Investigating the utility of human embryonic stem cell-derived neurons to model ageing and neurodegenerative disease using whole-genome gene expression and splicing analysis

A major goal in regenerative medicine is the predictable manipulation of human embryonic stem cells (hESCs) to defined cell fates that faithfully represent their somatic counterparts. Directed differentiation of hESCs into neuronal populations has galvanized much interest into their potential application in modelling neurodegenerative disease. However, neurodegenerative diseases are age-related, and therefore establishing the maturational comparability of hESC-derived neural derivatives is critical to generating accurate in vitro model systems. We address this issue by comparing genome-wide, exon-specific expression analyses of pluripotent hESCs, multipotent neural precursor cells and a terminally differentiated enriched neuronal population to expression data from post-mortem foetal and adult human brain samples. We show that hESC-derived neuronal cultures (using a midbrain differentiation protocol as a prototypic example of lineage restriction), while successful in generating physiologically functional neurons, are closer to foetal than adult human brain in terms of molecular maturation. These findings suggest that developmental stage has a more dominant influence on the cellular transcriptome than regional identity. In addition, we demonstrate that developmentally regulated gene splicing is common, and potentially a more sensitive measure of maturational state than gene expression profiling alone. In summary, this study highlights the value of genomic indices in refining and validating optimal cell populations appropriate for modelling ageing and neurodegeneration

PGC-1α Negatively Regulates Extrasynaptic NMDAR Activity and Excitotoxicity

Underexpression of the transcriptional coactivator PGC-1α is causally linked to certain neurodegenerative disorders, including Huntington's Disease (HD). HD pathoprogression is also associated with aberrant NMDAR activity, in particular an imbalance between synaptic versus extrasynaptic (NMDAR(EX)) activity. Here we show that PGC-1α controls NMDAR(EX) activity in neurons and that its suppression contributes to mutant Huntingtin (mHtt)-induced increases in NMDAR(EX) activity and vulnerability to excitotoxic insults. We found that knock-down of endogenous PGC-1α increased NMDAR(EX) activity and vulnerability to excitotoxic insults in rat cortical neurons. In contrast, exogenous expression of PGC-1α resulted in a neuroprotective reduction of NMDAR(EX) currents without affecting synaptic NMDAR activity. Since HD models are associated with mHtt-mediated suppression of PGC-1α expression, as well as increased NMDAR(EX) activity, we investigated whether these two events were linked. Expression of mHtt (148Q) resulted in a selective increase in NMDAR(EX) activity, compared with wild-type Htt (18Q), and increased vulnerability to NMDA excitotoxicity. Importantly, we observed that the effects of mHtt and PGC-1α knockdown on NMDAR(EX) activity and vulnerability to excitotoxicity were nonadditive and occluded each other, consistent with a common mechanism. Moreover, exogenous expression of PGC-1α reversed mtHtt-mediated increases in NMDAR(EX) activity and protected neurons against excitotoxic cell death. The link between mHtt, PGC-1α, and NMDAR activity was also confirmed in rat striatal neurons. Thus, targeting levels of PGC-1α expression may help reduce aberrant NMDAR(EX) activity in disorders where PGC-1α is underexpressed

