Important shapeshifter: mechanisms allowing astrocytes to respond to the changing nervous system during development, injury and disease

Astrocytes are the most prevalent glial cells in the brain. Historically considered as "merely supporting" neurons, recent research has shown that astrocytes actively participate in a large variety of central nervous system (CNS) functions including synaptogenesis, neuronal transmission and synaptic plasticity. During disease and injury, astrocytes efficiently protect neurons by various means, notably by sealing them off from neurotoxic factors and repairing the blood-brain barrier. Their ramified morphology allows them to perform diverse tasks by interacting with synapses, blood vessels and other glial cells. In this review article, we provide an overview of how astrocytes acquire their complex morphology during development. We then move from the developing to the mature brain, and review current research on perisynaptic astrocytic processes, with a particular focus on how astrocytes engage synapses and modulate their formation and activity. Comprehensive changes have been reported in astrocyte cell shape in many CNS pathologies. Factors influencing these morphological changes are summarized in the context of brain pathologies, such as traumatic injury and degenerative conditions. We provide insight into the molecular, cellular and cytoskeletal machinery behind these shape changes which drive the dynamic remodeling in astrocyte morphology during injury and the development of pathologies

Analyzing the functional diversity of profilin1 and profilin 2a

Profiline sind kleine Aktin-bindende Proteine, die den Nukleotidaustausch der Aktin-Monomere beschleunigen und diese an die Plus-Enden der Aktin-Filamente befördern. Da diese Funktionen für die Aktin-Dynamik essentiell sind, ist die Existenz gewebsspezifischer Profilin-Isoformen weitgehend unverstanden. In dieser Arbeit wurde untersucht, ob sich das in Mammalia ubiquitäre Profilin 1 (PFN1) und das ZNS-spezifische Profilin 2a (PFN2a) redundant zueinander verhalten oder ob sie unterschiedliche Funktionen besitzen. Trotz ähnlicher biochemischer Eigenschaften lokalisieren PFN1 und PFN2a unterschiedlich in Neuronen: Während PFN1 überwiegend homogen in den Neuriten verteilt ist, akkumuliert PFN2a in synaptischen Strukturen. Zudem wurde in dieser Arbeit ein RNAi-System entwickelt, mit dem endogene Proteine gegen exogene Varianten substituiert werden können. Mit diesem „Knock down & Knock in“-System wurde der Einfluss von PFN2a auf organotypisch kultivierte CA1-Neurone untersucht. Der Verlust von PFN2a verringert die Anzahl von dendritischen Verzweigungen sowie von „dendritic spines“. Die Substitution des endogenen PFN2a mit einer Profilin-Mutante mit reduzierter Aktin-Bindung hat gezeigt, dass die dendritische Morphologie auf der Interaktion von PFN2a mit Aktin beruht. Hingegen bewirkt die Expression einer Poly-Prolin-Bindungsmutante von PFN2a eine normale dendritische Komplexität und eine signifikant erhöhte Anzahl der „dendritic spines”. Sequenzvergleiche ergaben, dass PFN2a höher konserviert ist als PFN1. Darüber hinaus belegten Expressionsanalysen von PFN1 und PFN2a, dass PFN2a im Huhn ubiquitär exprimiert wird. RNAi-Experimente haben zudem gezeigt, dass in Hühnerfibroblasten vornehmlich PFN2a anstelle von PFN1 die Zelladhäsion und Zellmotilität moduliert. Die Ergebnisse dieser Arbeit bestätigen, dass PFN2a neuro-spezifische Funktionen ausübt. Andererseits weisen die Befunde an Hühnerzellen daraufhin, dass PFN2a prinzipiell auch als Aktin-Regulator fungieren kann.Profilins are small actin-binding proteins that accelerate the nucleotide-exchange of G-actin and deliver it to plus-ends of actin-filaments. According to the substantial contribution of profilin to actin dynamics, the functions of tissue-specific profilin-isoforms are poorly understood. Here, the functional diversity of the in Mammals ubiquitously expressed profilin 1 (PFN1) and the CNS-restricted profilin 2a (PFN2a) were studied. Although possessing similar biochemical properties, PFN1 and PFN2a exhibit differential localizations in neurons. Whereas PFN1 is homogenously distributed, PFN2a accumulates at synaptic structures. Furthermore, a RNAi-system were also developed that enables the simultaneous depletion of endogenous target proteins and the expression of exogenous variants. This so-called ôKnock down & knock inö-system were utilized to study the functions of PFN2a in organotypic-cultivated CA1-neurons. RNAi-mediated gene silencing of PFN2a leads to loss of dendritic arborizations and reduces significantly the number of dendritic spines. The substitution of endogenous PFN2a against a mutant deficient in actin binding demonstrates the modulation of dendritic morphology by the direct interaction of PFN2a and G-actin. However, the expression of PFN2a with disrupted poly-proline binding site restores dendritic complexity and increases significantly the density of dendritic spines in CA1-neurons. Phylogenetic analysis revealed that PFN2a is better conserved among vertebrates than PFN1. However, analysis of expression pattern showed that PFN2a is ubiquitously expressed in chicken tissues. Additionally, knock down experiments in chicken fibroblasts revealed the predominant involvement of PFN2a in processes of actin-based motility. In summary, the results of this work demonstrate specific functions of PFN2a in the mammalian CNS, whereas experiments in chicken show that PFN2a can principally serve as regulator of actin dynamics in non-neuronal cells

