Heterogeneity in astrocyte responses after acute injury in vitro and in vivo

Astrocytes present a major population of glial cells in the adult mammalian brain. The heterogeneity of astrocytes in different regions of the healthy central nervous system (CNS) and their physiological functions are well understood. In contrast, rather little is known about the diversity of astrocyte reactions under pathological conditions. After CNS injury the reaction of astrocytes, also termed ‘reactive astrogliosis’, is characterized by morphological and molecular changes such as hypertrophy, polarization, migration and up-regulation of intermediate filaments. So far, it was unknown whether all astrocytes undergo these changes, or whether only specific subpopulations of reactive astrocytes possess special plasticity. Since some quiescent, postmitotic astrocytes in the cortical gray matter apparently de-differentiate and re-enter the cell cycle upon injury, reactive astrocytes have the ability to acquire restrictive stem cell potential. However, the mechanisms leading to increased astrocyte numbers after acute injury, e.g. proliferation and migration, had not been investigated live in vivo. For the first time, recently established in vivo imaging using 2-photon laser scanning microscopy (2pLSM) allowed to follow single GFP-labeled astrocytes for days and weeks after cortical stab wound injury. Tracing morphological changes during the transition from a quiescent to reactive state, these live observations revealed a heterogeneous behavior of reactive astrocytes depending on the lesion size. Different subsets of astrocytes either became hypertrophic, polarized and/ or divided, but never migrated towards the injury. Intriguingly, the lack of astrocyte migration was not only contradictory to what had been predicted based on in vitro and in situ studies, but was also in stark contrast to the motility of other glial cells. Additionally, live imaging provided first evidence that only a small subset of reactive astrocytes in juxtavascular positions re-gains proliferative capacity after injury. While astrocyte proliferation was affected by conditional deletion of RhoGTPase Cdc42 – a key regulator of cell polarity –, the vascular niche was preserved, indicating that juxtavascular astrocytes are uniquely suited for proliferation after injury. Following the behavior of cdc42-deficient astrocytes by live imaging using an in vitro scratch wound assay, cell-autonomous effects including disturbed polarity and impaired directional migration confirmed a crucial role of Cdc42 signaling in reactive astrocytes after acute injury in vitro and in vivo. These novel insights revise current concepts of reactive astrocytes involved in glial scar formation by assigning regenerative potential to a minor pool of proliferative, juxtavascular astrocytes, and suggesting specific functions of different astrocyte subsets after CNS trauma.Astrozyten bilden die größte Gruppe von Gliazellen im Gehirn erwachsener Säugetiere. Die Heterogenität von Astrozyten in verschiedenen Regionen des zentralen Nervensystems (ZNS), sowie deren physiologischen Funktionen sind relativ gut untersucht. Hingegen ist die Diversität der astrozytären Reaktion unter pathologischen Bedingungen bis jetzt wenig verstanden. In Folge einer Verletzung des ZNS reagieren Astrozyten mit bestimmten molekularen und morphologischen Veränderungen wie Hypertrophie, Polarisierung, Wanderung und Hochregulation bestimmter Intermediärfilamente. Diese Veränderungen werden insgesamt als „reaktive Gliose“ zusammengefasst. Darüber hinaus scheinen nach akuten Gehirnverletzungen einige reife, postmitotische Astrozyten in der Großhirnrinde zu de-differenzieren, in den Zellzyklus einzutreten und begrenzt Stammzelleigenschaften zu erlangen. Es ist bisher nicht bekannt, ob alle Astrozyten auf Verletzungen reagieren, oder ob Teilpopulationen verschiedener Plastizität existieren. Weiterhin sind die Mechanismen, die zum Anstieg von Astrozyten nach akuter Verletzung führen, z.B. Proliferation und Wanderung, in vivo bislang nicht verstanden. Deshalb wurde hier erstmals das Verhalten von Fluoreszenz-markierten Astrozyten im Gehirn der Maus nach akuter kortikaler Verletzung live über einen längeren Zeitraum mittels 2-Photonenmikroskopie untersucht. Die Beobachtungen zeigten morphologische Veränderungen und heterogene Verhaltensmuster reaktiver Astrozyten, d.h. hypertrophe, polarisierende und sich teilende Astrozyten in Abhängigkeit von der Läsionsgröße. Im Gegensatz zu in vitro und in situ Studien, sowie bekannter Motilität anderer Typen von Gliazellen, wurde die Wanderung von Astrozyten zum Ort der Verletzung in vivo nicht beobachtet. Allerdings wurde entdeckt, dass sich nur eine kleine Teilpopulation von Astrozyten teilt, und diese vorrangig in direktem Kontakt mit Blutgefäßen (juxtavaskulär) liegt. Selbst nach Verlust der RhoGTPase Cdc42 – einem Schlüsselfaktor für Zellpolarität –, der zu einem Proliferationsdefekt der Astrozyten führte, blieb die vaskuläre Nische erhalten. In einem in vitro Verletzungsmodell zeigten cdc42-defizienten Astrozyten Polaritätsdefekte, verbunden mit desorientierter Wanderung und verminderter Zellteilung. Schlussfolgernd, spielen Cdc42-vermittelte Signalwege eine wichtige Rolle für die Reaktion von Astrozyten auf eine akute Verletzung in vitro und in vivo. Die hier präsentierte Studie trägt bedeutend zum Verständnis reaktiver Astrozyten in Bezug auf deren Rolle in der Narbenbildung und Regeneration von geschädigtem Hirngewebe bei. Insbesondere wurden neue Erkenntnisse über verschiedene Teilpopulationen von Astrozyten mit vermutlich unterschiedlichen Funktionen gewonnen. Hierbei konnte vor allem den juxtavaskulär proliferierenden Astrozyten nach einer traumatischen Hirnverletzung hohe Plastizität zugesprochen werden

