Modern In Vitro Techniques for Modeling Hearing Loss.

Sensorineural hearing loss (SNHL) is a prevalent and growing global health concern, especially within operational medicine, with limited therapeutic options available. This review article explores the emerging field of in vitro otic organoids as a promising platform for modeling hearing loss and developing novel therapeutic strategies. SNHL primarily results from the irreversible loss or dysfunction of cochlear mechanosensory hair cells (HCs) and spiral ganglion neurons (SGNs), emphasizing the need for innovative solutions. Current interventions offer symptomatic relief but do not address the root causes. Otic organoids, three-dimensional multicellular constructs that mimic the inner ear's architecture, have shown immense potential in several critical areas. They enable the testing of gene therapies, drug discovery for sensory cell regeneration, and the study of inner ear development and pathology. Unlike traditional animal models, otic organoids closely replicate human inner ear pathophysiology, making them invaluable for translational research. This review discusses methodological advances in otic organoid generation, emphasizing the use of human pluripotent stem cells (hPSCs) to replicate inner ear development. Cellular and molecular characterization efforts have identified key markers and pathways essential for otic organoid development, shedding light on their potential in modeling inner ear disorders. Technological innovations, such as 3D bioprinting and microfluidics, have further enhanced the fidelity of these models. Despite challenges and limitations, including the need for standardized protocols and ethical considerations, otic organoids offer a transformative approach to understanding and treating auditory dysfunctions. As this field matures, it holds the potential to revolutionize the treatment landscape for hearing and balance disorders, moving us closer to personalized medicine for inner ear conditions.

Dual effects of ARX poly-alanine mutations in human cortical and interneuron development.

Mutations in ARX , an X-linked gene, are implicated in a wide spectrum of neurological disorders including patients who have intellectual disability and epilepsy. Mouse models have shown that Arx is critical for cortical development and interneuron migration, however they do not recapitulate the full phenotype observed in patients. Moreover, the epilepsy in many patients with poly-alanine tract expansion (PAE) mutations in ARX show pharmacoresistance, emphasizing the need to develop new treatments. Here, we used human neural organoid models to study the consequences of PAE mutations, one of the most prevalent mutations in ARX . We found that PAE mutations result in an early increase in radial glia cells and intermediate progenitor cells, and premature differentiation leading to a loss of cortical neurons at later timepoints. Moreover, ARX expression is upregulated in CO derived from patient at 30 DIV which alters the expression of CDKN1C , SFRP1 , DLK1 and FABP7 , among others. We also found a cell autonomously enhanced interneuron migration, which can be rescued by CXCR4 inhibition. Furthermore, ARX PAE assembloids had hyper-activity and synchrony evident from the early stages. These data provide novel insights to the pathogenesis of these and likely related human neurological disorders and identifies a critical window for therapeutic interventions.

Understanding Primary Blast Injury: High Frequency Pressure Acutely Disrupts Neuronal Network Dynamics in Cerebral Organoids.

Blast exposure represents a common occupational risk capable of generating mild to severe traumatic brain injuries (TBI). During blast exposure, a pressure shockwave passes through the skull and exposes brain tissue to complex pressure waveforms. The primary neurophysiological response to blast-induced pressure waveforms remains poorly understood. Here, we use a computer-controlled table-top pressure chamber to expose human stem cell-derived cerebral organoids to varied frequency of pressure waves and characterize the neurophysiological response. Pressure waves that reach a maximum amplitude of 250 kPa were used to model a less severe TBI and 350 kPa for a more severe blast TBI event. With each amplitude, a frequency range of 500 Hz, 3000 Hz, and 5000 Hz was tested. Following the 250 kPa overpressure a multi-electrode array recorded organoid neural activity. We observed an acute suppression neuronal activity in single unit events, population events, and network oscillations that recovered within 24 h. Additionally, we observed a network desynchronization after exposure higher frequency waveforms. Conversely, organoids exposed to higher amplitude pressure (350k Pa) displayed drastic neurophysiological differences that failed to recover within 24 h. Further, lower amplitude "blast" (250 kPa) did not induce cellular damage whereas the higher amplitude "blast" (350 kPa) generated greater apoptosis throughout each organoid. Our data indicate that specific features of pressure waves found intracranially during blast TBI have varied effects on neurophysiological activity that can occur even without cellular damage.