Protein interacting with C kinase 1 suppresses invasion and anchorage independent growth of astrocytic tumour cells

Astrocytic tumors are the most common form of primary brain tumor. Astrocytic tumor cells infiltrate the surrounding CNS tissue, allowing them to evade removal upon surgical resection of the primary tumor. Dynamic changes to the actin cytoskeleton are crucial to cancer cell invasion, but the specific mechanisms that underlie the particularly invasive phenotype of astrocytic tumor cells are unclear. Protein interacting with C kinase 1 (PICK1) is a PDZ and BAR domain–containing protein that inhibits actin-related protein 2/3 (Arp2/3)-dependent actin polymerization and is involved in regulating the trafficking of a number of cell-surface receptors. Here we report that, in contrast to other cancers, PICK1 expression is down-regulated in grade IV astrocytic tumor cell lines and also in clinical cases of the disease in which grade IV tumors have progressed from lower-grade tumors. Exogenous expression of PICK1 in the grade IV astrocytic cell line U251 reduces their capacity for anchorage-independent growth, two-dimensional migration, and invasion through a three-dimensional matrix, strongly suggesting that low PICK1 expression plays an important role in astrocytic tumorigenesis. We propose that PICK1 negatively regulates neoplastic infiltration of astrocytic tumors and that manipulation of PICK1 is an attractive possibility for therapeutic intervention

Testis-expressed profilins 3 and 4 show distinct functional characteristics and localize in the acroplaxome-manchette complex in spermatids

Background Multiple profilin isoforms exist in mammals; at least four are expressed in the mammalian testis. The testis-specific isoforms profilin-3 (PFN3) and profilin-4 (PFN4) may have specialized roles in spermatogenic cells which are distinct from known functions fulfilled by the somatic profilins, profilin-1 (PFN1) and profilin-2 (PFN2). Results Ligand interactions and spatial distributions of PFN3 and PFN4 were compared by biochemical, molecular and immunological methods; PFN1 and PFN2 were employed as controls. β-actin, phosphoinositides, poly-L-proline and mDia3, but not VASP, were confirmed as in vitro interaction partners of PFN3. In parallel experiments, PFN4 bound to selected phosphoinositides but not to poly-L-proline, proline-rich proteins, or actin. Immunofluorescence microscopy of PFN3 and PFN4 revealed distinct subcellular locations in differentiating spermatids. Both were associated first with the acroplaxome and later with the transient manchette. Predicted 3D structures indicated that PFN3 has the actin-binding site conserved, but retains only approximately half of the common poly-L-proline binding site. PFN4, in comparison, has lost both, polyproline and actin binding sites completely, which is well in line with the experimental data. Conclusion The testis-specific isoform PFN3 showed major hallmarks of the well characterized somatic profilin isoforms, albeit with distinct binding affinities. PFN4, on the other hand, did not interact with actin or polyproline in vitro. Rather, it seemed to be specialized for phospholipid binding, possibly providing cellular functions which are distinct from actin dynamics regulation

