ISL1 is necessary for auditory neuron development and contributes toward tonotopic organization.

A cardinal feature of the auditory pathway is frequency selectivity, represented in a tonotopic map from the cochlea to the cortex. The molecular determinants of the auditory frequency map are unknown. Here, we discovered that the transcription factor ISL1 regulates the molecular and cellular features of auditory neurons, including the formation of the spiral ganglion and peripheral and central processes that shape the tonotopic representation of the auditory map. We selectively knocked out Isl1 in auditory neurons using Neurod1Cre strategies. In the absence of Isl1, spiral ganglion neurons migrate into the central cochlea and beyond, and the cochlear wiring is profoundly reduced and disrupted. The central axons of Isl1 mutants lose their topographic projections and segregation at the cochlear nucleus. Transcriptome analysis of spiral ganglion neurons shows that Isl1 regulates neurogenesis, axonogenesis, migration, neurotransmission-related machinery, and synaptic communication patterns. We show that peripheral disorganization in the cochlea affects the physiological properties of hearing in the midbrain and auditory behavior. Surprisingly, auditory processing features are preserved despite the significant hearing impairment, revealing central auditory pathway resilience and plasticity in Isl1 mutant mice. Mutant mice have a reduced acoustic startle reflex, altered prepulse inhibition, and characteristics of compensatory neural hyperactivity centrally. Our findings show that ISL1 is one of the obligatory factors required to sculpt auditory structural and functional tonotopic maps. Still, upon Isl1 deletion, the ensuing central plasticity of the auditory pathway does not suffice to overcome developmentally induced peripheral dysfunction of the cochlea.

Glioblastoma and cerebral organoids: development and analysis of an in vitro model for glioblastoma migration.

It is currently challenging to adequately model the growth and migration of glioblastoma using two-dimensional (2D) in vitro culture systems as they quickly lose the original, patient-specific identity and heterogeneity. However, with the advent of three-dimensional (3D) cell cultures and human-induced pluripotent stem cell (iPSC)-derived cerebral organoids (COs), studies demonstrate that the glioblastoma-CO (GLICO) coculture model helps to preserve the phenotype of the patient-specific tissue. Here, we aimed to set up such a model using mature COs and develop a pipeline for subsequent analysis of cocultured glioblastoma. Our data demonstrate that the growth and migration of the glioblastoma cell line within the mature COs are significantly increased in the presence of extracellular matrix proteins, shortening the time needed for glioblastoma to initiate migration. We also describe in detail the method for the visualization and quantification of these migrating cells within the GLICO model. Lastly, we show that this coculture model (and the human brain-like microenvironment) can significantly transform the gene expression profile of the established U87 glioblastoma cell line into proneural and classical glioblastoma cell types.

Human iPSC-Derived Neural Models for Studying Alzheimer's Disease: from Neural Stem Cells to Cerebral Organoids.

During the past two decades, induced pluripotent stem cells (iPSCs) have been widely used to study mechanisms of human neural development, disease modeling, and drug discovery in vitro. Especially in the field of Alzheimer's disease (AD), where this treatment is lacking, tremendous effort has been put into the investigation of molecular mechanisms behind this disease using induced pluripotent stem cell-based models. Numerous of these studies have found either novel regulatory mechanisms that could be exploited to develop relevant drugs for AD treatment or have already tested small molecules on in vitro cultures, directly demonstrating their effect on amelioration of AD-associated pathology. This review thus summarizes currently used differentiation strategies of induced pluripotent stem cells towards neuronal and glial cell types and cerebral organoids and their utilization in modeling AD and potential drug discovery.

Generation of three human iPSC lines from patients with a spontaneous late-onset Alzheimer's disease and three sex- and age-matched healthy controls.

Human induced pluripotent stem cell (iPSC) lines were generated from patients with spontaneous late-onset Alzheimer's disease (AD) and three healthy control individuals. Peripheral blood mononuclear cells were reprogrammed with Yamanaka factors (OSKM) using a commercially available Epi5 Reprogramming Kit. The pluripotency of iPSCs was confirmed by the expression of pluripotency factors and by their ability to differentiate to all three germ layers in vitro. Newly derived cell lines can be used to model Alzheimer's disease in vitro.

Generation of six human iPSC lines from patients with a familial Alzheimer's disease (n = 3) and sex- and age-matched healthy controls (n = 3).

Human induced pluripotent stem cell (iPSC) lines were generated from primary human fibroblasts isolated from three patients with a familial form of Alzheimer's disease (AD) and three healthy control individuals. Two AD-iPSC lines carry a PSEN1 mutation A246E; the third cell line carries a PSEN2 mutation N141I. The fibroblasts were reprogrammed with Yamanaka factors (OSKM) using a commercially available Epi5 Reprogramming Kit. The pluripotency of iPSCs was confirmed by the expression of pluripotency factors and by their ability to differentiate to all three germ layers in vitro. Newly derived cell lines can be used to model Alzheimer's disease in vitro.

Combined Atoh1 and Neurod1 Deletion Reveals Autonomous Growth of Auditory Nerve Fibers.

Ear development requires the transcription factors ATOH1 for hair cell differentiation and NEUROD1 for sensory neuron development. In addition, NEUROD1 negatively regulates Atoh1 gene expression. As we previously showed that deletion of the Neurod1 gene in the cochlea results in axon guidance defects and excessive peripheral innervation of the sensory epithelium, we hypothesized that some of the innervation defects may be a result of abnormalities in NEUROD1 and ATOH1 interactions. To characterize the interdependency of ATOH1 and NEUROD1 in inner ear development, we generated a new Atoh1/Neurod1 double null conditional deletion mutant. Through careful comparison of the effects of single Atoh1 or Neurod1 gene deletion with combined double Atoh1 and Neurod1 deletion, we demonstrate that NEUROD1-ATOH1 interactions are not important for the Neurod1 null innervation phenotype. We report that neurons lacking Neurod1 can innervate the flat epithelium without any sensory hair cells or supporting cells left after Atoh1 deletion, indicating that neurons with Neurod1 deletion do not require the presence of hair cells for axon growth. Moreover, transcriptome analysis identified genes encoding axon guidance and neurite growth molecules that are dysregulated in the Neurod1 deletion mutant. Taken together, we demonstrate that much of the projections of NEUROD1-deprived inner ear sensory neurons are regulated cell-autonomously.