A critical period of neuronal activity results in aberrant neurogenesis rewiring hippocampal circuitry in a mouse model of epilepsy.

In the mammalian hippocampus, adult-born granule cells (abGCs) contribute to the function of the dentate gyrus (DG). Disruption of the DG circuitry causes spontaneous recurrent seizures (SRS), which can lead to epilepsy. Although abGCs contribute to local inhibitory feedback circuitry, whether they are involved in epileptogenesis remains elusive. Here, we identify a critical window of activity associated with the aberrant maturation of abGCs characterized by abnormal dendrite morphology, ectopic migration, and SRS. Importantly, in a mouse model of temporal lobe epilepsy, silencing aberrant abGCs during this critical period reduces abnormal dendrite morphology, cell migration, and SRS. Using mono-synaptic tracers, we show silencing aberrant abGCs decreases recurrent CA3 back-projections and restores proper cortical connections to the hippocampus. Furthermore, we show that GABA-mediated amplification of intracellular calcium regulates the early critical period of activity. Our results demonstrate that aberrant neurogenesis rewires hippocampal circuitry aggravating epilepsy in mice.

Novel Targets of SARS-CoV-2 Spike Protein in Human Fetal Brain Development Suggest Early Pregnancy Vulnerability.

Pregnant women are at greater risk of infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), because of their altered immunity and strained cardiovascular system. Emerging studies of placenta, embryos, and cerebral organoids suggest that fetal organs including brain could also be vulnerable to coronavirus disease 2019 (COVID-19). Additionally, a case study from Paris has reported transient neurological complications in neonates born to pregnant mothers. However, it remains poorly understood whether the fetal brain expresses cellular components that interact with Spike protein (S) of coronaviruses, which facilitates fusion of virus and host cell membrane and is the primary protein in viral entry. To address this question, we analyzed the expression of known (ACE2, TMPRSS2, and FURIN) and novel (ZDHHC5, GOLGA7, and ATP1A1) S protein interactors in publicly available fetal brain bulk and single cell RNA sequencing datasets. Bulk RNA sequencing analysis across multiple regions of fetal brain spanning 8 weeks post conception (wpc)-37wpc indicates that two of the known S protein interactors are expressed at low levels with median normalized gene expression values ranging from 0.08 to 0.06 (ACE2) and 0.01-0.02 (TMPRSS2). However, the third known S protein interactor FURIN is highly expressed (11.1-44.09) in fetal brain. Interestingly, all three novel S protein interactors are abundantly expressed throughout fetal brain development with median normalized gene expression values ranging from 20.38-21.60 (ZDHHC5), 92.47-68.35 (GOLGA7), and 65.45-194.5 (ATP1A1). Moreover, the peaks of expression of novel interactors is around 12-26wpc. Using publicly available single cell RNA sequencing datasets, we further show that novel S protein interactors show higher co-expression with neurons than with neural progenitors and astrocytes. These results suggest that even though two of the known S protein interactors are present at low levels in fetal brain, novel S protein interactors are abundantly present and could play a direct or indirect role in SARS-CoV-2 fetal brain pathogenesis, especially during the 2nd and 3rd trimesters of pregnancy.

Stem cells: A path towards improved epilepsy therapies.