Drebrin controls scar formation and astrocyte reactivity upon traumatic brain injury by regulating membrane trafficking

The brain of mammals lacks a significant ability to regenerate neurons and is thus particularly vulnerable. To protect the brain from injury and disease, damage control by astrocytes through astrogliosis and scar formation is vital. Here, we show that brain injury in mice triggers an immediate upregulation of the actin-binding protein Drebrin (DBN) in astrocytes, which is essential for scar formation and maintenance of astrocyte reactivity. In turn, DBN loss leads to defective astrocyte scar formation and excessive neurodegeneration following brain injuries. At the cellular level, we show that DBN switches actin homeostasis from ARP2/3-dependent arrays to microtubule-compatible scaffolds, facilitating the formation of RAB8-positive membrane tubules. This injury-specific RAB8 membrane compartment serves as hub for the trafficking of surface proteins involved in astrogliosis and adhesion mediators, such as β1-integrin. Our work shows that DBN-mediated membrane trafficking in astrocytes is an important neuroprotective mechanism following traumatic brain injury in mice

The antagonistic modulation of Arp2/3 activity by N-WASP/WAVE2 and PICK1 defines dynamic changes in astrocyte morphology

Astrocytes exhibit a complex, branched morphology, allowing them to functionally interact with numerous blood vessels, neighboring glial processes and neuronal elements, including synapses. They also respond to central nervous system (CNS) injury by a process known as astrogliosis, which involves morphological changes, including cell body hypertrophy and thickening of major processes. Following severe injury, astrocytes exhibit drastically reduced morphological complexity and collectively form a glial scar. The mechanistic details behind these morphological changes are unknown. Here, we investigate the regulation of the actin-nucleating Arp2/3 complex in controlling dynamic changes in astrocyte morphology. In contrast to other cell types, Arp2/3 inhibition drives the rapid expansion of astrocyte cell bodies and major processes. This intervention results in a reduced morphological complexity of astrocytes in both dissociated culture and in brain slices. We show that this expansion requires functional myosin II downstream of ROCK and RhoA. Knockdown of the Arp2/3 subunit Arp3 or the Arp2/3 activator N-WASP by siRNA also results in cell body expansion and reduced morphological complexity, whereas depleting WAVE2 specifically reduces the branching complexity of astrocyte processes. By contrast, knockdown of the Arp2/3 inhibitor PICK1 increases astrocyte branching complexity. Furthermore, astrocyte expansion induced by ischemic conditions is delayed by PICK1 knockdown or N-WASP overexpression. Our findings identify a new morphological outcome for Arp2/3 activation in restricting rather than promoting outwards movement of the plasma membrane in astrocytes. The Arp2/3 regulators PICK1, and N-WASP and WAVE2 function antagonistically to control the complexity of astrocyte branched morphology, and this mechanism underlies the morphological changes seen in astrocytes during their response to pathological insult

The Small GTPase Arf1 Modulates Arp2/3-Mediated Actin Polymerization via PICK1 to Regulate Synaptic Plasticity

SummaryInhibition of Arp2/3-mediated actin polymerization by PICK1 is a central mechanism to AMPA receptor (AMPAR) internalization and long-term depression (LTD), although the signaling pathways that modulate this process in response to NMDA receptor (NMDAR) activation are unknown. Here, we define a function for the GTPase Arf1 in this process. We show that Arf1-GTP binds PICK1 to limit PICK1-mediated inhibition of Arp2/3 activity. Expression of mutant Arf1 that does not bind PICK1 leads to reduced surface levels of GluA2-containing AMPARs and smaller spines in hippocampal neurons, which occludes subsequent NMDA-induced AMPAR internalization and spine shrinkage. In organotypic slices, NMDAR-dependent LTD of AMPAR excitatory postsynaptic currents is abolished in neurons expressing mutant Arf1. Furthermore, NMDAR stimulation downregulates Arf1 activation and binding to PICK1 via the Arf-GAP GIT1. This study defines Arf1 as a critical regulator of actin dynamics and synaptic function via modulation of PICK1

