV) infusion. The main goal of this study is to determine if O3FA administered by inhalation of a nebulized formulation mitigates LPS-induced acute lung inflammation in male Wistar rats. Inflammation was triggered by intraperitoneal injection of LPS once a day for 14 days. One hour later, rats received nebulized treatments consisting of egg lecithin emulsified O3, budesonide and Montelukast, and blends of O3 and melatonin or Montelukast or Cannabidiol; O3 was in the form of free fatty acids for all groups except one group with ethyl esters. Lung histology and cytokines were determined in n=3 rats per group at day 8 and day 15. All groups had alveolar histiocytosis severity scores half or less than that of the disease control (Cd) treated with LPS and saline only inhalation. IL-6, TNF-, TGF-{beta}, and IL-10 were attenuated in all O3 groups. IL-1{beta} was attenuated in most but not all O3 groups. O3 administered as ethyl ester was overall most effective in mitigating LPS effects. No evidence of lipid pneumonia or other chronic distress was observed. These preclinical data suggest that O3FA formulations should be further investigated as treatments in lung inflammation and thrombosis related lung disorders, including asthma, chronic obstructive pulmonary disease, lung cancer and acute respiratory distress like COVID-19.

physiology↗

bioRxiv2020

Role for the Na+/K+ ATPase α 3 subunit in folding and lamination of the human neocortex

Osmotic equilibrium and membrane potential in animal cells depend on concentration gradients of sodium (Na+) and potassium (K+) ions across the plasma membrane, a function that is catalyzed by the Na,K-ATPase alpha subunit. In vertebrates, four paralogous genes, ATP1A1-4, encode distinct alpha subunit isoforms (a1-a4), three of which (a1, a2, a3) are expressed in the brain, and two (a1, a3) predominantly in neurons. The a3 isoform, encoded by ATP1A3, is critical to neuronal physiology, and a growing spectrum of neurological diseases are associated with ATP1A3 pathogenic variants, with ages of onset ranging from early childhood to adulthood. Here, we describe ATP1A3 variants encoding dysfunctional a3 subunits in children affected by polymicrogyria, a developmental malformation of the cerebral cortex characterized by abnormal folding and laminar organization. To gain cell-biological insights into the spatiotemporal dynamics of prenatal ATP1A3 expression, we established a transcriptional atlas of ATP1A3 expression during cortical development using mRNA in situ hybridization and transcriptomic profiling of ~125,000 individual cells with single-cell RNA sequencing (Drop-Seq) from various areas of the midgestational human neocortex. We find that fetal expression of ATP1A3 is restricted to a subset of excitatory neurons carrying transcriptional signatures of neuronal activity and maturation characteristic of the developing subplate. Furthermore, by performing Drop-Seq on ~52,000 nuclei from four different areas of an infant human neocortex, we show that ATP1A3 expression persists throughout early postnatal development, not only within excitatory neurons across all cortical layers, but also and more predominantly in inhibitory neurons, with specific enrichment in fast-spiking basket cells. In addition, we show that ATP1A3 expression, both in fetal and postnatal neurons, tends to be higher in frontal cortical areas than in occipital areas, in a pattern consistent with the rostro-caudal maturation gradient of the human neocortex. Finally, we discover distinct co-expression patterns linking catalytic subunit isoforms (ATP1A1,2,3) and auxiliary isoforms (ATP1B1,2,3), suggesting the ATPase pump may form non-redundant, cell-type specific -{beta} combinations. Together, the importance of the developmental phenotypes and dynamic expression patterns of ATP1A3 point to a key role for a3 in the development and function of human cortex.

neuroscience↗

bioRxiv2019

The trajectory of gait development in mice

Gait irregularities are prevalent in neurodevelopmental diseases and disorders (NDDs). However, there is a paucity of information on gait phenotypes in the NDD experimental models. This is in part due to the lack of understanding of the normal developmental trajectory of gait maturation in the mouse. Using the DigiGait system, we have developed a quantitative, standardized, and reproducible assay of developmental gait metrics in commonly used mouse strains that can be added to the battery of mouse model phenotyping. With this assay, we characterized the trajectory of gait in the developing C57BL/6J and FVB/AntJ mouse lines. In both lines, maturation of mouse gait between P21 and P30 was best reflected by aspects of the stance phase of the stride. Additionally, in C57BL/6J mice, gait maturation was observed through how the paw is loaded and unloaded during the stance phase of a stride. In FVB/AntJ mice, gait maturity during this developmental window was best observed through the decreased variability in paw area on the belt during stance. Our results also underscore the importance of considering body length when interpreting gait metrics. Further, our results show that background strain needs to be considered when comparing gait across models. Overall, our results show that aspects of mouse gait development parallel a timeline of normal human gait development, such as the percent of stride that is stance phase and swing phase. This study may be used as a standard reference for developmental gait phenotyping of a variety of murine models, such as models of neurodevelopmental disease.

animal behavior and cognition↗

bioRxiv2017

Maternal SSRI treatment during offspring development results in long-term behavioral, cellular, and neuroimaging disruptions

Serotonergic dysregulation is implicated in psychiatric disorders, including autism spectrum disorders (ASD). Epidemiological studies suggest selective serotonin reuptake inhibitor (SSRI) treatment during pregnancy may increase ASD risk in offspring, however it is unclear from these studies whether ASD susceptibility is related to the maternal diagnosis or if treatment poses additional risk. Here, we exposed mouse dams to fluoxetine and characterized the offspring to isolate possible effects of SSRI exposure on ASD-relevant behaviors. We demonstrate social communication and interaction deficits and repetitive behaviors, with corresponding dendritic morphology changes in pertinent brain regions. Also, using a novel application of optical intrinsic signal imaging, we show altered stimulus-evoked cortical response and region-specific decreases in functional connectivity. These findings indicate drug exposure alone is sufficient to induce long-term behavioral, cellular, and hemodynamic-response disruptions in offspring, thus contributing to our understanding of ASD pathogenesis, risk and mechanism, as well as the developmental role of serotonin.

neuroscience↗