Biology subjects

Walker, J. C.

Publications and source records attributed to Walker, J. C..

11 recordsLinked to original sources

bioRxiv2025

Neuroprotective Parkinson's Disease Therapeutic: Transition Metal Dichalcogenide Nanoflower Treatments Alleviate Pathological Cell Stress

Parkinsons disease (PD) is triggered by irreversible degeneration of dopaminergic neurons in the midbrain, hypothalamus, and thalamus. Although the underlying molecular etiology of these pathological processes remains unclear, progressive aggregation of alpha-synuclein (-syn) and mitochondrial dysfunction are two expected mechanisms implicated in neuronal degeneration. Accumulating evidence indicates that transition metal dichalcogenide (TMD) nanoflowers (NFs), a novel class of nanomaterials, can restore mitochondrial health by the activation of mitochondrial biogenesis. However, therapeutic potential of TMD NFs in PD remains unclear. The current study investigates the neuroprotective properties of molybdenum disulfide (MoS2) and molybdenum diselenide (MoSe2) nanoflowers (NFs) in neurons and astrocytes exposed to -syn aggregates. It was found that MoS2 and MoSe2 suppressed -syn-induced unfolded protein response (UPR) in the endoplasmic reticulum, and upregulated autophagy and exocytosis of -syn fibrils. TMD NFs also reversed -syn-induced damage of cell mitochondria, simultaneously stimulating mitochondrial biogenesis. As a result, a drastic decrease in ROS levels in both neurons and astrocytes was observed. These results show that MoS2 or MoSe2 NFs could fully rescue neurons and astrocytes from the cytotoxic effects of -syn fibrils. Neuroprotective properties of these novel nanomaterials were further explored in Caenorhabditis elegans that overexpress -syn. Nematodes that received NFs experienced a drastic reduction in the amount of aggregated -syn which resulted in a significant increase in C. elegans lifespan. These findings indicated that MoS2 or MoSe2 NFs could be used as novel therapeutic to decelerate the progression of PD.

neuroscience↗

bioRxiv2025

Delayed forebrain excitatory and inhibitory neurogenesis inSTRADA-related megalencephaly via mTOR hyperactivity

Biallelic pathogenic variants in STRADA, an upstream regulator of the mechanistic target of rapamycin (mTOR) pathway, result in megalencephaly, drug-resistant epilepsy, and severe intellectual disability. This study explores how mTOR pathway hyperactivity alters cell fate specification in dorsal and ventral forebrain development using STRADA knock-out human stem cell derived brain organoids. In both dorsal and ventral forebrain STRADA knock-out organoids, neurogenesis is delayed, with a predilection for progenitor renewal and proliferation and an increase in outer radial glia. Ventrally, interneuron subtypes shift to an increase in neuropeptide-Y expressing cells. Inhibition of the mTOR pathway with rapamycin results in rescue for most phenotypes. When mTOR pathway variants are present in all cells of the developing brain, overproduction of interneurons and altered interneuron cell fate may underlie mechanisms of megalencephaly, epilepsy, and cognitive impairment. Our findings suggest mTOR inhibition during fetal brain development as a potential therapeutic strategy in STRADA deficiency.

neuroscience↗

bioRxiv2023

Genome-wide CRISPRi Screen in Human iNeurons to Identify Novel Focal Cortical Dysplasia Genes

Focal cortical dysplasia (FCD) is a common cause of focal epilepsy that typically results from brain mosaic mutations in the mTOR cell signaling pathway. To identify new FCD genes, we developed an in vitro CRISPRi screen in human neurons and used FACS enrichment based on the FCD biomarker, phosphorylated S6 ribosomal protein (pS6). Using whole-genome (110,000 gRNAs) and candidate (129 gRNAs) libraries, we discovered 12 new genes that significantly increase pS6 levels. Interestingly, positive hits were enriched for brain-specific genes, highlighting the effectiveness of using human iPSC-derived induced neurons (iNeurons) in our screen. We investigated the signaling pathways of six candidate genes: LRRC4, EIF3A, TSN, HIP1, PIK3R3, and URI1. All six genes increased phosphorylation of S6. However, only two genes, PIK3R3 and HIP1, caused hyperphosphorylation more proximally in the AKT/mTOR/S6 signaling pathway. Importantly, these two genes have recently been found independently to be mutated in resected brain tissue from FCD patients, supporting the predictive validity of our screen. Knocking down each of the other four genes (LRRC4, EIF3A, TSN, and URI1) in iNeurons caused them to become resistant to the loss of growth factor signaling; without growth factor stimulation, pS6 levels were comparable to growth factor stimulated controls. Our data markedly expand the set of genes that are likely to regulate mTOR pathway signaling in neurons and provide additional targets for identifying somatic gene variants that cause FCD. Significance StatementFocal cortical dysplasia (FCD) is a common cause of focal epilepsy due to somatic variants in mTOR pathway genes in FCD brain tissue. Unbiased sequencing to identify novel FCD genes is challenging since these variants are often in a small subset of neurons. To overcome this challenge, we used an in vitro genetic screen in human neurons using an FCD biomarker, uncovering genes that increase neuronal mTOR signaling when their expression is lost. Two candidate genes were mutated in patient FCD brain tissues in a recent study, supporting a causative role for these genes in FCD. Our work suggests gene candidates for somatic variant analysis in FCD tissue and indicates added value for genetic screening in neurons over cell lines.

