A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells.

Autism spectrum disorders (ASD) are complex neurodevelopmental diseases in which different combinations of genetic mutations may contribute to the phenotype. Using Rett syndrome (RTT) as an ASD genetic model, we developed a culture system using induced pluripotent stem cells (iPSCs) from RTT patients' fibroblasts. RTT patients' iPSCs are able to undergo X-inactivation and generate functional neurons. Neurons derived from RTT-iPSCs had fewer synapses, reduced spine density, smaller soma size, altered calcium signaling and electrophysiological defects when compared to controls. Our data uncovered early alterations in developing human RTT neurons. Finally, we used RTT neurons to test the effects of drugs in rescuing synaptic defects. Our data provide evidence of an unexplored developmental window, before disease onset, in RTT syndrome where potential therapies could be successfully employed. Our model recapitulates early stages of a human neurodevelopmental disease and represents a promising cellular tool for drug screening, diagnosis and personalized treatment.

Dopaminergic inputs in the dentate gyrus direct the choice of memory encoding.

Rewarding experiences are often well remembered, and such memory formation is known to be dependent on dopamine modulation of the neural substrates engaged in learning and memory; however, it is unknown how and where in the brain dopamine signals bias episodic memory toward preceding rather than subsequent events. Here we found that photostimulation of channelrhodopsin-2-expressing dopaminergic fibers in the dentate gyrus induced a long-term depression of cortical inputs, diminished theta oscillations, and impaired subsequent contextual learning. Computational modeling based on this dopamine modulation indicated an asymmetric association of events occurring before and after reward in memory tasks. In subsequent behavioral experiments, preexposure to a natural reward suppressed hippocampus-dependent memory formation, with an effective time window consistent with the duration of dopamine-induced changes of dentate activity. Overall, our results suggest a mechanism by which dopamine enables the hippocampus to encode memory with reduced interference from subsequent experience.

Probing sporadic and familial Alzheimer's disease using induced pluripotent stem cells.

Our understanding of Alzheimer's disease pathogenesis is currently limited by difficulties in obtaining live neurons from patients and the inability to model the sporadic form of the disease. It may be possible to overcome these challenges by reprogramming primary cells from patients into induced pluripotent stem cells (iPSCs). Here we reprogrammed primary fibroblasts from two patients with familial Alzheimer's disease, both caused by a duplication of the amyloid-ß precursor protein gene (APP; termed APP(Dp)), two with sporadic Alzheimer's disease (termed sAD1, sAD2) and two non-demented control individuals into iPSC lines. Neurons from differentiated cultures were purified with fluorescence-activated cell sorting and characterized. Purified cultures contained more than 90% neurons, clustered with fetal brain messenger RNA samples by microarray criteria, and could form functional synaptic contacts. Virtually all cells exhibited normal electrophysiological activity. Relative to controls, iPSC-derived, purified neurons from the two APP(Dp) patients and patient sAD2 exhibited significantly higher levels of the pathological markers amyloid-ß(1-40), phospho-tau(Thr 231) and active glycogen synthase kinase-3ß (aGSK-3ß). Neurons from APP(Dp) and sAD2 patients also accumulated large RAB5-positive early endosomes compared to controls. Treatment of purified neurons with ß-secretase inhibitors, but not γ-secretase inhibitors, caused significant reductions in phospho-Tau(Thr 231) and aGSK-3ß levels. These results suggest a direct relationship between APP proteolytic processing, but not amyloid-ß, in GSK-3ß activation and tau phosphorylation in human neurons. Additionally, we observed that neurons with the genome of one sAD patient exhibited the phenotypes seen in familial Alzheimer's disease samples. More generally, we demonstrate that iPSC technology can be used to observe phenotypes relevant to Alzheimer's disease, even though it can take decades for overt disease to manifest in patients.

Selective Degeneration of Entorhinal-CA1 Synapses in Alzheimer's Disease via Activation of DAPK1.

