Martha Constantine-Paton

Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease

The long-term goal of nuclear transfer or alternative reprogramming approaches is to create patient-specific donor cells for transplantation therapy, avoiding immunorejection, a major complication in current transplantation medicine. It was recently shown that the four transcription factors Oct4, Sox2, Klf4, and c-Myc induce pluripotency in mouse fibroblasts. However, the therapeutic potential of induced pluripotent stem (iPS) cells for neural cell replacement strategies remained unexplored. Here, we show that iPS cells can be efficiently differentiated into neural precursor cells, giving rise to neuronal and glial cell types in culture. Upon transplantation into the fetal mouse brain, the cells migrate into various brain regions and differentiate into glia and neurons, including glutamatergic, GABAergic, and catecholaminergic subtypes. Electrophysiological recordings and morphological analysis demonstrated that the grafted neurons had mature neuronal activity and were functionally integrated in the host brain. Furthermore, iPS cells were induced to differentiate into dopamine neurons of midbrain character and were able to improve behavior in a rat model of Parkinsons disease upon transplantation into the adult brain. We minimized the risk of tumor formation from the grafted cells by separating contaminating pluripotent cells and committed neural cells using fluorescence-activated cell sorting. Our results demonstrate the therapeutic potential of directly reprogrammed fibroblasts for neuronal cell replacement in the animal model.

Microarray analysis of microRNA expression in the developing mammalian brain

BackgroundMicroRNAs are a large new class of tiny regulatory RNAs found in nematodes, plants, insects and mammals. MicroRNAs are thought to act as post-transcriptional modulators of gene expression. In invertebrates microRNAs have been implicated as regulators of developmental timing, neuronal differentiation, cell proliferation, programmed cell death and fat metabolism. Little is known about the roles of microRNAs in mammals.ResultsWe isolated 18-26 nucleotide RNAs from developing rat and monkey brains. From the sequences of these RNAs and the sequences of the rat and human genomes we determined which of these small RNAs are likely to have derived from stem-loop precursors typical of microRNAs. Next, we developed a microarray technology suitable for detecting microRNAs and printed a microRNA microarray representing 138 mammalian microRNAs corresponding to the sequences of the microRNAs we cloned as well as to other known microRNAs. We used this microarray to determine the profile of microRNAs expressed in the developing mouse brain. We observed a temporal wave of expression of microRNAs, suggesting that microRNAs play important roles in the development of the mammalian brain.ConclusionWe describe a microarray technology that can be used to analyze the expression of microRNAs and of other small RNAs. MicroRNA microarrays offer a new tool that should facilitate studies of the biological roles of microRNAs. We used this method to determine the microRNA expression profile during mouse brain development and observed a temporal wave of gene expression of sequential classes of microRNAs.

Postsynaptic BDNF‐TrkB signaling in synapse maturation, plasticity, and disease

Brain‐derived neurotrophic factor (BDNF) is a prototypic neurotrophin that regulates diverse developmental events from the selection of neural progenitors to the terminal dendritic differentiation and connectivity of neurons. We focus here on activity‐dependent synaptic regulation by BDNF and its receptor, full length TrkB. BDNF‐TrkB signaling is involved in transcription, translation, and trafficking of proteins during various phases of synaptic development and has been implicated in several forms of synaptic plasticity. These functions are carried out by a combination of the three signaling cascades triggered when BDNF binds TrkB: The mitogen‐activated protein kinase (MAPK), the phospholipase Cγ (PLC PLCγ), and the phosphatidylinositol 3‐kinase (PI3K) pathways. MAPK and PI3K play crucial roles in both translation and/or trafficking of proteins induced by synaptic activity, whereas PLCγ regulates intracellular Ca2+ that can drive transcription via cyclic AMP and a protein kinase C. Conversely, the abnormal regulation of BDNF is implicated in various developmental and neurodegenerative diseases that perturb neural development and function. We will discuss the current state of understanding BDNF signaling in the context of synaptic development and plasticity with a focus on the postsynaptic cell and close with the evidence that basic mechanisms of BDNF function still need to be understood to effectively treat genetic disruptions of these pathways that cause devastating neurodevelopmental diseases.