neuroscience↗

bioRxiv2023

A shared pathogenic mechanism for valproic acid and SHROOM3 knockout in a brain organoid model of neural tube defects

Neural tube defects (NTDs) including anencephaly and spina bifida are common major malformations of fetal development resulting from incomplete closure of the neural tube. These conditions lead to either universal death (anencephaly) or life-long severe complications (spina bifida). Despite hundreds of genetic mouse models having neural tube defect phenotypes, the genetics of human NTDs are poorly understood. Furthermore, pharmaceuticals such as antiseizure medications have been found clinically to increase the risk of NTDs when administered during pregnancy. Therefore, a model that recapitulates human neurodevelopment would be of immense benefit to understand the genetics underlying NTDs and identify teratogenic mechanisms. Using our self-organizing single rosette spheroid (SOSRS) brain organoid system, we have developed a high-throughput image analysis pipeline for evaluating SOSRS structure for NTD-like phenotypes. Similar to small molecule inhibition of apical constriction, the antiseizure medication valproic acid (VPA), a known cause of NTDs, increases the apical lumen size and apical cell surface area in a dose-responsive manner. This expansion was mimicked by GSK3{beta} and HDAC inhibitors; however, RNA sequencing suggests VPA does not inhibit GSK3{beta} at these concentrations. Knockout of SHROOM3, a well-known NTD-related gene, also caused expansion of the lumen as well as reduced f-actin polarization. The increased lumen sizes were caused by reduced cell apical constriction suggesting that impingement of this process is a shared mechanism for VPA treatment and SHROOM3-KO, two well-known causes of NTDs. Our system allows the rapid identification of NTD-like phenotypes for both compounds and genetic variants and should prove useful for understanding specific NTD mechanisms and predicting drug teratogenicity.

neuroscience↗

bioRxiv2022

Single-cell transcriptomics of the Arabidopsis floral abscission zone

Abscission is the programmed separation of plant organs. It is widespread in the plant kingdom with important functions in development and environmental response. In Arabidopsis, abscission of floral organs (sepals, petals, and stamens) is controlled by two receptor-like protein kinases HAESA (HAE) and HAESA LIKE-2 (HSL2), which orchestrate the programmed dissolution of the abscission zone connecting floral organs to the developing fruit. In this work, we use single-cell RNA-Sequencing to characterize the core HAE/HSL2 abscission gene expression program. We identify the MAP KINASE PHOSPHATASE-1/MKP1 gene as a negative regulator of this pathway. MKP1 acts prior to activation of HAE/HSL2 signaling to establish a signaling threshold required for the initiation of abscission. By analogy to electrical circuit control, we liken MKP1 to a molecular grounding mechanism that dissipates errant pathway activation absent HAE/HSL2 signaling. Furthermore, we use single-cell data to identify genes expressed in two sub-populations of abscission zone cells: those proximal and those distal to the plane of separation. We identify INFLORESCENCE DEFICIENT IN ABSCISSION/IDA, encoding the activating ligand of HAE/HSL2, as one of the mRNAs most highly enriched in distal abscission zone cells at the base of the abscising organs. We show how this expression pattern forms a biophysical circuit breaker whereby, when the organ is shed, the source of the IDA peptide is removed, leading to cessation of HAE/HSL2 signaling. Overall, this work provides insight into the multiple control mechanisms acting on the abscission-signaling pathway.