Excitatory pyramidal neurons in the entorhinal cortical layer II region (ECIIPN) form functional excitatory synapses with CA1 parvalbumin inhibitory neurons (CA1PV) and undergo selective degeneration in the early stages of Alzheimer's disease (AD). Here, we show that death-associated protein kinase 1 (DAPK1) is selectively activated in ECIIPN of AD mice. Inhibition of DAPK1 by deleting a catalytic domain or a death domain of DAPK1 rescues the ECIIPN-CA1PV synaptic loss and improves spatial learning and memory in AD mice. This study demonstrates that activation of DAPK1 in ECIIPN contributes to a memory loss in AD and hence warrants a promising target for the treatment of AD. SIGNIFICANCE STATEMENT: Our recent study reported that excitatory pyramidal neurons in the entorhinal cortical layer II region (ECIIPN) target to CA1 parvalbumin-type inhibitory neurons (CA1PV) at a direct pathway and are one of the most vulnerable brain cells that are selectively degenerated in the early stage of Alzheimer's disease (AD). Our present study shows that death-associated protein kinase 1 (DAPK1) is selectively activated in ECIIPN of AD mice. Inhibition of DAPK1 by deleting a catalytic domain or a death domain of DAPK1 rescues the ECIIPN-CA1PV synaptic loss and improves spatial learning and memory in the early stage of AD. These data not only demonstrate a crucial molecular event for synaptic degeneration but also provide a therapeutic target for the treatment of AD.

Modelling schizophrenia using human induced pluripotent stem cells.

Schizophrenia (SCZD) is a debilitating neurological disorder with a world-wide prevalence of 1%; there is a strong genetic component, with an estimated heritability of 80-85%. Although post-mortem studies have revealed reduced brain volume, cell size, spine density and abnormal neural distribution in the prefrontal cortex and hippocampus of SCZD brain tissue and neuropharmacological studies have implicated dopaminergic, glutamatergic and GABAergic activity in SCZD, the cell types affected in SCZD and the molecular mechanisms underlying the disease state remain unclear. To elucidate the cellular and molecular defects of SCZD, we directly reprogrammed fibroblasts from SCZD patients into human induced pluripotent stem cells (hiPSCs) and subsequently differentiated these disorder-specific hiPSCs into neurons (Supplementary Fig. 1). SCZD hiPSC neurons showed diminished neuronal connectivity in conjunction with decreased neurite number, PSD95-protein levels and glutamate receptor expression. Gene expression profiles of SCZD hiPSC neurons identified altered expression of many components of the cyclic AMP and WNT signalling pathways. Key cellular and molecular elements of the SCZD phenotype were ameliorated following treatment of SCZD hiPSC neurons with the antipsychotic loxapine. To date, hiPSC neuronal pathology has only been demonstrated in diseases characterized by both the loss of function of a single gene product and rapid disease progression in early childhood. We now report hiPSC neuronal phenotypes and gene expression changes associated with SCZD, a complex genetic psychiatric disorder.

L1 retrotransposition in human neural progenitor cells.

Long interspersed element 1 (LINE-1 or L1) retrotransposons have markedly affected the human genome. L1s must retrotranspose in the germ line or during early development to ensure their evolutionary success, yet the extent to which this process affects somatic cells is poorly understood. We previously demonstrated that engineered human L1s can retrotranspose in adult rat hippocampus progenitor cells in vitro and in the mouse brain in vivo. Here we demonstrate that neural progenitor cells isolated from human fetal brain and derived from human embryonic stem cells support the retrotransposition of engineered human L1s in vitro. Furthermore, we developed a quantitative multiplex polymerase chain reaction that detected an increase in the copy number of endogenous L1s in the hippocampus, and in several regions of adult human brains, when compared to the copy number of endogenous L1s in heart or liver genomic DNAs from the same donor. These data suggest that de novo L1 retrotransposition events may occur in the human brain and, in principle, have the potential to contribute to individual somatic mosaicism.

Cord blood-derived neuronal cells by ectopic expression of Sox2 and c-Myc.

The finding that certain somatic cells can be directly converted into cells of other lineages by the delivery of specific sets of transcription factors paves the way to novel therapeutic applications. Here we show that human cord blood (CB) CD133(+) cells lose their hematopoietic signature and are converted into CB-induced neuronal-like cells (CB-iNCs) by the ectopic expression of the transcription factor Sox2, a process that is further augmented by the combination of Sox2 and c-Myc. Gene-expression analysis, immunophenotyping, and electrophysiological analysis show that CB-iNCs acquire a distinct neuronal phenotype characterized by the expression of multiple neuronal markers. CB-iNCs show the ability to fire action potentials after in vitro maturation as well as after in vivo transplantation into the mouse hippocampus. This system highlights the potential of CB cells and offers an alternative means to the study of cellular plasticity, possibly in the context of drug screening research and of future cell-replacement therapies.

Ataxia telangiectasia mutated (ATM) modulates long interspersed element-1 (L1) retrotransposition in human neural stem cells.