z-stack of the postcentral gyrus of the mouse brain in vivo.

<p>(A)–(E) show blood vessels (arrows), stained by Texas Red 70 kDa Dextran in 5, 60, 120, 200 and 300 µm underneath the brain surface. Internal detectors were used with maintained settings for gain and offset. a, left column: Without laser adjustment all blood vessels within the 300 µm range were visible, but appeared darker and blurry below a depth of 200 µm. b, right column: the same region acquired with adjustment of the laser power. In contrast to the liver and kidney a much deeper penetration into the tissue was possible.</p

Depth of glomeruli in 10-week-old adult mice.

<p>Box-plot shows mean±25% (boxes) and the 5–95% percentile (whiskers). (A) Box-plot of all ten strains arranged according to the 25% percentile. (B) Box-plot of the peer group (129, CB6F1, C3H/N, NMRI, SJL, DBA/2, FVB, CD-1) vs. Bl/6 and BALB/c.</p

Depth of glomeruli in 4-week-old juvenile mice.

<p>Box-plot shows mean±25% (boxes) and the 5–95% percentile (whiskers). (A) Box-plot of all ten strains arranged according to the 25% percentile. (B) Box-plot of the peer group (129, CB6F1, NMRI, SJL, FVB, CD-1) vs. Bl/6, BALB/c, C3H/N and DBA/2.</p

MPM imaging conditions in a kidney of a MWF rat in vivo.

<p>(A) Image of a superficial glomerulus (g) and a distal tubule (dt) in the kidney of a MWF rat. Proximal tubules (pt) showed green autofluorescence. Intra- and extraglomerular capillaries (arrows) were visualized with Texas Red-labeled 70 kDa dextran. The glomerulus was located directly underneath the renal capsule allowing high resolution imaging. (B) Histology of a MWF rat kidney section with many superficial glomeruli (arrows).</p

Microglial roles in Alzheimer's disease: An agent‐based model to elucidate microglial spatiotemporal response to beta‐amyloid

Abstract Alzheimer's disease (AD) is characterized by beta‐amyloid (Aβ) plaques in the brain and widespread neuronal damage. Because of the high drug attrition rates in AD, there is increased interest in characterizing neuroimmune responses to Aβ plaques. In response to AD pathology, microglia are innate phagocytotic immune cells that transition into a neuroprotective state and form barriers around plaques. We seek to understand the role of microglia in modifying Aβ dynamics and barrier formation. To quantify the influence of individual microglia behaviors (activation, chemotaxis, phagocytosis, and proliferation) on plaque size and barrier coverage, we developed an agent‐based model to characterize the spatiotemporal interactions between microglia and Aβ. Our model qualitatively reproduces mouse data trends where the fraction of microglia coverage decreases as plaques become larger. In our model, the time to microglial arrival at the plaque boundary is significantly negatively correlated (p < 0.0001) with plaque size, indicating the importance of the time to microglial activation for regulating plaque size. In addition, in silico behavioral knockout simulations show that phagocytosis knockouts have the strongest impact on plaque size, but modest impacts on microglial coverage and activation. In contrast, the chemotaxis knockouts had a strong impact on microglial coverage with a more modest impact on plaque volume and microglial activation. These simulations suggest that phagocytosis, chemotaxis, and replication of activated microglia have complex impacts on plaque volume and coverage, whereas microglial activation remains fairly robust to perturbations of these functions. Thus, our work provides insights into the potential and limitations of targeting microglial activation as a pharmacological strategy for the treatment of AD

