NMDA Receptor Stimulation Induces Reversible Fission of the Neuronal Endoplasmic Reticulum

With few exceptions the endoplasmic reticulum (ER) is considered a continuous system of endomembranes within which proteins and ions can move. We have studied dynamic structural changes of the ER in hippocampal neurons in primary culture and organotypic slices. Fluorescence recovery after photobleaching (FRAP) was used to quantify and model ER structural dynamics. Ultrastructure was assessed by electron microscopy. In live cell imaging experiments we found that, under basal conditions, the ER of neuronal soma and dendrites was continuous. The smooth and uninterrupted appearance of the ER changed dramatically after glutamate stimulation. The ER fragmented into isolated vesicles in a rapid fission reaction that occurred prior to overt signs of neuronal damage. ER fission was found to be independent of ER calcium levels. Apart from glutamate, the calcium ionophore ionomycin was able to induce ER fission. The N-methyl, D-aspartate (NMDA) receptor antagonist MK-801 inhibited ER fission induced by glutamate as well as by ionomycin. Fission was not blocked by either ifenprodil or kinase inhibitors. Interestingly, sub-lethal NMDA receptor stimulation caused rapid ER fission followed by fusion. Hence, ER fission is not strictly associated with cellular damage or death. Our results thus demonstrate that neuronal ER structure is dynamically regulated with important consequences for protein mobility and ER luminal calcium tunneling

REST suppression mediates neural conversion of adult human fibroblasts via microRNA-dependent and -independent pathways.

Direct conversion of human fibroblasts into mature and functional neurons, termed induced neurons (iNs), was achieved for the first time 6 years ago. This technology offers a promising shortcut for obtaining patient- and disease-specific neurons for disease modeling, drug screening, and other biomedical applications. However, fibroblasts from adult donors do not reprogram as easily as fetal donors, and no current reprogramming approach is sufficiently efficient to allow the use of this technology using patient-derived material for large-scale applications. Here, we investigate the difference in reprogramming requirements between fetal and adult human fibroblasts and identify REST as a major reprogramming barrier in adult fibroblasts. Via functional experiments where we overexpress and knockdown the REST-controlled neuron-specific microRNAs miR-9 and miR-124, we show that the effect of REST inhibition is only partially mediated via microRNA up-regulation. Transcriptional analysis confirmed that REST knockdown activates an overlapping subset of neuronal genes as microRNA overexpression and also a distinct set of neuronal genes that are not activated via microRNA overexpression. Based on this, we developed an optimized one-step method to efficiently reprogram dermal fibroblasts from elderly individuals using a single-vector system and demonstrate that it is possible to obtain iNs of high yield and purity from aged individuals with a range of familial and sporadic neurodegenerative disorders including Parkinson's, Huntington's, as well as Alzheimer's disease

Mechanisms controlling striatal projection neurone generation, from patterning to early differentiation

The striatum is part of the telencephalon, the most anterior part of the vertebrate brain. From when it first can be identified, telencephalic morphology is highly complex and a wide range of mechanisms has been suggested to participate in its induction, patterning and neurogenesis. Sonic hedgehog (shh), a gene coding for a secreted protein has been suggested to be required for ventral telencephalic, including striatal, development. The first study in this thesis demonstrates that shh is important for striatal development but that aspects of this process can occur in shh mutant mice. Once the lateral ganglionic eminence (LGE, the source of striatal projection neurones) has been defined, mechanisms are required to ensure correct identity of striatal progenitor cells. The second study shows that two homeodomain transcription factor genes control aspects of this: Pax6, which is expressed in cortical progenitors and Gsh2 found in striatal progenitors. By analysing mice, mutant for either one or both of these genes, we concluded that they oppositely regulate the identity of cortical and striatal progenitors. Interestingly, we were able to demonstrate that in the absence of Gsh2, the highly similar gene Gsh1 rescues aspects of striatal development (paper III) while other aspects appear Gsh gene-independent.. When striatal neurogenesis has started, post-mitotic cells most likely rely on cell-intrinsic as well as cell-extrinsic mechanisms to ensure correct differentiation. In study four we describe the identification of candidates to the first category; homeodomain genes of the Meis and Pbx family. Finally, in the fifth study, retinoids are presented as a candidate cell-extrinsic regulator of striatal projection neurone development

Rapid fragmentation of the endoplasmic reticulum in cortical neurons of the mouse brain in situ following cardiac arrest.