NMDA receptor-mediated control of protein synthesis at developing synapses

We demonstrate a rapid and complex effect of N-methyl-d-aspartate receptor (NMDAR) activation on synaptic protein synthesis in the superior colliculi of young rats. Within minutes of receptor activation, translation of alpha Ca2+/calmodulin dependent kinase II (αCamK II) was increased, whereas total protein synthesis was reduced. NMDAR activation also increased phosphorylation of eukaryotic elongation factor 2 (eEF2), a process known to inhibit protein translation by reducing peptide chain elongation. Low doses of cycloheximide, which reduce elongation rate independently of eEF2 phosphorylation, decreased overall protein synthesis but increased αCaMK II synthesis. These observations suggest that regulation of peptide elongation via eEF2 phosphorylation can link NMDAR activation to local increases in the synthesis of specific proteins during activity-dependent synaptic change.

Modulation of NMDA receptor function: implications for vertebrate neural development.

The NMDA subtype of glutamate receptor is hypothesized to mediate synaptic competition in the developing brain by stabilizing converging synapses that have correlated activity patterns. Disruption of NMDA receptor function during development interferes with synapse elimination and sensory map formation. Moreover, many studies indicate that NMDA receptor function is high during times of synaptic rearrangement. In this review, a corollary of the NMDA receptor hypothesis for activity‐dependent synapse stabilization is proposed. As developing inputs increase in number and strength, the increasing excitatory synaptic activity in young neurons should lead to increases in postsynaptic Ca2+ influx through NMDA receptors. This Ca2+ flux is postulated to trigger a feedback system that changes the subunit composition of the NMDA receptor complex so that less Ca2+ enters postsynaptic cells upon NMDA receptor activation. Changes in NMDA receptor effectiveness resulting from manipulations of activity are consistent with the idea that NMDA receptor function is under the control of activity. This postulate of activity‐dependent control of NMDA receptor expression has implications for the control of brain plasticity. If particular combinations of NMDA receptor subunits typically expressed in young animals are better than adult receptor types at maintaining synapses in regions where they are not well correlated with other inputs, then expression of these juvenile subunit combinations could facilitate synaptic rearrangements in the mature brain after the normal end of synaptic plasticity. Thus, understanding the regulation of NMDA receptor function during development could provide a novel approach to restructuring circuitry in the adult brain to compensate for damage produced by trauma or disease.—Scheetz, A. J., Constantine‐Paton, M. Modulation of NMDA receptor function: implications for vertebrate neural development. FASEB J. 8: 745‐752; 1994.

NMDA receptor antagonists disrupt the retinotectal topographic map

We tested the effect of two NMDA receptor antagonists, APV or MK801 (with NMDA), and the receptor agonist NMDA on the maintenance of retinal topography in frogs. Topography was assayed by measuring the dispersion of retrogradely labeled ganglion cells following a local HRP injection into the tectum. In untreated tadpoles, labeled cells covered about 5% of the retinal area. In APV- or MK801/NMDA-treated tadpoles, labeled ganglion cells covered 17% and 10% of the retinal area, respectively. Neither treatment with L-APV nor with NMDA disrupts the fidelity of the retinotectal projection. Neither APV- nor NMDA-treated ganglion cell terminals differed from untreated terminals with respect to tangential area, branch number, or branch density. These data support a role for the NDMA receptor in visual system development.

LTP and activity-dependent synaptogenesis: the more alike they are, the more different they become

Recent data suggest that long-term potentiation and activity-dependent synaptogenesis share the same mechanism at the initiation stage during which NMDA receptor activity is necessary to increase the postsynaptic response via AMPA receptor currents. However, several fundamental differences between the environments of young and mature synapses and the neurons that support them suggest that the same cellular mechanism is facilitated by very different parameters in the young versus the mature brain.

Receptor compartmentalization and trafficking at glutamate synapses: a developmental proposal.

