live-imaging-followed-single-cell-tracking-to-monitor-cell-biology

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

The adult mouse subependymal zone (SEZ) harbors neural stem cells that are thought to exclusively generate GABAergic interneurons of the olfactory bulb. We examined the adult generation of glutamatergic juxtaglomerular neurons, which had dendritic arborizations that projected into adjacent glomeruli, identifying them as short-axon cells. Fate mapping revealed that these originate from Neurog2- and Tbr2-expressing progenitors located in the dorsal region of the SEZ. Examination of the progenitors of these glutamatergic interneurons allowed us to determine the sequential expression of transcription factors in these cells that are thought to be hallmarks of glutamatergic neurogenesis in the developing cerebral cortex and adult hippocampus. Indeed, the molecular specification of these SEZ progenitors allowed for their recruitment into the cerebral cortex after a lesion was induced. Taken together, our data indicate that SEZ progenitors not only produce a population of adult-born glutamatergic juxtaglomerular neurons, but may also provide a previously unknown source of progenitors for endogenous repair.

Adult generation of glutamatergic olfactory bulb interneurons.

Die adulten Maus Subependymal Zone (SEZ) birgt neuralen Stammzellen, der man ausschlie&szlig;lich GABAergic Interneuronen der Riechkolben generieren. Wir untersucht die Erwachsene Generation Glutamat Juxtaglomerul&auml;rer Neuronen, die dendritische Arborizations, die in angrenzenden Glomerula projiziert hatte, sie als kurz-Axon Zellen identifiziert. Schicksal Zuordnung ergab, dass diese vom Neurog2 und Tbr2-Ausdruck Vorl&auml;uferzellen befindet sich in der dorsalen Region das SEZ stammen. Pr&uuml;fung der die Vorl&auml;ufer der diese Glutamat-Interneuronen konnten wir den sequenziellen Ausdruck der Transkriptionsfaktoren in diesen Zellen zu bestimmen, die als Markenzeichen der Neurogenese Glutamat in den Entwicklungsl&auml;ndern Hirnrinde und Erwachsenen Hippocampus gelten. In der Tat die molekulare Spezifikation der diese SEZ-Vorl&auml;uferzellen zul&auml;ssig, f&uuml;r deren Einstellung in der Hirnrinde, nachdem eine L&auml;sion verursacht wurde. Zusammengenommen zeigen unsere Daten, dass SEZ Vorl&auml;uferzellen produzieren nicht nur eine Bev&ouml;lkerung von Erwachsenen-US-amerikanischer Glutamat Juxtaglomerul&auml;rer Neuronen, sondern k&ouml;nnen auch endogene Reparatur eine bisher unbekannte Quelle der Vorfahre vorsehen.

Erwachsene Generation von Glutamat Riechkolben Interneuronen.

Astroglia from the postnatal cerebral cortex can be reprogrammed in vitro to generate neurons following forced expression of neurogenic transcription factors, thus opening new avenues towards a potential use of endogenous astroglia for brain repair. However, in previous attempts astroglia-derived neurons failed to establish functional synapses, a severe limitation towards functional neurogenesis. It remained therefore also unknown whether neurons derived from reprogrammed astroglia could be directed towards distinct neuronal subtype identities by selective expression of distinct neurogenic fate determinants. Here we show that strong and persistent expression of neurogenic fate determinants driven by silencing-resistant retroviral vectors instructs astroglia from the postnatal cortex in vitro to mature into fully functional, synapse-forming neurons. Importantly, the neurotransmitter fate choice of astroglia-derived neurons can be controlled by selective expression of distinct neurogenic transcription factors: forced expression of the dorsal telencephalic fate determinant neurogenin-2 (Neurog2) directs cortical astroglia to generate synapse-forming glutamatergic neurons; in contrast, the ventral telencephalic fate determinant Dlx2 induces a GABAergic identity, although the overall efficiency of Dlx2-mediated neuronal reprogramming is much lower compared to Neurog2, suggesting that cortical astroglia possess a higher competence to respond to the dorsal telencephalic fate determinant. Interestingly, however, reprogramming of astroglia towards the generation of GABAergic neurons was greatly facilitated when the astroglial cells were first expanded as neurosphere cells prior to transduction with Dlx2. Importantly, this approach of expansion under neurosphere conditions and subsequent reprogramming with distinct neurogenic transcription factors can also be extended to reactive astroglia isolated from the adult injured cerebral cortex, allowing for the selective generation of glutamatergic or GABAergic neurons. These data provide evidence that cortical astroglia can undergo a conversion across cell lineages by forced expression of a single neurogenic transcription factor, stably generating fully differentiated neurons. Moreover, neuronal reprogramming of astroglia is not restricted to postnatal stages but can also be achieved from terminally differentiated astroglia of the adult cerebral cortex following injury-induced reactivation.

