Atlastin-1 regulates endosomal tubulation and lysosomal proteolysis in human cortical neurons.

Mutation of the ATL1 gene is one of the most common causes of hereditary spastic paraplegia (HSP), a group of genetic neurodegenerative conditions characterised by distal axonal degeneration of the corticospinal tract axons. Atlastin-1, the protein encoded by ATL1, is one of three mammalian atlastins, which are homologous dynamin-like GTPases that control endoplasmic reticulum (ER) morphology by fusing tubules to form the three-way junctions that characterise ER networks. However, it is not clear whether atlastin-1 is required for correct ER morphology in human neurons and if so what the functional consequences of lack of atlastin-1 are. Using CRISPR-inhibition we generated human cortical neurons lacking atlastin-1. We demonstrate that ER morphology was altered in these neurons, with a reduced number of three-way junctions. Neurons lacking atlastin-1 had longer endosomal tubules, suggestive of defective tubule fission. This was accompanied by reduced lysosomal proteolytic capacity. As well as demonstrating that atlastin-1 is required for correct ER morphology in human neurons, our results indicate that lack of a classical ER-shaping protein such as atlastin-1 may cause altered endosomal tubulation and lysosomal proteolytic dysfunction. Furthermore, they strengthen the idea that defective lysosome function contributes to the pathogenesis of a broad group of HSPs, including those where the primary localisation of the protein involved is not at the endolysosomal system.

Co-culture of Human Stem Cell Derived Neurons and Oligodendrocyte Progenitor Cells.

Crosstalk between neurons and oligodendrocytes is important for proper brain functioning. Multiple co-culture methods have been developed to study oligodendrocyte maturation, myelination or the effect of oligodendrocytes on neurons. However, most of these methods contain cells derived from animal models. In the current protocol, we co-culture human neurons with human oligodendrocytes. Neurons and oligodendrocyte precursor cells (OPCs) were differentiated separately from pluripotent stem cells according to previously published protocols. To study neuron-glia cross-talk, neurons and OPCs were plated in co-culture mode in optimized conditions for additional 28 days, and prepared for OPC maturation and neuronal morphology analysis. To our knowledge, this is one of the first neuron-OPC protocols containing all human cells. Specific neuronal abnormalities not observed in mono-cultures of Tuberous Sclerosis Complex (TSC) neurons, became apparent when TSC neurons were co-cultured with TSC OPCs. These results show that this co-culture system can be used to study human neuron-OPC interactive mechanisms involved in health and disease.

Neuron-Glia Interactions Increase Neuronal Phenotypes in Tuberous Sclerosis Complex Patient iPSC-Derived Models.

Tuberous sclerosis complex (TSC) is a rare neurodevelopmental disorder resulting from autosomal dominant mutations in the TSC1 or TSC2 genes, leading to a hyperactivated mammalian target of rapamycin (mTOR) pathway, and gray and white matter defects in the brain. To study the involvement of neuron-glia interactions in TSC phenotypes, we generated TSC patient induced pluripotent stem cell (iPSC)-derived cortical neuronal and oligodendrocyte (OL) cultures. TSC neuron mono-cultures showed increased network activity, as measured by calcium transients and action potential firing, and increased dendritic branching. However, in co-cultures with OLs, neuronal defects became more apparent, showing cellular hypertrophy and increased axonal density. In addition, TSC neuron-OL co-cultures showed increased OL cell proliferation and decreased OL maturation. Pharmacological intervention with the mTOR regulator rapamycin suppressed these defects. Our patient iPSC-based model, therefore, shows a complex cellular TSC phenotype arising from the interaction of neuronal and glial cells and provides a platform for TSC disease modeling and drug development.

Patterning factors during neural progenitor induction determine regional identity and differentiation potential in vitro.