Superficial Nephrons in BALB/c and C57BL/6 Mice Facilitate In Vivo Multiphoton Microscopy of the Kidney

Multiphoton microscopy (MPM) offers a unique approach for addressing both the function and structure of an organ in near-real time in the live animal. The method however is limited by the tissue-specific penetration depth of the excitation laser. In the kidney, structures in the range of 100 µm from the surface are accessible for MPM. This limitation of MPM aggravates the investigation of the function of structures located deeper in the renal cortex, like the glomerulus and the juxtaglomerular apparatus. In view of the relevance of gene-targeted mice for investigating the function of these structures, we aimed to identify a mouse strain with a high percentage of superficially located glomeruli. The mean distance of the 30 most superficial glomeruli from the kidney surface was determined in 10 commonly used mouse strains. The mean depth of glomeruli was 118.4±3.4, 123.0±2.7, 133.7±3.0, 132.3±2.6, 141.0±4.0, 145.3±4.3, 148.9±4.2, 151.6±2.7, 167.7±3.9, and 207.8±3.2 µm in kidney sections from 4-week-old C3H/HeN, BALB/cAnN, SJL/J, C57BL/6N, DBA/2N, CD1 (CRI), 129S2/SvPas, CB6F1, FVB/N and NMRI (Han) mice, respectively (n = 5 animals from each strain). The mean distance from the kidney surface of the most superficial glomeruli was significantly lower in the strains C3H/HeN Crl, BALB/cAnN, DBA/2NCrl, and C57BL/6N when compared to a peer group consisting of all the other strains (p<.0001). In 10-week-old mice, the most superficial glomeruli were located deeper in the cortex when compared to 4-week-old animals, with BALB/cAnN and C57BL/6N being the strains with the highest percentage of superficial glomeruli (25% percentile 116.7 and 121.9 µm, respectively). In summary, due to significantly more superficial glomeruli compared to other commonly used strains, BALB/cAnN and C57BL/6N mice appear to be particularly suitable for the investigation of glomerular function using MPM

Microglial Gi-dependent dynamics regulate brain network hyperexcitability.

Microglial surveillance is a key feature of brain physiology and disease. Here, we found that Gi-dependent microglial dynamics prevent neuronal network hyperexcitability. By generating MgPTX mice to genetically inhibit Gi in microglia, we show that sustained reduction of microglia brain surveillance and directed process motility induced spontaneous seizures and increased hypersynchrony after physiologically evoked neuronal activity in awake adult mice. Thus, Gi-dependent microglia dynamics may prevent hyperexcitability in neurological diseases

Microglial Gi-dependent dynamics regulate brain network hyperexcitability.

Microglial surveillance is a key feature of brain physiology and disease. Here, we found that Gi-dependent microglial dynamics prevent neuronal network hyperexcitability. By generating MgPTX mice to genetically inhibit Gi in microglia, we show that sustained reduction of microglia brain surveillance and directed process motility induced spontaneous seizures and increased hypersynchrony after physiologically evoked neuronal activity in awake adult mice. Thus, Gi-dependent microglia dynamics may prevent hyperexcitability in neurological diseases

Fibrinogen Activates BMP Signaling in Oligodendrocyte Progenitor Cells and Inhibits Remyelination after Vascular Damage.

Blood-brain barrier (BBB) disruption alters the composition of the brain microenvironment by allowing blood proteins into the CNS. However, whether blood-derived molecules serve as extrinsic inhibitors of remyelination is unknown. Here we show that the coagulation factor fibrinogen activates the bone morphogenetic protein (BMP) signaling pathway in oligodendrocyte progenitor cells (OPCs) and suppresses remyelination. Fibrinogen induces phosphorylation of Smad 1/5/8 and inhibits OPC differentiation into myelinating oligodendrocytes (OLs) while promoting an astrocytic fate in&nbsp;vitro. Fibrinogen effects are rescued by BMP type I receptor inhibition using dorsomorphin homolog 1 (DMH1) or CRISPR/Cas9 activin A receptor type I (ACVR1) knockout in OPCs. Fibrinogen and the BMP target Id2 are increased in demyelinated multiple sclerosis (MS) lesions. Therapeutic depletion of fibrinogen decreases BMP signaling and enhances remyelination in&nbsp;vivo. Targeting fibrinogen may be an upstream therapeutic strategy to promote the regenerative potential of CNS progenitors in diseases with remyelination failure