Suppression of the intrinsic apoptosis pathway by sinaptic activity

Synaptic activity promotes resistance to diverse apoptotic insults, the mechanism behind which is incompletely understood. We show here that a coordinated downregulation of core components of the intrinsic apoptosis pathway by neuronal activity forms a key part of the underlying mechanism. Activity-dependent protection against apoptotic insults is associated with inhibition of cytochrome c release in most but not all neurons, indicative of anti-apoptotic signaling both upstream and downstream of this step. We find that enhanced firing activity suppresses expression of the proapoptotic BH3-only member gene Puma in a NMDA receptor-dependent, p53-independent manner. Puma expression is sufficient to induce cytochrome c loss and neuronal apoptosis. Puma deficiency protects neurons against apoptosis and also occludes the protective effect of synaptic activity, while blockade of physiological NMDA receptor activity in the developing mouse brain induces neuronal apoptosis that is preceded by upregulation of Puma. However, enhanced activity can also confer resistance to Puma-induced apoptosis, acting downstream of cytochrome c release. This mechanism is mediated by transcriptional suppression of apoptosome components Apaf-1 and procaspase-9, and limiting caspase-9 activity, since overexpression of procaspase-9 accelerates the rate of apoptosis in active neurons back to control levels. Synaptic activity does not exert further significant anti-apoptotic effects downstream of caspase-9 activation, since an inducible form of caspase-9 overrides the protective effect of synaptic activity, despite activity-induced transcriptional suppression of caspase-3. Thus, suppression of apoptotic gene expression may synergize with other activity-dependent events such as enhancement of antioxidant defenses to promote neuronal survival

Global epigenomic reconfiguration during mammalian brain development

DNA methylation is implicated in mammalian brain development and plasticity underlying learning and memory. We report the genome-wide composition, patterning, cell specificity, and dynamics of DNA methylation at single-base resolution in human and mouse frontal cortex throughout their lifespan. Widespread methylome reconfiguration occurs during fetal to young adult development, coincident with synaptogenesis. During this period, highly conserved non-CG methylation (mCH) accumulates in neurons, but not glia, to become the dominant form of methylation in the human neuronal genome. Moreover, we found an mCH signature that identifies genes escaping X-chromosome inactivation. Last, whole-genome single-base resolution 5-hydroxymethylcytosine (hmC) maps revealed that hmC marks fetal brain cell genomes at putative regulatory regions that are CG-demethylated and activated in the adult brain and that CG demethylation at these hmC-poised loci depends on Tet2 activity

Activin/Nodal Inhibition Alone Accelerates Highly Efficient Neural Conversion from Human Embryonic Stem Cells and Imposes a Caudal Positional Identity

Background Neural conversion from human embryonic stem cells (hESCs) has been demonstrated in a variety of systems including chemically defined suspension culture, not requiring extrinsic signals, as well as in an adherent culture method that involves dual SMAD inhibition using Noggin and SB431542 (an inhibitor of activin/nodal signaling). Previous studies have also determined a role for activin/nodal signaling in development of the neural plate and anterior fate specification. We therefore sought to investigate the independent influence of SB431542 both on neural commitment of hESCs and positional identity of derived neural progenitors in chemically defined substrate-free conditions. Methodology/Principal Findings We show that in non-adherent culture conditions, treatment with SB431542 alone for 8 days is sufficient for highly efficient and accelerated neural conversion from hESCs with negligible mesendodermal, epidermal or trophectodermal contamination. In addition the resulting neural progenitor population has a predominantly caudal identity compared to the more anterior positional fate of non-SB431542 treated cultures. Finally we demonstrate that resulting neurons are electro-physiologically competent. Conclusions This study provides a platform for the efficient generation of caudal neural progenitors under defined conditions for experimental study

Mechanisms of Inhibition and Potentiation of α4β2 Nicotinic Acetylcholine Receptors by Members of the Ly6 Protein Family*

α4β2 nicotinic acetylcholine receptors (nAChRs) are abundantly expressed throughout the central nervous system and are thought to be the primary target of nicotine, the main addictive substance in cigarette smoking. Understanding the mechanisms by which these receptors are regulated may assist in developing compounds to selectively interfere with nicotine addiction. Here we report previously unrecognized modulatory properties of members of the Ly6 protein family on α4β2 nAChRs. Using a FRET-based Ca(2+) flux assay, we found that the maximum response of α4β2 receptors to agonist was strongly inhibited by Ly6h and Lynx2 but potentiated by Ly6g6e. The mechanisms underlying these opposing effects appear to be fundamentally distinct. Receptor inhibition by Lynx2 was accompanied by suppression of α4β2 expression at the cell surface, even when assays were preceded by chronic exposure of cells to an established chaperone, nicotine. Receptor inhibition by Lynx2 also was resistant to pretreatment with extracellular phospholipase C, which cleaves lipid moieties like those that attach Ly6 proteins to the plasma membrane. In contrast, potentiation of α4β2 activity by Ly6g6e was readily reversible by pretreatment with phospholipase C. Potentiation was also accompanied by slowing of receptor desensitization and an increase in peak currents. Collectively our data support roles for Lynx2 and Ly6g6e in intracellular trafficking and allosteric potentiation of α4β2 nAChRs, respectively