Directing astroglia from the cerebral cortex into subtype specific functional neurons.

Astroglia aus der postnatalen Hirnrinde kann in-vitro Neuronen erzeugen umprogrammiert werden folgende Ausdruck der neurogenen Transkriptionsfaktoren, damit er&ouml;ffnet neue Wege f&uuml;r eine m&ouml;gliche Verwendung von endogenen Astroglia f&uuml;r Gehirn Reparatur gezwungen. Allerdings versucht in fr&uuml;heren Astroglia abgeleitete Neuronen, die nicht funktionale Synapsen, eine schwere Einschr&auml;nkung gegen&uuml;ber funktionellen Neurogenese nachgewiesen. Es blieb daher auch unbekannt, ob Neuronen abgeleitet umprogrammiert Astroglia unterschiedliche neuronale Untertyp Identit&auml;ten durch selektive Ausdruck unterschiedliche neurogene Schicksal Determinanten zugeleitet werden k&ouml;nnte. Hier zeigen wir, dass starke und best&auml;ndige Ausdruck neurogene Schicksal Determinanten getrieben Unterdr&uuml;ckender-resistenten retrovirale Vektoren weist Astroglia aus der postnatalen Kortex in-vitro ausreifen in voll funktionsf&auml;hige, Synapse-bildenden Neuronen. Wichtig ist, die Neurotransmitter-Schicksal-Wahl von Astroglia abgeleitete Neuronen gesteuert werden durch selektive Ausdruck der unterschiedlichen neurogene Transkriptionsfaktoren: gezwungen Ausdruck des dorsalen Teleenzephalischer Schicksal bestimmende Neurogenin-2 (Neurog2) leitet die kortikale Astroglia zum Generieren von Synapse-bildenden Glutamat Nervenzellen; im Gegensatz dazu induziert die Determinante der ventralen Teleenzephalischer Schicksal Dlx2 eine GABAergic Identit&auml;t, obwohl die Gesamteffizienz des Dlx2-vermittelte neuronale Neuprogrammierung viel niedriger im Vergleich zu Neurog2 ist, was darauf hindeutet, dass kortikale Astroglia besitzen eine h&ouml;here Kompetenz zur Bew&auml;ltigung der dorsalen Teleenzephalischer Schicksal bestimmende Faktor. Interessanterweise jedoch eine Anpassung der Astroglia gegen&uuml;ber der Generation GABAergic Neuronen wurde erheblich erleichtert, wenn die Astroglial-Zellen zun&auml;chst wichtiger ist als Neurosphere Zellen vor Transduktion mit Dlx2. erweitert wurden, diesen Ansatz der Expansion unter Neurosphere Bedingungen und nachfolgende Neuprogrammierung mit unterschiedlichen neurogene Transkriptionsfaktoren auch reaktive Astroglia isoliert von Erwachsenen verletzten Gro&szlig;hirnrinde, zulassend die selektive Erzeugung von Glutamat oder GABAergic Neuronen ausgezogen werden kann. Diese Daten belegen, dass kortikale Astroglia kann eine Konvertierung &uuml;ber Zelle &Uuml;bertragungslinien durch erzwungene Ausdruck des einzigen neurogene Transkriptionsfaktor unterziehen, stabil generieren vollst&auml;ndig differenzierten Neuronen. Dar&uuml;ber hinaus neuronale Neuprogrammierung der Astroglia beschr&auml;nkt sich nicht auf postnatale B&uuml;hnen, sondern auch von unheilbar differenzierte Astroglia der Erwachsenen Gro&szlig;hirnrinde nach Sch&auml;digung induziert Reaktivierung erreicht werden kann.

Regie Astroglia aus der Hirnrinde in Untertyp spezifische funktionelle Neuronen.

