Astrocyte lineage cells are essential for functional neuronal differentiation and synapse maturation in human iPSC-derived neural networks.

Human astrocytes differ dramatically in cell morphology and gene expression from murine astrocytes. The latter are well known to be of major importance in the formation of neuronal networks by promoting synapse maturation. However, whether human astrocyte lineage cells have a similar role in network formation has not been firmly established. Here, we investigated the impact of human astrocyte lineage cells on the functional maturation of neural networks that were derived from human induced pluripotent stem cells (hiPSCs). Initial in vitro differentiation of hiPSC-derived neural progenitor cells and immature neurons (glia+ cultures) resulted in spontaneously active neural networks as indicated by synchronous neuronal Ca2+ transients. Depleting proliferating neural progenitors from these cultures by short-term antimitotic treatment resulted in strongly astrocyte lineage cell-depleted neuronal networks (glia- cultures). Strikingly, in contrast to glia+ cultures, glia- cultures did not exhibit spontaneous network activity. Detailed analysis of the morphological and electrophysiological properties of neurons by patch clamp recordings revealed reduced dendritic arborization in glia- cultures. In addition, a reduced action potential frequency upon current injection in pyramidal-like neurons was observed, whereas the electrical excitability of multipolar neurons was unaltered. Furthermore, we found a reduced dendritic density of PSD95-positive excitatory synapses, and more immature properties of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) miniature excitatory postsynaptic currents (mEPSCs) in glia- cultures, suggesting that the maturation of glutamatergic synapses depends on the presence of hiPSC-derived astrocyte lineage cells. Intriguingly, addition of the astrocyte-derived synapse maturation inducer cholesterol increased the dendritic density of PSD95-positive excitatory synapses in glia- cultures.

C-terminal fragment of N-cadherin accelerates synapse destabilization by amyloid-ß.

The aetiology of Alzheimer's disease is thought to include functional impairment of synapses and synapse loss as crucial pathological events leading to cognitive dysfunction and memory loss. Oligomeric amyloid-ß peptides are well known to induce functional damage, destabilization and loss of brain synapses. However, the complex molecular mechanisms of amyloid-ß action resulting ultimately in synapse elimination are incompletely understood, thus limiting knowledge of potential therapeutic targets. Under physiological conditions, long-term synapse stability is mediated by trans-synaptically interacting adhesion molecules such as the homophilically binding N-cadherin/catenin complexes. In this study, we addressed whether inhibition of N-cadherin function affects amyloid-ß-induced synapse impairment. We found that blocking N-cadherin function, both by specific peptides interfering with homophilic binding and by expression of a dominant-negative, ectodomain-deleted N-cadherin mutant, resulted in a strong acceleration of the effect of amyloid-ß on synapse function in cultured cortical neurons. The frequency of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor-mediated miniature excitatory postsynaptic currents was reduced upon amyloid-ß application much earlier than observed in controls. We further hypothesized that ectodomain-shed, transmembrane C-terminal fragments that are generated during N-cadherin proteolytic processing might similarly enhance amyloid-ß-induced synapse damage. Indeed, expression of human N-cadherin C-terminal fragment 1 strongly accelerated amyloid-ß-triggered synapse impairment. Ectodomain-shed N-cadherin C-terminal fragment 1 is further proteolytically cleaved by γ-secretase. Therefore, both pharmacological inhibition of γ-secretase and expression of the dominant-negative presenilin 1 mutant L166P were used to increase the presence of endogeneous N-cadherin C-terminal fragment 1. Under these conditions, we again found a strong acceleration of amyloid-ß-induced synapse impairment, which could be compensated by over-expression of full-length N-cadherin. Intriguingly, western blot analysis of post-mortem brains from patients with Alzheimer's disease revealed an enhanced presence of N-cadherin C-terminal fragment 1. Thus, an inhibition of N-cadherin function by proteolytically generated N-cadherin C-terminal fragment 1 might play an important role in Alzheimer's disease progression by accelerating amyloid-ß-triggered synapse damage.

Induced Neurons for the Study of Neurodegenerative and Neurodevelopmental Disorders.

Patient-derived or genomically modified human induced pluripotent stem cells (iPSCs) offer the opportunity to study neurodevelopmental and neurodegenerative disorders. Overexpression of certain neurogenic transcription factors (TFs) in iPSCs can induce efficient differentiation into homogeneous populations of the disease-relevant neuronal cell types. Here we provide protocols for genomic manipulations of iPSCs by CRISPR/Cas9. We also introduce two methods, based on lentiviral delivery and the piggyBac transposon system, to stably integrate neurogenic TFs into human iPSCs. Furthermore, we describe the TF-mediated neuronal differentiation and maturation in combination with astrocyte cocultures.

Combined Experimental and System-Level Analyses Reveal the Complex Regulatory Network of miR-124 during Human Neurogenesis.

Non-coding RNAs regulate many biological processes including neurogenesis. The brain-enriched miR-124 has been assigned as a key player of neuronal differentiation via its complex but little understood regulation of thousands of annotated targets. To systematically chart its regulatory functions, we used CRISPR/Cas9 gene editing to disrupt all six miR-124 alleles in human induced pluripotent stem cells. Upon neuronal induction, miR-124-deleted cells underwent neurogenesis and became functional neurons, albeit with altered morphology and neurotransmitter specification. Using RNA-induced-silencing-complex precipitation, we identified 98 high-confidence miR-124 targets, of which some directly led to decreased viability. By performing advanced transcription-factor-network analysis, we identified indirect miR-124 effects on apoptosis, neuronal subtype differentiation, and the regulation of previously uncharacterized zinc finger transcription factors. Our data emphasize the need for combined experimental- and system-level analyses to comprehensively disentangle and reveal miRNA functions, including their involvement in the neurogenesis of diverse neuronal cell types found in the human brain.

On-demand optogenetic activation of human stem-cell-derived neurons.

The widespread application of human stem-cell-derived neurons for functional studies is impeded by complicated differentiation protocols, immaturity, and deficient optogene expression as stem cells frequently lose transgene expression over time. Here we report a simple but precise Cre-loxP-based strategy for generating conditional, and thereby stable, optogenetic human stem-cell lines. These cells can be easily and efficiently differentiated into functional neurons, and optogene expression can be triggered by administering Cre protein to the cultures. This conditional expression system may be applied to stem-cell-derived neurons whenever timed transgene expression could help to overcome silencing at the stem-cell level.

Vision Restoration Becomes Druggable.

In this issue of Neuron, Tochitsky et al. (2016) have identified the mechanism by which small-molecule photoswitches enter and specifically activate retinal OFF-ganglion cells in degenerated retinas. This drug development is a tremendous step toward the treatment of blindness, regardless of the underlying mutation.

Biophysical Properties of Optogenetic Tools and Their Application for Vision Restoration Approaches.

Optogenetics is the use of genetically encoded light-activated proteins to manipulate cells in a minimally invasive way using light. The most prominent example is channelrhodopsin-2 (ChR2), which allows the activation of electrically excitable cells via light-dependent depolarization. The combination of ChR2 with hyperpolarizing-light-driven ion pumps such as the Cl(-) pump halorhodopsin (NpHR) enables multimodal remote control of neuronal cells in culture, tissue, and living animals. Very soon, it became obvious that this method offers a chance of gene therapy for many diseases affecting vision. Here, we will give a brief introduction to retinal function and retinal diseases; optogenetic vision restoration strategies will be highlighted. We will discuss the functional and structural properties of rhodopsin-based optogenetic tools and analyze the potential for the application of vision restoration.