PGC-1α negatively regulates extrasynaptic NMDAR activity and excitotoxicity

Underexpression of the transcriptional coactivator PGC-1α is causally linked to certain neurodegenerative disorders, including Huntington's Disease (HD). HD pathoprogression is also associated with aberrant NMDAR activity, in particular an imbalance between synaptic versus extrasynaptic (NMDAR(EX)) activity. Here we show that PGC-1α controls NMDAR(EX) activity in neurons and that its suppression contributes to mutant Huntingtin (mHtt)-induced increases in NMDAR(EX) activity and vulnerability to excitotoxic insults. We found that knock-down of endogenous PGC-1α increased NMDAR(EX) activity and vulnerability to excitotoxic insults in rat cortical neurons. In contrast, exogenous expression of PGC-1α resulted in a neuroprotective reduction of NMDAR(EX) currents without affecting synaptic NMDAR activity. Since HD models are associated with mHtt-mediated suppression of PGC-1α expression, as well as increased NMDAR(EX) activity, we investigated whether these two events were linked. Expression of mHtt (148Q) resulted in a selective increase in NMDAR(EX) activity, compared with wild-type Htt (18Q), and increased vulnerability to NMDA excitotoxicity. Importantly, we observed that the effects of mHtt and PGC-1α knockdown on NMDAR(EX) activity and vulnerability to excitotoxicity were nonadditive and occluded each other, consistent with a common mechanism. Moreover, exogenous expression of PGC-1α reversed mtHtt-mediated increases in NMDAR(EX) activity and protected neurons against excitotoxic cell death. The link between mHtt, PGC-1α, and NMDAR activity was also confirmed in rat striatal neurons. Thus, targeting levels of PGC-1α expression may help reduce aberrant NMDAR(EX) activity in disorders where PGC-1α is underexpressed

Mechanisms of Inhibition and Potentiation of α4β2 Nicotinic Acetylcholine Receptors by Members of the Ly6 Protein Family

α4β2 nicotinic acetylcholine receptors (nAChRs) are abundantly expressed throughout the central nervous system and are thought to be the primary target of nicotine, the main addictive substance in cigarette smoking. Understanding the mechanisms by which these receptors are regulated may assist in developing compounds to selectively interfere with nicotine addiction. Here we report previously unrecognized modulatory properties of members of the Ly6 protein family on α4β2 nAChRs. Using a FRET-based Ca(2+) flux assay, we found that the maximum response of α4β2 receptors to agonist was strongly inhibited by Ly6h and Lynx2 but potentiated by Ly6g6e. The mechanisms underlying these opposing effects appear to be fundamentally distinct. Receptor inhibition by Lynx2 was accompanied by suppression of α4β2 expression at the cell surface, even when assays were preceded by chronic exposure of cells to an established chaperone, nicotine. Receptor inhibition by Lynx2 also was resistant to pretreatment with extracellular phospholipase C, which cleaves lipid moieties like those that attach Ly6 proteins to the plasma membrane. In contrast, potentiation of α4β2 activity by Ly6g6e was readily reversible by pretreatment with phospholipase C. Potentiation was also accompanied by slowing of receptor desensitization and an increase in peak currents. Collectively our data support roles for Lynx2 and Ly6g6e in intracellular trafficking and allosteric potentiation of α4β2 nAChRs, respectively