Instructing glial cells to generate neurons may prove to be a strategy to replace neurons that have degenerated. Here, we describe a robust protocol for the efficient in vitro conversion of postnatal astroglia from the mouse cerebral cortex into functional, synapse-forming neurons. This protocol involves two steps: (i) expansion of astroglial cells (7 d) and (ii) astroglia-to-neuron conversion induced by persistent and strong retroviral expression of Neurog2 (encoding neurogenin-2) or Mash1 (also referred to as achaete-scute complex homolog 1 or Ascl1) and/or distal-less homeobox 2 (Dlx2) for generation of glutamatergic or GABAergic neurons, respectively (7-21 d for different degrees of maturity). Our protocol of astroglia-to-neuron conversion by a single neurogenic transcription factor provides a stringent experimental system to study the specification of a selective neuronal subtype, thus offering an alternative to the use of embryonic or neural stem cells. Moreover, it can be a useful model for studies of lineage conversion from non-neuronal cells, with potential for brain regenerative medicine.

Generation of subtype-specific neurons from postnatal astroglia of the mouse cerebral cortex.

Instruktion Gliazellen Neuronen erzeugen k&ouml;nnte sich als eine Strategie, um Neuronen zu ersetzen, die entartet sind. Hier beschreiben wir eine robuste Protokoll f&uuml;r effiziente in-vitro-Umwandlung der postnatalen Astroglia aus die Maus-Hirnrinde in funktionelle, Synapse-bildenden Neuronen. Dieses Protokoll besteht aus zwei Schritten: (i) Ausbau der Astroglial Zellen (7D) und (Ii) Astroglia Neuron Konvertierung induziert durch anhaltende und starke retrovirale Ausdruck des Neurog2 (Neurogenin-2-Codierung) oder Mash1 (auch ASCL2-Scute komplizierte Homolog 1 oder Ascl1 genannt) und/oder distalen weniger Homeobox 2 (Dlx2) f&uuml;r die Erzeugung von Glutamat oder GABAergic Neuronen, bzw. (7-21 d f&uuml;r verschiedene Grade der Reife). Unser Protokoll Astroglia-Neuron-Konvertierung durch einen einzigen neurogene Transkriptionsfaktor bietet eine strenge experimentelle System um die Spezifikation der selektiven neuronale Subtyp, und bieten damit eine Alternative zur Verwendung von embryonalen oder neurale Stammzellen zu studieren. Es kann dar&uuml;ber hinaus ein n&uuml;tzliches Modell f&uuml;r Studien der Herkunfts-Konvertierung von nicht-neuronale Zellen, mit Potential f&uuml;r die regenerative Medizin Gehirn.

Generation der Subtyp-spezifische Neuronen aus postnatale Astroglia der Gro&szlig;hirnrinde Maus.

Reprogramming of somatic cells into neurons provides a new approach toward cell-based therapy of neurodegenerative diseases. A major challenge for the translation of neuronal reprogramming into therapy is whether the adult human brain contains cell populations amenable to direct somatic cell conversion. Here we show that cells from the adult human cerebral cortex expressing pericyte hallmarks can be reprogrammed into neuronal cells by retrovirus-mediated coexpression of the transcription factors Sox2 and Mash1. These induced neuronal cells acquire the ability of repetitive action potential firing and serve as synaptic targets for other neurons, indicating their capability of integrating into neural networks. Genetic fate-mapping in mice expressing an inducible Cre recombinase under the tissue-nonspecific alkaline phosphatase promoter corroborated the pericytic origin of the reprogrammed cells. Our results raise the possibility of functional conversion of endogenous cells in the adult human brain to induced neuronal fates.

Reprogramming of pericyte-derived cells of the adult human brain into induced neuronal cells.

The adult mouse subependymal zone (SEZ) harbours adult neural stem cells (aNSCs) that give rise to neuronal and oligodendroglial progeny. However it is not known whether the same aNSC can give rise to neuronal and oligodendroglial progeny or whether these distinct progenies constitute entirely separate lineages. Continuous live imaging and single-cell tracking of aNSCs and their progeny isolated from the mouse SEZ revealed that aNSCs exclusively generate oligodendroglia or neurons, but never both within a single lineage. Moreover, activation of canonical Wnt signalling selectively stimulated proliferation within the oligodendrogliogenic lineage, resulting in a massive increase in oligodendrogliogenesis without changing lineage choice or proliferation within neurogenic clones. In vivo activation or inhibition of canonical Wnt signalling respectively increased or decreased the number of Olig2 and PDGFR- α positive cells, suggesting that this pathway contributes to the fine tuning of oligodendrogliogenesis in the adult SEZ.