plant biology↗

bioRxiv2022

Self-organizing Single-Rosette Brain Organoids from Human Pluripotent Stem Cells

The field of brain organoid research is complicated by morphological variability with multiple neural rosette structures per organoid. We have developed a new human brain organoid technique that generates self-organizing, single-rosette spheroids (SOSRS) with reproducible size, cortical-like lamination, and cell diversity. Rather than patterning a 3-dimensional embryoid body, we initiate brain organoid formation from a 2-dimensional monolayer of human pluripotent stem cells (hPSCs) that is patterned with small molecules into neuroepithelium and differentiated to cells of the developing dorsal cerebral cortex. This approach recapitulates the 2D to 3D transition from neural plate to neural tube that occurs during neurodevelopment. The vast majority of monolayer fragments form spheres with a single central lumen and consistent growth rates. Over time, the SOSRS develop appropriately ordered lamination consistent with six cortical layers by immunocytochemistry and single cell RNA-sequencing. The reproducibility of this method has allowed us to demonstrate robust structural phenotypes arising from chemical teratogen exposure or when modeling a genetic neurodevelopmental epileptic disorder. This platform should advance studies of human cortical development, brain disorder mechanisms, and precision therapies. SUMMARY STATEMENTSimple procedure for generating reproducible single rosette cortical brain organoids used to identify robust structural phenotypes with neuroteratogen exposure and in a genetic neurodevelopmental disease model.

neuroscience↗

bioRxiv2020

Default Mode and Salience Network Alterations in Suicidal and Non-Suicidal Self-Injurious Thoughts and Behaviors in Adolescents with Depression

Suicidal ideation (SI) and non-suicidal self-injury (NSSI) are two distinct yet often co-occurring risk factors for suicide in adolescents. Elucidating the neurobiological patterns that specifically characterize SI and NSSI in adolescents is needed to inform the use of these markers in intervention studies and to develop brain-based treatment targets. Here, we clinically assessed 70 adolescents--49 adolescents with depression and 21 healthy controls--to determine SI and NSSI history. Twenty-eight of the depressed adolescents had a history of SI and 29 had a history of NSSI (20 overlapping). All participants underwent a resting-state fMRI scan. We compared groups in network coherence of subdivisions of the central executive network (CEN), default mode network (DMN), and salience network (SN). We also examined group differences in between-network connectivity and explored brain-behavior correlations. Depressed adolescents with SI and with NSSI had lower coherence in the ventral DMN compared to those without SI or NSSI, respectively, and healthy controls (all ps<0.043). Depressed adolescents with NSSI had lower coherence in the anterior DMN and in insula-SN (all ps<0.030), and higher CEN-DMN connectivity compared to those without NSSI and healthy controls (all ps<0.030). Lower network coherence in all DMN subnetworks and insula-SN were associated with higher SI and NSSI (all ps<0.001). Thus, SI and NSSI are related to brain networks associated with difficulties in self-referential processing and future planning, while NSSI specifically is related to brain networks associated with disruptions in interoceptive awareness. Intrinsic network patterns may be reliable biomarkers of SI and NSSI in adolescents.

neuroscience↗

bioRxiv2020

Variant-specific changes in persistent or resurgent Na+ current in SCN8A-EIEE13 iPSC-derived neurons

Missense variants in the voltage-gated sodium channel (VGSC) gene, SCN8A, are linked to early-infantile epileptic encephalopathy type 13 (EIEE13). EIEE13 patients exhibit a wide spectrum of intractable seizure types, severe developmental delay, movement disorders, and elevated risk of sudden unexpected death in epilepsy (SUDEP). The mechanisms by which SCN8A variants lead to epilepsy are poorly understood, although heterologous expression systems and mouse models have demonstrated altered sodium current (INa) properties. To investigate these mechanisms using a patient-specific model system, we generated induced pluripotent stem cells (iPSCs) from three patients with missense variants in SCN8A: p.R1872>L (P1); p.V1592>L (P2); and p.N1759>S (P3). Using small molecule differentiation into excitatory neurons, iPSC-derived neurons from all three patients displayed altered INa. P1 and P2 had elevated persistent INa, while P3 had increased resurgent INa compared to controls. Further analyses focused on one of the patients with increased persistent INa (P1) and the patient with increased resurgent INa (P3). Excitatory cortical neurons from both patients had prolonged action potential (AP) repolarization and shorter axon initial segment lengths compared to controls, the latter analyzed by immunostaining for ankyrin-G. Using doxycycline-inducible expression of the neuronal transcription factors Neurogenin 1 and 2 to synchronize differentiation of induced excitatory cortical-like neurons (iNeurons), we investigated network activity and response to pharmacotherapies. Both patient neurons and iNeurons displayed similar abnormalities in AP repolarization. Patient iNeurons showed increased burstiness that was sensitive to phenytoin, currently a standard treatment for EIEE patients, or riluzole, an FDA-approved drug used in amyotrophic lateral sclerosis and known to block persistent and resurgent INa, at pharmacologically relevant concentrations. Patch-clamp recordings showed that riluzole suppressed spontaneous firing and increased the AP firing threshold of patient-derived neurons to more depolarized potentials. Our results indicate that patient-specific neurons are useful for modeling EIEE13 and demonstrate SCN8A variant-specific mechanisms. Moreover, these findings suggest that patient-specific iPSC neuronal disease modeling offers a useful platform for discovering precision epilepsy therapies.