The neural tube consists of neural progenitors (NPs) that acquire different characteristics during gestation due to patterning factors. However, the influence of such patterning factors on human pluripotent stem cells (hPSCs) during in vitro neural differentiation is often unclear. This study compared neural induction protocols involving in vitro patterning with single SMAD inhibition (SSI), retinoic acid (RA) administration and dual SMAD inhibition (DSI). While the derived NP cells expressed known NP markers, they differed in their NP expression profile and differentiation potential. Cortical neuronal cells generated from 1) SSI NPs exhibited less mature neuronal phenotypes, 2) RA NPs exhibited an increased GABAergic phenotype, and 3) DSI NPs exhibited greater expression of glutamatergic lineage markers. Further, although all NPs generated astrocytes, astrocytes derived from the RA-induced NPs had the highest GFAP expression. Differences between NP populations included differential expression of regional identity markers HOXB4, LBX1, OTX1 and GSX2, which persisted into mature neural cell stages. This study suggests that patterning factors regulate how potential NPs may differentiate into specific neuronal and glial cell types in vitro. This challenges the utility of generic neural induction procedures, while highlighting the importance of carefully selecting specific NP protocols.

Multi-level characterization of balanced inhibitory-excitatory cortical neuron network derived from human pluripotent stem cells.

Generation of neuronal cultures from induced pluripotent stem cells (hiPSCs) serve the studies of human brain disorders. However we lack neuronal networks with balanced excitatory-inhibitory activities, which are suitable for single cell analysis. We generated low-density networks of hPSC-derived GABAergic and glutamatergic cortical neurons. We used two different co-culture models with astrocytes. We show that these cultures have balanced excitatory-inhibitory synaptic identities using confocal microscopy, electrophysiological recordings, calcium imaging and mRNA analysis. These simple and robust protocols offer the opportunity for single-cell to multi-level analysis of patient hiPSC-derived cortical excitatory-inhibitory networks; thereby creating advanced tools to study disease mechanisms underlying neurodevelopmental disorders.

Differential Maturation of the Two Regulated Secretory Pathways in Human iPSC-Derived Neurons.

Neurons communicate by regulated secretion of chemical signals from synaptic vesicles (SVs) and dense-core vesicles (DCVs). Here, we investigated the maturation of these two secretory pathways in micro-networks of human iPSC-derived neurons. These micro-networks abundantly expressed endogenous SV and DCV markers, including neuropeptides. DCV transport was microtubule dependent, preferentially anterograde in axons, and 2-fold faster in axons than in dendrites. SV and DCV secretion were strictly Ca2+ and SNARE dependent. DCV secretion capacity matured until day in vitro (DIV) 36, with intense stimulation releasing 6% of the total DCV pool, and then plateaued. This efficiency is comparable with mature mouse neurons. In contrast, SV secretion capacity continued to increase until DIV50, with substantial further increase in secretion efficiency and decrease in silent synapses. These data show that the two secretory pathways can be studied in human neurons and that they mature differentially, with DCV secretion reaching maximum efficiency when that of SVs is still low.

Extracellular ezrin: a novel biomarker for traumatic brain injury.

Traumatic brain injury (TBI) is a heterogeneous disease, and the discovery of diagnostic and prognostic TBI biomarkers is highly desirable in order to individualize patient care. We have previously published a study in which we identified possible TBI biomarkers by mass spectrometry 24 h after injury in a cell culture model. Ezrin-radixin-moesin (ERM) proteins were found abundantly in the medium after trauma, and in the present study we have identified extracellular ezrin as a possible biomarker for brain trauma by analyzing cell culture medium from injured primary neurons and glia and by measuring ezrin in cerebrospinal fluid (CSF) from both rats and humans. Our results show that extracellular ezrin concentration was substantially increased in cell culture medium after injury, but that the intracellular expression of the protein remained stable over time. Controlled cortical impact injured rats showed an increased amount of ezrin in CSF at both day 3 and day 7 after trauma. Moreover, ezrin was present in all ventricular CSF samples from seven humans with severe TBI. In contrast to intracellular ezrin, which is distinctly activated following TBI, extracellular ezrin is nonphosphorylated. This is the first report of extracellular ERM proteins in human and experimental models of TBI, providing a scientific foundation for further assessment of ezrin as a potential biomarker.