Functional properties of in vitro excitatory cortical neurons derived from human pluripotent stem cells

The in vitro derivation of regionally defined human neuron types from patient‐derived stem cells is now established as a resource to investigate human development and disease. Characterization of such neurons initially focused on the expression of developmentally regulated transcription factors and neural markers, in conjunction with the development of protocols to direct and chart the fate of differentiated neurons. However, crucial to the understanding and exploitation of this technology is to determine the degree to which neurons recapitulate the key functional features exhibited by their native counterparts, essential for determining their usefulness in modelling human physiology and disease in vitro. Here, we review the emerging data concerning functional properties of human pluripotent stem cell‐derived excitatory cortical neurons, in the context of both maturation and regional specificity. [Image: see text

Importin 13-dependent axon diameter growth regulates conduction speeds along myelinated CNS axons

Axon diameter influences the conduction properties of myelinated axons, both directly, and indirectly through effects on myelin. However, we have limited understanding of mechanisms controlling axon diameter growth in the central nervous system, preventing systematic dissection of how manipulating diameter affects myelination and conduction along individual axons. Here we establish zebrafish to study axon diameter. We find that importin 13b is required for axon diameter growth, but does not affect cell body size or axon length. Using neuron-specific ipo13b mutants, we assess how reduced axon diameter affects myelination and conduction, and find no changes to myelin thickness, precision of action potential propagation, or ability to sustain high frequency firing. However, increases in conduction speed that occur along single myelinated axons with development are tightly linked to their growth in diameter. This suggests that axon diameter growth is a major driver of increases in conduction speeds along myelinated axons over time.<br/

Importin 13-dependent axon diameter growth regulates conduction speeds along myelinated CNS axons

Axon diameter influences the conduction properties of myelinated axons, both directly, and indirectly through effects on myelin. However, we have limited understanding of mechanisms controlling axon diameter growth in the central nervous system, preventing systematic dissection of how manipulating diameter affects myelination and conduction along individual axons. Here we establish zebrafish to study axon diameter. We find that importin 13b is required for axon diameter growth, but does not affect cell body size or axon length. Using neuron-specific ipo13b mutants, we assess how reduced axon diameter affects myelination and conduction, and find no changes to myelin thickness, precision of action potential propagation, or ability to sustain high frequency firing. However, increases in conduction speed that occur along single myelinated axons with development are tightly linked to their growth in diameter. This suggests that axon diameter growth is a major driver of increases in conduction speeds along myelinated axons over time

SRSF1-dependent inhibition of C9ORF72-repeat RNA nuclear export: genome-wide mechanisms for neuroprotection in amyotrophic lateral sclerosis.

BACKGROUND: Loss of motor neurons in amyotrophic lateral sclerosis (ALS) leads to progressive paralysis and death. Dysregulation of thousands of RNA molecules with roles in multiple cellular pathways hinders the identification of ALS-causing alterations over downstream changes secondary to the neurodegenerative process. How many and which of these pathological gene expression changes require therapeutic normalisation remains a fundamental question. METHODS: Here, we investigated genome-wide RNA changes in C9ORF72-ALS patient-derived neurons and Drosophila, as well as upon neuroprotection taking advantage of our gene therapy approach which specifically inhibits the SRSF1-dependent nuclear export of pathological C9ORF72-repeat transcripts. This is a critical study to evaluate (i) the overall safety and efficacy of the partial depletion of SRSF1, a member of a protein family involved itself in gene expression, and (ii) a unique opportunity to identify neuroprotective RNA changes. RESULTS: Our study shows that manipulation of 362 transcripts out of 2257 pathological changes, in addition to inhibiting the nuclear export of repeat transcripts, is sufficient to confer neuroprotection in C9ORF72-ALS patient-derived neurons. In particular, expression of 90 disease-altered transcripts is fully reverted upon neuroprotection leading to the characterisation of a human C9ORF72-ALS disease-modifying gene expression signature. These findings were further investigated in vivo in diseased and neuroprotected Drosophila transcriptomes, highlighting a list of 21 neuroprotective changes conserved with 16 human orthologues in patient-derived neurons. We also functionally validated the high neuroprotective potential of one of these disease-modifying transcripts, demonstrating that inhibition of ALS-upregulated human KCNN1-3 (Drosophila SK) voltage-gated potassium channel orthologs mitigates degeneration of human motor neurons and Drosophila motor deficits. CONCLUSIONS: Strikingly, the partial depletion of SRSF1 leads to expression changes in only a small proportion of disease-altered transcripts, indicating that not all RNA alterations need normalization and that the gene therapeutic approach is safe in the above preclinical models as it does not disrupt globally gene expression. The efficacy of this intervention is also validated at genome-wide level with transcripts modulated in the vast majority of biological processes affected in C9ORF72-ALS. Finally, the identification of a characteristic signature with key RNA changes modified in both the disease state and upon neuroprotection also provides potential new therapeutic targets and biomarkers.This work was initiated with the Medical Research Council (MRC) grant MR/M010864/1 (KN, GMH, PJS) and the MND Association grant Hautbergue/Apr16/846–791 (GMH, LF, AJW, PJS, LMC). This research was further supported by the MRC New Investigator research grant MR/R024162/1 (GMH) and the Biotechnology and Biological Sciences Research Council (BBSRC) grant BB/S005277/1 (GMH). LC was supported by H2020-EU EU Marie Curie fellowship CONTESSA (ID: 660388). CDSS is funded by an AstraZeneca Post-Doctoral award. LF was funded by the Thierry Latran Foundation (FTLAAP2016/ Astrocyte secretome) and is currently supported by the MND Association grant Apr16/848–791 and the Academy of Medical Sciences Springboard Award. AJW was supported by MRC core funding (MC_UU_00015/6) and ERC Starting grant (DYNAMITO; 309742). GMH also reports grants Apr17/854–791 from the MND Association, Thierry Latran FTLAAP2016/ Astrocyte secretome and Royal Society International Exchanges grant IEC\R3\17010 during the course of this study. MA acknowledge grants from Alzheimer’s Research UK (ARUK-PG2018B-005), European Research Council (ERC Advanced Award 294745) and MRC DPFS (129016). PJS is supported as an NIHR Senior Investigator Investigator (NF-SI-0617–10077) and by the MND Association (AMBRoSIA 972–797) and MRC grant MR/S004920/1