Oligodendrogliogenic and neurogenic adult subependymal zone neural stem cells constitute distinct lineages and exhibit differential responsiveness to Wnt signalling.

The adult cerebral cortex lacks the capacity to replace degenerated neurons following traumatic injury. Conversion of nonneuronal cells into induced neurons has been proposed as an innovative strategy toward brain repair. Here, we show that retrovirus-mediated expression of the transcription factors Sox2 and Ascl1, but strikingly also Sox2 alone, can induce the conversion of genetically fate-mapped NG2 glia into induced doublecortin (DCX)(+) neurons in the adult mouse cerebral cortex following stab wound injury in vivo. In contrast, lentiviral expression of Sox2 in the unlesioned cortex failed to convert oligodendroglial and astroglial cells into DCX(+) cells. Neurons induced following injury mature morphologically and some acquire NeuN while losing DCX. Patch-clamp recording of slices containing Sox2- and/or Ascl1-transduced cells revealed that a substantial fraction of these cells receive synaptic inputs from neurons neighboring the injury site. Thus, NG2 glia represent a potential target for reprogramming strategies toward cortical repair.

Sox2-mediated conversion of NG2 glia into induced neurons in the injured adult cerebral cortex.

Astrocytes react to brain injury in a heterogeneous manner with only a subset resuming proliferation and acquiring stem cell properties in vitro. In order to identify novel regulators of this subset, we performed genomewide expression analysis of reactive astrocytes isolated 5 days after stab wound injury from the gray matter of adult mouse cerebral cortex. The expression pattern was compared with astrocytes from intact cortex and adult neural stem cells (NSCs) isolated from the subependymal zone (SEZ). These comparisons revealed a set of genes expressed at higher levels in both endogenous NSCs and reactive astrocytes, including two lectins-Galectins 1 and 3. These results and the pattern of Galectin expression in the lesioned brain led us to examine the functional significance of these lectins in brains of mice lacking Galectins 1 and 3. Following stab wound injury, astrocyte reactivity including glial fibrillary acidic protein expression, proliferation and neurosphere-forming capacity were found significantly reduced in mutant animals. This phenotype could be recapitulated in vitro and was fully rescued by addition of Galectin 3, but not of Galectin 1. Thus, Galectins 1 and 3 play key roles in regulating the proliferative and NSC potential of a subset of reactive astrocytes.

Astrocyte reactivity after brain injury-: The role of galectins 1 and 3.

Reactive astrocytes (RAs) have been reported to convert to multipotent neural stem cells (NSCs) capable of neurosphere (NS) formation and multilineage differentiation in vitro. Using genetic tagging, we determined that subventricular zone (SVZ) NSCs give rise to NSs derived from the stroke-injured cortex. We demonstrate that these cells can be isolated from the cortex in two different models of stroke and from different stroke-lesioned cortical regions. Interestingly, SVZ NSCs give rise to a subpopulation of RAs in the cortex that contribute to astrogliosis and scar formation. Last, we show that these SVZ derived RAs can be converted to neurons in vivo by forced expression of Ascl1. Identifying the contribution of cells originating from the SVZ to injury repair has implications for neural regeneration strategies.

Adult Neural Stem Cells from the Subventricular Zone Give Rise to Reactive Astrocytes in the Cortex after Stroke.

Despite the widespread interest in direct neuronal reprogramming, the mechanisms underpinning fate conversion remain largely unknown. Our study revealed a critical time point after which cells either successfully convert into neurons or succumb to cell death. Co-transduction with Bcl-2 greatly improved negotiation of this critical point by faster neuronal differentiation. Surprisingly, mutants with reduced or no affinity for Bax demonstrated that Bcl-2 exerts this effect by an apoptosis-independent mechanism. Consistent with a caspase-independent role, ferroptosis inhibitors potently increased neuronal reprogramming by inhibiting lipid peroxidation occurring during fate conversion. Genome-wide expression analysis confirmed that treatments promoting neuronal reprogramming elicit an anti-oxidative stress response. Importantly, co-expression of Bcl-2 and anti-oxidative treatments leads to an unprecedented improvement in glial-to-neuron conversion after traumatic brain injury in vivo, underscoring the relevance of these pathways in cellular reprograming irrespective of cell type in vitro and in vivo.