Long interspersed element-1 (L1) retrotransposons compose ∼20% of the mammalian genome, and ongoing L1 retrotransposition events can impact genetic diversity by various mechanisms. Previous studies have demonstrated that endogenous L1 retrotransposition can occur in the germ line and during early embryonic development. In addition, recent data indicate that engineered human L1s can undergo somatic retrotransposition in human neural progenitor cells and that an increase in human-specific L1 DNA content can be detected in the brains of normal controls, as well as in Rett syndrome patients. Here, we demonstrate an increase in the retrotransposition efficiency of engineered human L1s in cells that lack or contain severely reduced levels of ataxia telangiectasia mutated, a serine/threonine kinase involved in DNA damage signaling and neurodegenerative disease. We demonstrate that the increase in L1 retrotransposition in ataxia telangiectasia mutated-deficient cells most likely occurs by conventional target-site primed reverse transcription and generate either longer, or perhaps more, L1 retrotransposition events per cell. Finally, we provide evidence suggesting an increase in human-specific L1 DNA copy number in postmortem brain tissue derived from ataxia telangiectasia patients compared with healthy controls. Together, these data suggest that cellular proteins involved in the DNA damage response may modulate L1 retrotransposition.

Basal Forebrain Cholinergic Innervation Induces Depression-Like Behaviors Through Ventral Subiculum Hyperactivation.

Malfunction of the ventral subiculum (vSub), the main subregion controlling the output connections from the hippocampus, is associated with major depressive disorder (MDD). Although the vSub receives cholinergic innervation from the medial septum and diagonal band of Broca (MSDB), whether and how the MSDB-to-vSub cholinergic circuit is involved in MDD is elusive. Here, we found that chronic unpredictable mild stress (CUMS) induced depression-like behaviors with hyperactivation of vSub neurons, measured by c-fos staining and whole-cell patch-clamp recording. By retrograde and anterograde tracing, we confirmed the dense MSDB cholinergic innervation of the vSub. In addition, transient restraint stress in CUMS increased the level of ACh in the vSub. Furthermore, chemogenetic stimulation of this MSDB-vSub innervation in ChAT-Cre mice induced hyperactivation of vSub pyramidal neurons along with depression-like behaviors; and local infusion of atropine, a muscarinic receptor antagonist, into the vSub attenuated the depression-like behaviors induced by chemogenetic stimulation of this pathway and CUMS. Together, these findings suggest that activating the MSDB-vSub cholinergic pathway induces hyperactivation of vSub pyramidal neurons and depression-like behaviors, revealing a novel circuit underlying vSub pyramidal neuronal hyperactivation and its associated depression.

Dopaminergic modulation of cortical inputs during maturation of adult-born dentate granule cells.

Adult neurogenesis, a particular form of plasticity in the adult brain, is under dynamic control of neuronal activity mediated by various neurotransmitters. Despite accumulating evidence suggesting that the neurotransmitter dopamine (DA) regulates proliferation of neural precursor cells in the neurogenic zones, whether and how it acts on newly generated neurons that integrate into the established network remains unknown. Using patch-clamp recordings from retrovirus-labeled newborn hippocampal dentate granule cells (DGCs) in acute mouse brain slices, we found that DA not only caused a long-lasting attenuation of medial perforant path (MPP) inputs to the young DGCs, but also decreased their capacity to express long-term potentiation (LTP). In contrast, DA suppressed MPP transmission to mature DGCs to a similar extent but did not influence their LTP expression. This difference was linked to activation of distinct subtypes of DA receptors in DGCs at different developmental stages. Our observations suggest that DA is particularly effective in modulating the activities of hyperexcitable young neurons, which may have important implications for the dentate function as a filter for incoming information to the hippocampus.

The antidepressant effect of nucleus accumbens deep brain stimulation is mediated by parvalbumin-positive interneurons in the dorsal dentate gyrus.

The nucleus accumbens (NAc) is a crucial region in the reward circuit and is related to anhedonia, the pivotal symptom of major depression disorder (MDD). Deep brain stimulation (DBS) of NAc has been identified as an effective treatment for severe refractory major depression; however, the underlying mechanism of NAc-DBS in MDD treatment remains elusive. Using the chronic unpredictable mild stress (CUMS) mouse model, we found NAc-DBS rescued depression-like behaviors, and reversed high gamma oscillation reduction and neurogenesis impairment in the dorsal dentate gyrus. Inactivation of parvalbumin (PV)-positive interneurons (PVI) in the dorsal DG led to depression-like behavior and decreased adult neurogenesis. Further investigation elucidated the VTA-DG GABAergic projection and CA1-NAc projection might jointly participate in NAc-DBS therapeutic mechanism. Disinhibition of the VTA-DG GABAergic projection had an antidepressant effect, and inhibition of the CA1-NAc projection reduced the antidepressant effect of DBS-NAc. Moreover, disinhibiting the VTA-DG GABAergic projection or activating the CA1-NAc projection could increase PVI activity in the dorsal DG. These results showed PVI in the dorsal DG as an essential target in depression and NAc-DBS antidepressant mechanisms.