Neuronal endoplasmic reticulum (ER), continuous from soma to dendritic spines, undergoes rapid fragmentation in response to N-methyl-D-aspartate (NMDA) receptor stimulation in hippocampal slices and neuronal primary cultures. Here, we show that ER fragments in the mouse brain following cardiac arrest (CA) induced brain ischemia. The ER structure was assessed in vivo in cortical pyramidal neurons in transgenic mice expressing ER-targeted GFP using two-photon laser scanning microscopy with fluorescence recovery after photobleaching (FRAP). Endoplasmic reticulum fragmentation occurred 1 to 2 minutes after CA and once induced, fragmentation was rapid (<15 seconds). We propose that acute ER fragmentation may be a protective response against severe ischemic stress.Journal of Cerebral Blood Flow & Metabolism advance online publication, 6 April 2011; doi:10.1038/jcbfm.2011.37

Endoplasmic reticulum dynamics in hippocampal dendritic spines induced by agonists of type I metabotropic glutamate but not by muscarinic acetylcholine receptors.

Neurons in the hippocampus exhibit subpopulations of dendritic spines that contain endoplasmic reticulum (ER). ER in spines is important for synaptic activity and its associated Ca(2+) signaling. The dynamic distribution of ER to spines is regulated by diacylglycerol and partly mediated by protein kinase C, metalloproteinases and γ-secretase. In this study, we explored whether pharmacological activation of type I metabotropic glutamate receptors (mGluRs) and muscarinic acetylcholine receptors (mAChRs) known to activate phospholipase C would have any effect on spine ER content. We found that DHPG (100 μM) but not carbachol (10 μM) caused a reduction in the number of spines with ER. We further found that ER Ca(2+) depletion triggered by thapsigargin (200 nM) had no effect on ER localization in spines

Expression of Meis and Pbx genes and their protein products in the developing telencephalon : Implications for regional differentiation

The Meis and Pbx genes encode for homeodomain proteins of the TALE class and have been shown to act as co-factors for other homeodomain transcription factors (Mann and Affolter, 1998. Curr. Opin. Genet. Dev. 8, 423-429). We have studied the expression of these genes in the mouse telencephalon and found that Meis1 and Meis2 display region-specific patterns of expression from embryonic day (E)10.5 until birth, defining distinct subterritories in the developing telencephalon. The expression of the Meis genes and their proteins is highest in the subventricular zone (SVZ) and mantle regions of the ventral telencephalon. Compared to the Meis genes, Pbx genes show a broader expression within the telencephalon. However, as is the case in Drosophila (Rieckhof et al., 1997. Cell 91, 171-183; Kurrant et al., 1998. Development 125, 1037-1048; Pai et al., 1998. Genes Dev. 12, 435-446), nuclear localized PBX proteins were found to correlate highly with Meis expression. In addition, DLX proteins co-localize with nuclear PBX in distinct regions of the ventral telencephalon. (C) 2000 Elsevier Science Ireland Ltd. (C) 2000 Elsevier Science Ireland Ltd

Dynamic distribution of endoplasmic reticulum in hippocampal neuron dendritic spines

The role of the endoplasmic reticulum (ER) localized in dendritic spines has become a subject of intense interest because of its potential functions in local protein synthesis and signal transduction. Although it is recognized from electron microscopic studies that not all spines contain ER, little is know of its dynamic regulation or turnover. Here, we report a surprising degree of turnover of ER within spines. Using confocal microscopy imaging we observed continuity of spine-ER with dendritic ER in hippocampal primary neurons. Over 24 h, less than 50% of spine ER was stable. Despite this high degree of turn over, we identified a significant subset of spines that maintained ER for at least 4 days. These results indicate that within a single neuron, the organelle composition of a spine is unexpectedly dynamic and may explain aspects of the spine-to-spine variation in calcium spike magnitude and localized protein synthesis and trafficking