Identification and Successful Negotiation of a Metabolic Checkpoint in Direct Neuronal Reprogramming.

The key signalling pathways and transcriptional programmes that instruct neuronal diversity during development have largely been identified. In this Review, we discuss how this knowledge has been used to successfully reprogramme various cell types into an amazing array of distinct types of functional neurons. We further discuss the extent to which direct neuronal reprogramming recapitulates embryonic development, and examine the particular barriers to reprogramming that may exist given a cell's unique developmental history. We conclude with a recently proposed model for cell specification called the 'Cook Islands' model, and consider whether it is a fitting model for cell specification based on recent results from the direct reprogramming field.

Direct neuronal reprogramming: learning from and for development.

The ability to directly reprogram mature cells to alternative fates challenges concepts of how cell identities are maintained, erased, and acquired. Recent advances in understanding and overcoming hurdles to direct neuronal conversion have provided new insights into mechanisms that maintain cell identity programs and have enabled high efficiency reprogramming in vivo. We discuss key cell-intrinsic molecular and metabolic constraints that influence the establishment of a new identity as well as environmental inputs from injured brains that favor or harm the conversion process. Finally, we outline the challenges ahead with a particular focus on direct neuronal reprogramming in vivo.

Direct Neuronal Reprogramming: Achievements, Hurdles, and New Roads to Success.

The low-density lipoprotein receptor-related protein 4 (LRP4) is critical in muscle fibers for the establishment of the neuromuscular junction. Here, we show that LRP4 is also expressed by embryonic cortical and hippocampal neurons, and that downregulation of LRP4 in these neurons reduced density of synapses and number of primary dendrites. Accordingly, overexpression of LRP4 in cultured neurons had the opposite effect inducing more but shorter primary dendrites with an increased number of spines. Transsynaptic tracing mediated by rabies virus revealed a reduced number of neurons presynaptic to cortical neurons in which LRP4 was knocked down. Moreover, neuron-specific knock down of LRP4 by in utero electroporation of LRP4 miRNA in vivo also resulted in neurons with fewer primary dendrites and a lower density of spines in the developing cortex and hippocampus. Collectively, our results demonstrate an essential and novel role of neuronal LRP4 in dendritic development and synaptogenesis in the CNS.

Article	Year
Adult generation of glutamatergic olfactory bulb interneurons. Nature neuroscience\| PubMed ID: 19881504	2009
Directing astroglia from the cerebral cortex into subtype specific functional neurons. PLoS biology\| PubMed ID: 20502524	2010
Generation of subtype-specific neurons from postnatal astroglia of the mouse cerebral cortex. Nature protocols\| PubMed ID: 21293461	2011
Reprogramming of pericyte-derived cells of the adult human brain into induced neuronal cells. Cell stem cell\| PubMed ID: 23040476	2012
Oligodendrogliogenic and neurogenic adult subependymal zone neural stem cells constitute distinct lineages and exhibit differential responsiveness to Wnt signalling. Nature cell biology\| PubMed ID: 23644466	2013
Sox2-mediated conversion of NG2 glia into induced neurons in the injured adult cerebral cortex. Stem cell reports\| PubMed ID: 25458895	2014
Astrocyte reactivity after brain injury-: The role of galectins 1 and 3. Glia\| PubMed ID: 26250529	2015
Adult Neural Stem Cells from the Subventricular Zone Give Rise to Reactive Astrocytes in the Cortex after Stroke. Cell stem cell\| PubMed ID: 26456685	2015
Identification and Successful Negotiation of a Metabolic Checkpoint in Direct Neuronal Reprogramming. Cell stem cell\| PubMed ID: 26748418	2016
Direct neuronal reprogramming: learning from and for development. Development (Cambridge, England)\| PubMed ID: 27436039	2016
Direct Neuronal Reprogramming: Achievements, Hurdles, and New Roads to Success. Cell stem cell\| PubMed ID: 28686866	2017
Neuronal LRP4 regulates synapse formation in the developing CNS. Development (Cambridge, England)\| PubMed ID: 29061639	2017

Sergio Gascon

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