neuroscience↗

bioRxiv2018

Hypermorphic SERK1 mutations function via a SOBIR1 pathway to activate floral abscission signaling

In Arabidopsis, the abscission of floral organs is regulated by two related receptor-like protein kinases (RLKs), HAESA and HAESA-like 2 (HAE/HSL2). HAE/HSL2, in complex with members of the SERK family of coreceptor protein kinases, are activated by the binding of the proteolytically processed peptide ligand IDA. This leads to expression of genes encoding secreted cell wall remodeling and hydrolase enzymes. hae hsl2 mutants fail to induce expression of these genes and retain floral organs indefinitely. In this paper we report identification of an allelic series of hae hsl2 suppressor mutations in the SERK1 coreceptor protein kinase gene. Genetic and transcriptomic evidence indicates these alleles represent a novel class of gain of function mutations that activate signaling independent of HAE/HSL2. We show that the suppression effect surprisingly does not rely on protein kinase activity of SERK1, and that activation of signaling relies on the RLK gene SOBIR1. The effect of these mutations can be mimicked by loss of function of BIR1, a known negative regulator of SERK-SOBIR1 signaling. These results suggest BIR1 functions to negatively regulate SERK-SOBIR1 signaling during abscission, and that the identified SERK1 mutations likely interfere with this negative regulation.

plant biology↗

bioRxiv2017

Leaf shedding as a bacterial defense in Arabidopsis cauline leaves

Plants utilize an innate immune system to protect themselves from disease. While many molecular components of plant innate immunity resemble the innate immunity of animals, plants also have evolved a number of truly unique defense mechanisms, particularly at the physiological level. Plants flexible developmental program allows them the unique ability to simply produce new organs as needed, affording them the ability to replace damaged organs. Here we develop a system to study pathogen-triggered leaf abscission in Arabidopsis. Cauline leaves infected with the bacterial pathogen Pseudomonas syringae abscise as part of the defense mechanism. Pseudomonas syringae lacking a functional type III secretion system fail to elicit an abscission response, suggesting that the abscission response is a novel form of immunity triggered by effectors. HAESA/HAESA-like 2, INFLORESCENCE DEFICIENT IN ABSCISSION, and NEVERSHED are all required for pathogen-triggered abscission to occur. Additionally phytoalexin deficient 4, enhanced disease susceptibility 1, salicylic acid induction deficient 2, and senescence-associated gene 101 plants with mutations in genes necessary for bacterial defense and salicylic acid signaling, and NahG transgenic plants with low levels of salicylic acid fail to abscise cauline leaves normally. Bacteria that physically contact abscission zones trigger a strong abscission response; however, long distance signals are also sent from distal infected tissue to the abscission zone, alerting the abscission zone of looming danger. We propose a threshold model regulating cauline leaf defense where minor infections are handled by limiting bacterial growth, but when an infection is deemed out of control, cauline leaves are shed. Together with previous results our findings suggest that salicylic acid may regulate both pathogen- and drought-triggered leaf abscission.\n\nAuthor SummaryPlants have a flexible development program that determine their form. We describe an organ level defense response in Arabidopsis to bacterial attack where plants simply shed heavily infected leaves. The genetics regulating this defense mechanism are comprised of both classical defense genes and floral organ abscission genes working together. Long distance signals are transmitted from infected areas to abscission zones which activate the abscission receptor. Salicylic acid, a defense hormone, signaling is necessary for cauline leaf abscission.

genetics↗

bioRxiv2017

Advances in abscission signaling

Abscission is a process in plants for shedding unwanted organs such as leaves, flowers, fruits, or floral organs. Shedding of leaves in the fall is the most visually obvious display of abscission in nature. The very shape plants take is forged by the processes of growth and abscission. Mankind manipulates abscission in modern agriculture to do things like prevent pre-harvest fruit drop prior to mechanical harvesting in fruit orchards. Abscission occurs specifically at abscission zones that are laid down as the organ that will one day abscise is developed. A sophisticated signaling network initiates abscission when it is time to shed the unwanted organ. In this article, we review recent advances in understanding the signaling mechanisms that activate abscission. Physiological advances and roles for hormones in abscission are also addressed. Finally, we discuss current avenues for basic abscission research and potentially lucrative future directions for its application to modern agriculture.

plant biology↗