Fission and Fusion of the Neuronal Endoplasmic Reticulum

The endoplasmic reticulum (ER) is central for protein synthesis and is the largest intracellular Ca2+ store in neurons. The neuronal ER is classically described to have a continuous lumen spanning all cellular compartments. This allows neuronal ER to integrate spatially separate events in the cell. Recent in vitro as well as in vivo findings, however, demonstrate that the neuronal ER is a structurally dynamic entity, capable of rapid fragmentation, i.e., ER fission. The ER fragments can fuse back together and reinstate ER continuity. This reversible phenomenon can be induced repeatedly within the same cell, is temperature-dependent, and compatible with cell survival. The key trigger for dendritic ER fission is N-methyl D-aspartate (NMDA) receptor stimulation in the presence of extracellular Ca2+. However, the exact molecular machinery responsible for the fission and fusion of neuronal ER remains unknown. Reversible ER fission represents a new cell biological event downstream of NMDA receptor-gated Ca2+ influx and may thus influence many aspects of neuronal function in physiology and disease. Hence, it constitutes a new field for exploration in neuroscience that will benefit greatly from recent advances in light microscopy imaging techniques allowing dynamic characterization of cellular events in vitro and in vivo

Identification of two distinct progenitor populations in the lateral ganglionic eminence: Implications for striatal and olfactory bulb neurogenesis

The lateral ganglionic eminence (LGE) is known to give rise to striatal projection neurons as well as interneurons, which migrate in the rostral migratory stream (RMS) to populate the granule cell and glomerular layers of the olfactory bulb. Because all of these neuronal subtypes express Distalless-related (DLX) homeobox proteins during their differentiation, we set out to further characterize progenitors in the Dlx-positive domain of the LGE. Previous studies have shown that the LIM homeobox protein Islet1 (ISL1) marks the LGE subventricular zone (SVZ) and differentiating striatal projection neurons. However, ISL1 is not expressed in neurons of the developing olfactory bulb or the RMS. We show here that the dorsal-most portion of the Dlx-expressing region of the LGE SVZ lacks ISL1 cells. This dorsal domain, however, contains cells that express the ETS transcription factor Er81, which is also expressed in granule and periglomerular cells of the developing and adult olfactory bulb. Moreover, the adult SVZ and RMS contain numerous Er81-positive cells. Fate-mapping studies using Dlx5/6-cre transgenic mice demonstrate that Er81-positive cells in the granule cell and glomerular layers of the olfactory bulb derive from the Dlx-expressing SVZ region. These findings suggest that the LGE SVZ contains two distinct progenitor populations: a DLX+;ISL1(+) population representing striatal progenitors and a DLX+;Er81(+) population comprising olfactory bulb interneuron progenitors. In support of this, mice mutant for the homeobox genes Gsh2 and Gsh1/2, which show olfactory bulb defects, exhibit dramatically reduced numbers of Er81-positive cells in the LGE SVZ as well as in the olfactory bulb mantle

Potassium-induced structural changes of the endoplasmic reticulum in pyramidal neurons in murine organotypic hippocampal slices.

The endoplasmic reticulum (ER) structure is of central importance for the regulation of cellular anabolism, stress response, and signal transduction. Generally continuous, the ER can temporarily undergo dramatic structural rearrangements resulting in a fragmented appearance. In this study we assess the dynamic nature of ER fission in pyramidal neurons in organotypic hippocampal slice cultures stimulated by depolarizing concentration of potassium (50 mM). The slices were obtained from transgenic mice expressing fluorescent ER-targeted DsRed2 protein. We employed live tissue confocal microscopy imaging with fluorescence recovery after photobleaching (FRAP) to monitor the extent of structural rearrangements of the ER. In control slices, the ER structure was continuous. Potassium stimulation resulted in extensive fragmentation (fission), whereas return to basal potassium levels (2.5 mM) led to ER fusion and normalization of ER structure. This ER fission/fusion could be repeated several times in the same neuron, demonstrating the reversibility of the process. Blockade of the N-methyl-D-aspartate receptor (NMDAR) with the antagonist D-AP5 or removal of extracellular Ca(2+) prevented depolarization-induced ER fission. ER fission is sensitive to temperature, and decreasing temperature from 35°C to 30°C augments fission, implying that the altering of ER continuity may be a protective response against damage. We conclude that events that generate membrane depolarisation in brain tissue lead to the release of endogenous glutamate that may regulate neuronal ER continuity. The rapid and reversible NMDAR-mediated changes in ER structure reflect an adaptive, innate property of the ER for synaptic activation as well as response to tissue stress, injury, and disease. © 2011 Wiley-Liss, Inc