Maturation of AMPAR Composition and the GABAAR Reversal Potential in hPSC-Derived Cortical Neurons

Rodent-based studies have shown that neurons undergo major developmental changes to ion channel expression and ionic gradients that determine their excitation-inhibition balance. Neurons derived from human pluripotent stem cells theoretically offer the potential to study classical developmental processes in a human-relevant system, although this is currently not well explored. Here, we show that excitatory cortical-patterned neurons derived from multiple human pluripotent stem cell lines exhibit native-like maturation changes in AMPAR composition such that there is an increase in the expression of GluA2(R) subunits. Moreover, we observe a dynamic shift in intracellular Cl(−) levels, which determines the reversal potential of GABA(A)R-mediated currents and is influenced by neurotrophic factors. The shift is concomitant with changes in KCC2 and NKCC1 expression. Because some human diseases are thought to involve perturbations to AMPAR GluA2 content and others in the chloride reversal potential, human stem-cell-derived neurons represent a valuable tool for studying these fundamental properties

Ionotropic GABA and glycine receptor subunit composition in human pluripotent stem cell-derived excitatory cortical neurones

We have assessed, using whole-cell patch-clamp recording and RNA-sequencing (RNA-seq), the properties and composition of GABA(A) receptors (GABA(A)Rs) and strychnine-sensitive glycine receptors (GlyRs) expressed by excitatory cortical neurons derived from human embryonic stem cells (hECNs). The agonists GABA and muscimol gave EC(50) values of 278 μm and 182 μm, respectively, and the presence of a GABA(A)R population displaying low agonist potencies is supported by strong RNA-seq signals for α2 and α3 subunits. GABA(A)R-mediated currents, evoked by EC(50) concentrations of GABA, were blocked by bicuculline and picrotoxin with IC(50) values of 2.7 and 5.1 μm, respectively. hECN GABA(A)Rs are predominantly γ subunit-containing as assessed by the sensitivity of GABA-evoked currents to diazepam and insensitivity to Zn(2+), together with the weak direct agonist action of gaboxadol; RNA-seq indicated a predominant expression of the γ2 subunit. Potentiation of GABA-evoked currents by propofol and etomidate and the lack of inhibition of currents by salicylidine salycylhydrazide (SCS) indicate expression of the β2 or β3 subunit, with RNA-seq analysis indicating strong expression of β3 in hECN GABA(A)Rs. Taken together our data support the notion that hECN GABA(A)Rs have an α2/3β3γ2 subunit composition – a composition that also predominates in immature rodent cortex. GlyRs expressed by hECNs were activated by glycine with an EC(50) of 167 μm. Glycine-evoked (500 μm) currents were blocked by strychnine (IC(50) = 630 nm) and picrotoxin (IC(50) = 197 μm), where the latter is suggestive of a population of heteromeric receptors. RNA-seq indicates GlyRs are likely to be composed of α2 and β subunits

Neuronal activity disrupts myelinated axon integrity in the absence of NKCC1b

Through a genetic screen in zebrafish, we identified a mutant with disruption to myelin in both the CNS and PNS caused by a mutation in a previously uncharacterized gene, slc12a2b, predicted to encode a Na+, K+, and Cl− (NKCC) cotransporter, NKCC1b. slc12a2b/NKCC1b mutants exhibited a severe and progressive pathology in the PNS, characterized by dysmyelination and swelling of the periaxonal space at the axon–myelin interface. Cell-type–specific loss of slc12a2b/NKCC1b in either neurons or myelinating Schwann cells recapitulated these pathologies. Given that NKCC1 is critical for ion homeostasis, we asked whether the disruption to myelinated axons in slc12a2b/NKCC1b mutants is affected by neuronal activity. Strikingly, we found that blocking neuronal activity completely prevented and could even rescue the pathology in slc12a2b/NKCC1b mutants. Together, our data indicate that NKCC1b is required to maintain neuronal activity–related solute homeostasis at the axon–myelin interface, and the integrity of myelinated axons