This article focuses on NMDA receptor subunit changes that occur in the forebrain and midbrain during development, namely the switch from predominance of NMDA receptors rich in NR2B subunits to that of NMDA receptors rich in NR2A subunits. We review the potential roles in brain plasticity of two membrane-associated guanylate kinases (MAGUKs), SAP102 and PSD95, which form a scaffold for the ion-passing glutamate receptors at the postsynaptic density, and we consider the known functional significance of these molecules in subunit switching. In addition, based on recent analyses of the synaptic location of glutamate receptors, activity-dependent changes in developing visual neurons, and extensive data on MAGUKs, we propose a model of glutamatergic synaptic differentiation. In this model, different NMDA receptor scaffolding and signaling complexes effect the trafficking and synaptic localization of NR2A-rich and NR2B-rich receptors, leading to tangential compartmentalization of these receptors and their movement between synaptic and extrasynaptic compartments.

BDNF induces transport of PSD-95 to dendrites through PI3K-AKT signaling after NMDA receptor activation

The N-methyl-D-aspartate receptor (NMDAR), brain-derived neurotrophic factor (BDNF), postsynaptic density protein 95 (PSD-95) and phosphatidylinositol 3-kinase (PI3K) have all been implicated in long-term potentiation. Here we show that these molecules are involved in a single pathway for synaptic potentiation. In visual cortical neurons in young rodents, the neurotrophin receptor TrkB is associated with PSD-95. When BDNF is applied to cultured visual cortical neurons, PSD-95–labeled synaptic puncta enlarge, and fluorescent recovery after photobleaching (FRAP) reveals increased delivery of green fluorescent protein–tagged PSD-95 to the dendrites. The recovery of fluorescence requires TrkB, signaling through PI3K and the serine-threonine kinase Akt, and an intact Golgi apparatus. Stimulation of NMDARs mimics the PSD-95 trafficking that is induced by BDNF but requires active BDNF and PI3K. Furthermore, local dendritic contact with a BDNF-coated microsphere induces PSD-95 FRAP throughout the dendrites of the stimulated neuron, suggesting that this mechanism induces rapid neuron-wide synaptic increases in PSD-95 and refinement whenever a few robust inputs activate the NMDAR-BDNF-PI3K pathway.

Distinct roles of NR2A and NR2B cytoplasmic tails in long term potentiation

NMDA receptors (NMDARs) are critical mediators of activity-dependent synaptic plasticity, but the differential roles of NR2A- versus NR2B-containing NMDARs have been controversial. Here, we investigate the roles of NR2A and NR2B in long-term potentiation (LTP) in organotypic hippocampal slice cultures using RNA interference (RNAi) and overexpression, to complement pharmacological approaches. In young slices, when NR2B is the predominant subunit expressed, LTP is blocked by the NR2B-selective antagonist Ro25-6981 [R-(R,S)-α-(4-hydroxyphenyl)-β-methyl-4-(phenylmethyl)-1-piperidine propranol]. As slices mature and NR2A expression rises, activation of NR2B receptors became no longer necessary for LTP induction. LTP was blocked, however, by RNAi knockdown of NR2B, and this was rescued by coexpression of an RNAi-resistant NR2B (NR2B*) cDNA. Interestingly, a chimeric NR2B subunit in which the C-terminal cytoplasmic tail was replaced by that of NR2A failed to rescue LTP, whereas the reverse chimera, NR2A channel with NR2B tail, was able to restore LTP. Thus, expression of NR2B with its intact cytoplasmic tail is required for LTP induction, at an age when channel activity of NR2B–NMDARs is not required for LTP. Overexpression of wild-type NR2A failed to rescue LTP in neurons transfected with the NR2B–RNAi construct, despite restoring NMDA–EPSC amplitude to a similar level as NR2B*. Surprisingly, an NR2A construct lacking its entire C-terminal cytoplasmic tail regained its ability to restore LTP. Together, these data suggest that the NR2B subunit plays a critical role for LTP, presumably by recruiting relevant molecules important for LTP via its cytoplasmic tail. In contrast, NR2A is not essential for LTP, and its cytoplasmic tail seems to carry inhibitory factors for LTP.