Despite the immense growth of new anti-seizure drugs (ASDs), approximately one-third of epilepsy patients remain resistant to current treatment options. Advancements in whole genome sequencing technology continues to identify an increasing number of epilepsy-associated genes at a rate that is outpacing the development of in vivo animal models. Patient-derived induced pluripotent stem cells (iPSCs) show promise in providing a platform for modeling genetic epilepsies, high throughput drug screening, and personalized medicine. This is largely due to the ease of collecting donor cells for iPSC reprogramming, and their ability to be maintained in vitro, while preserving the patient's genetic background. In this review, we summarize the current state of iPSC research in epilepsy and closely related syndromes, discuss the growing need for high-throughput drug screening (HTS), and review the use of stem cell technology for the purpose of autologous transplantation for epilepsy stem cell therapy. Although the use of iPSC technology, as it applies to ASD discovery, is in its infancy, we highlight the significant progress that has been made in phenotype and assay development to facilitate systematic HTS for personalized medicine. This article is part of the special issue entitled 'New Epilepsy Therapies for the 21st Century - From Antiseizure Drugs to Prevention, Modification and Cure of Epilepsy'.

Coupled sensory interneurons mediate escape neural circuit processing in an aquatic annelid worm, Lumbriculus variegatus.

The interneurons associated with rapid escape circuits are adapted for fast pathway activation and rapid conduction. An essential aspect of fast activation is the processing of sensory information with limited delays. Although aquatic annelid worms have some of the fastest escape responses in nature, the sensory networks that mediate their escape behavior are not well defined. Here, we demonstrate that the escape circuit of the mud worm, Lumbriculus variegatus, is a segmentally arranged network of sensory interneurons electrically coupled to the central medial giant fiber (MGF), the command-like interneuron for head withdrawal. Electrical stimulation of the body wall evoked fast, short-duration spikelets in the MGF, which we suggest are the product of intermediate giant fiber activation coupled to MGF collateral dendrites. Since these contact sites have immunoreactivity with a glutamate receptor antibody, and the glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dion abolishes evoked MGF responses, we conclude that the afferent pathway for MGF-mediated escape is glutamatergic. This electrically coupled sensory network may facilitate rapid escape activation by enhancing the amplitude of giant axon depolarization.

Aberrant hippocampal neurogenesis contributes to epilepsy and associated cognitive decline.

Acute seizures after a severe brain insult can often lead to epilepsy and cognitive impairment. Aberrant hippocampal neurogenesis follows the insult but the role of adult-generated neurons in the development of chronic seizures or associated cognitive deficits remains to be determined. Here we show that the ablation of adult neurogenesis before pilocarpine-induced acute seizures in mice leads to a reduction in chronic seizure frequency. We also show that ablation of neurogenesis normalizes epilepsy-associated cognitive deficits. Remarkably, the effect of ablating adult neurogenesis before acute seizures is long lasting as it suppresses chronic seizure frequency for nearly 1 year. These findings establish a key role of neurogenesis in chronic seizure development and associated memory impairment and suggest that targeting aberrant hippocampal neurogenesis may reduce recurrent seizures and restore cognitive function following a pro-epileptic brain insult.

Rapid neural circuit switching mediated by synaptic plasticity during neural morphallactic regeneration.

The aquatic oligochaete, Lumbriculus variegatus (Lumbriculidae), undergoes a rapid regenerative transformation of its neural circuits following body fragmentation. This type of nervous system plasticity, called neural morphallaxis, involves the remodeling of the giant fiber pathways that mediate rapid head and tail withdrawal behaviors. Extra- and intracellular electrophysiological recordings demonstrated that changes in cellular properties and synaptic connections underlie neurobehavioral plasticity during morphallaxis. Sensory-to-giant interneuron connections, undetectable prior to body injury, emerged within hours of segment amputation. The appearance of functional synaptic transmission was followed by interneuron activation, coupling of giant fiber spiking to motor outputs and overt segmental shortening. The onset of morphallactic plasticity varied along the body axis and emerged more rapidly in segments closer to regions of sensory field overlap between the two giant fiber pathways. The medial and lateral giant fibers were simultaneously activated during a transient phase of network remodeling. Thus, synaptic plasticity at sensory-to-giant interneuron connections mediates escape circuit morphallaxis in this regenerating annelid worm.