Reprogramming astrocytes to motor neurons by activation of endogenous Ngn2 and Isl1.

Central nervous system injury and neurodegenerative diseases cause irreversible loss of neurons. Overexpression of exogenous specific transcription factors can reprogram somatic cells into functional neurons for regeneration and functional reconstruction. However, these practices are potentially problematic due to the integration of vectors into the host genome. Here, we showed that the activation of endogenous genes Ngn2 and Isl1 by CRISPRa enabled reprogramming of mouse spinal astrocytes and embryonic fibroblasts to motor neurons. These induced neurons showed motor neuronal morphology and exhibited electrophysiological activities. Furthermore, astrocytes in the spinal cord of the adult mouse can be converted into motor neurons by this approach with high efficiency. These results demonstrate that the activation of endogenous genes is sufficient to induce astrocytes into functional motor neurons in vitro and in vivo. This direct neuronal reprogramming approach may provide a novel potential therapeutic strategy for treating neurodegenerative diseases and spinal cord injury.

Spike timing-dependent LTP/LTD mediates visual experience-dependent plasticity in a developing retinotectal system.

Sensory experience plays an instructive role in the development of the nervous system. Here we showed that visual experience can induce persistent modification of developing retinotectal circuits via spike timing-dependent plasticity (STDP). Pairing light stimuli with spiking of the tectal cell induced persistent enhancement or reduction of light-evoked responses, with a dependence on the relative timing between light stimulus and postsynaptic spiking similar to that for STDP. Using precisely timed sequential three-bar stimulation to mimic a moving bar, we showed that spike timing-dependent LTP/LTD can account for the asymmetric modification of the tectal cell receptive field induced by moving bar. Furthermore, selective inhibition of signaling mediated by brain-derived neurotrophic factor and nitric oxide, which are respectively required for light-induced LTP and LTD, interfered with moving bar-induced temporally specific changes in the tectal cell responses. Together, these findings suggest that STDP can mediate sensory experience-dependent circuit refinement in the developing nervous system.

Visual stimuli-induced LTD of GABAergic synapses mediated by presynaptic NMDA receptors.

Local GABA (gamma-aminobutyric acid) circuits contribute to sensory experience-dependent refinement of neuronal connections in the developing nervous system, but whether GABAergic synapses themselves can be rapidly modified by sensory stimuli is largely unknown. Here we report that repetitive light stimuli or theta burst stimulation (TBS) of the optic nerve in the developing Xenopus retinotectal system induces long-term potentiation (LTP) of glutamatergic inputs but long-term depression (LTD) of GABAergic inputs to the same tectal neuron. The LTD is due to a reduction in presynaptic GABA release and requires activation of presynaptic NMDA (N-methyl-D-aspartate) receptors (NMDARs) and coincident high-level GABAergic activity. Thus, the presynaptic NMDAR may function as a coincidence detector for adjacent glutamatergic and GABAergic activities, leading to coordinated synaptic modification by sensory experience.

Development of Depotentiation in Adult-Born Dentate Granule Cells.

Activity-dependent synaptic plasticity, i.e., long-term potentiation (LTP), long-term depression (LTD) and LTP reversal, is generally thought to make up the cellular mechanism underlying learning and memory in the mature brain, in which N-methyl-D-aspartate subtype of glutamate (NMDA) receptors and neurogenesis play important roles. LTP reversal may be the mechanism of forgetting and may mediate many psychiatric disorders, such as schizophrenia, but the specific mechanisms underlying these disorders remain unclear. In addition, LTP reversal during the development of adult-born dentate granule cells (DGCs) remains unknown. We found that the expression of the NMDA receptor subunits NR2A and NR2B displayed dynamic changes during the development of postnatal individuals and the maturation of adult-born neurons and was coupled with the change in LTP reversal. The susceptibility of LTP reversal progressively increases with the rise in the expression of NR2A during the development of postnatal individual and adult-born neurons. In addition, NMDA receptor subunits NR2A, but not NR2B, mediated LTP reversal in the DGCs of the mouse hippocampus.

Corrigendum: Intense Exercise Promotes Adult Hippocampal Neurogenesis But Not Spatial Discrimination.

[This corrects the article DOI: 10.3389/fncel.2017.00013.].

Memory Susceptibility to Retroactive Interference Is Developmentally Regulated by NMDA Receptors.

Retroactive interference (RI) occurs when new incoming information impairs an existing memory, which is one of the primary sources of forgetting. Although long-term potentiation (LTP) reversal shows promise as the underlying neural correlate, the key molecules that control the sensitivity of memory circuits to RI are unknown, and the developmental trajectory of RI effects is unclear. Here we found that depotentiation in the hippocampal dentate gyrus (DG) depends on GluN2A-containing NMDA receptors (NMDARs). The susceptibility of LTP to disruption progressively increases with the rise in the GluN2A/GluN2B ratio during development. The vulnerability of hippocampus-dependent memory to interference from post-learning novelty exploration is subject to similar developmental regulation by NMDARs. Both GluN2A overexpression and GluN2B downregulation in the DG promote RI-induced forgetting. Altogether, our results suggest that a switch in GluN2 subunit predominance may confer age-related differences to depotentiation and underlie the developmental decline in memory resistance to RI.

Intense Exercise Promotes Adult Hippocampal Neurogenesis But Not Spatial Discrimination.

Hippocampal neurogenesis persists throughout adult life and plays an important role in learning and memory. Although the influence of physical exercise on neurogenesis has been intensively studied, there is controversy in regard to how the impact of exercise may vary with its regime. Less is known about how distinct exercise paradigms may differentially affect the learning behavior. Here we found that, chronic moderate treadmill running led to an increase of cell proliferation, survival, neuronal differentiation, and migration. In contrast, intense running only promoted neuronal differentiation and migration, which was accompanied with lower expressions of vascular endothelial growth factor, brain-derived neurotrophic factor, insulin-like growth factor 1, and erythropoietin. In addition, the intensely but not mildly exercised animals exhibited a lower mitochondrial activity in the dentate gyrus. Correspondingly, neurogenesis induced by moderate but not intense exercise was sufficient to improve the animal's ability in spatial pattern separation. Our data indicate that the effect of exercise on spatial learning is intensity-dependent and may involve mechanisms other than a simple increase in the number of new neurons.

Impairments of spatial memory in an Alzheimer's disease model via degeneration of hippocampal cholinergic synapses.

Choline acetyltransferase neurons in the vertical diagonal band of Broca (vChATs) degenerate in the early stage of Alzheimer's disease (AD). Here, we report that vChATs directly innervate newly generated immature neurons (NGIs) in the dorsal hippocampus (dNGIs) of adult mice and regulate both the dNGIs survival and spatial pattern separation. In a mouse model that exhibits amyloid-ß plaques similar to AD patients, cholinergic synaptic transmission, dNGI survival and spatial pattern separation are impaired. Activation of vChATs with theta burst stimulation (TBS) that alleviates the decay in cholinergic synaptic transmission effectively protects against spatial pattern separation impairments in the AD mice and this protection was completely abolished by inhibiting the dNGIs survival. Thus, the impairments of pattern separation-associated spatial memory in AD mice are in part caused by degeneration of cholinergic synaptic transmission that modulates the dNGIs survival.

Distinct roles of NMDA receptors at different stages of granule cell development in the adult brain.

NMDA receptor (NMDAR)-dependent forms of synaptic plasticity are thought to underlie the assembly of developing neuronal circuits and to play a crucial role in learning and memory. It remains unclear how NMDAR might contribute to the wiring of adult-born granule cells (GCs). Here we demonstrate that nascent GCs lacking NMDARs but rescued from apoptosis by overexpressing the pro-survival protein Bcl2 were deficient in spine formation. Insufficient spinogenesis might be a general cause of cell death restricted within the NMDAR-dependent critical time window for GC survival. NMDAR loss also led to enhanced mushroom spine formation and synaptic AMPAR activity throughout the development of newborn GCs. Moreover, similar elevated synapse maturation in the absence of NMDARs was observed in neonate-generated GCs and CA1 pyramidal neurons. Together, these data suggest that NMDAR operates as a molecular monitor for controlling the activity-dependent establishment and maturation rate of synaptic connections between newborn neurons and others.