Neuronal Profilin Isoforms Are Addressed by Different Signalling Pathways

Profilins are prominent regulators of actin dynamics. While most mammalian cells express only one profilin, two isoforms, PFN1 and PFN2a are present in the CNS. To challenge the hypothesis that the expression of two profilin isoforms is linked to the complex shape of neurons and to the activity-dependent structural plasticity, we analysed how PFN1 and PFN2a respond to changes of neuronal activity. Simultaneous labelling of rodent embryonic neurons with isoform-specific monoclonal antibodies revealed both isoforms in the same synapse. Immunoelectron microscopy on brain sections demonstrated both profilins in synapses of the mature rodent cortex, hippocampus and cerebellum. Both isoforms were significantly more abundant in postsynaptic than in presynaptic structures. Immunofluorescence showed PFN2a associated with gephyrin clusters of the postsynaptic active zone in inhibitory synapses of embryonic neurons. When cultures were stimulated in order to change their activity level, active synapses that were identified by the uptake of synaptotagmin antibodies, displayed significantly higher amounts of both isoforms than non-stimulated controls. Specific inhibition of NMDA receptors by the antagonist APV in cultured rat hippocampal neurons resulted in a decrease of PFN2a but left PFN1 unaffected. Stimulation by the brain derived neurotrophic factor (BDNF), on the other hand, led to a significant increase in both synaptic PFN1 and PFN2a. Analogous results were obtained for neuronal nuclei: both isoforms were localized in the same nucleus, and their levels rose significantly in response to KCl stimulation, whereas BDNF caused here a higher increase in PFN1 than in PFN2a. Our results strongly support the notion of an isoform specific role for profilins as regulators of actin dynamics in different signalling pathways, in excitatory as well as in inhibitory synapses. Furthermore, they suggest a functional role for both profilins in neuronal nuclei

Profilin Isoforms in Health and Disease – All the Same but Different

Profilins are small actin binding proteins, which are structurally conserved throughout evolution. They are probably best known to promote and direct actin polymerization. However, they also participate in numerous cell biological processes beyond the roles typically ascribed to the actin cytoskeleton. Moreover, most complex organisms express several profilin isoforms. Their cellular functions are far from being understood, whereas a growing number of publications indicate that profilin isoforms are involved in the pathogenesis of various diseases. In this review, we will provide an overview of the profilin family and "typical" profilin properties including the control of actin dynamics. We will then discuss the profilin isoforms of higher animals in detail. In terms of cellular functions, we will focus on the role of Profilin 1 (PFN1) and Profilin 2a (PFN2a), which are co-expressed in the central nervous system. Finally, we will discuss recent findings that link PFN1 and PFN2a to neurological diseases, such as amyotrophic lateral sclerosis (ALS), Fragile X syndrome (FXS), Huntington's disease and spinal muscular atrophy (SMA)

Testis-expressed profilins 3 and 4 show distinct functional characteristics and localize in the acroplaxome-manchette complex in spermatids

Abstract Background Multiple profilin isoforms exist in mammals; at least four are expressed in the mammalian testis. The testis-specific isoforms profilin-3 (PFN3) and profilin-4 (PFN4) may have specialized roles in spermatogenic cells which are distinct from known functions fulfilled by the "somatic" profilins, profilin-1 (PFN1) and profilin-2 (PFN2). Results Ligand interactions and spatial distributions of PFN3 and PFN4 were compared by biochemical, molecular and immunological methods; PFN1 and PFN2 were employed as controls. β-actin, phosphoinositides, poly-L-proline and mDia3, but not VASP, were confirmed as in vitro interaction partners of PFN3. In parallel experiments, PFN4 bound to selected phosphoinositides but not to poly-L-proline, proline-rich proteins, or actin. Immunofluorescence microscopy of PFN3 and PFN4 revealed distinct subcellular locations in differentiating spermatids. Both were associated first with the acroplaxome and later with the transient manchette. Predicted 3D structures indicated that PFN3 has the actin-binding site conserved, but retains only approximately half of the common poly-L-proline binding site. PFN4, in comparison, has lost both, polyproline and actin binding sites completely, which is well in line with the experimental data. Conclusion The testis-specific isoform PFN3 showed major hallmarks of the well characterized "somatic" profilin isoforms, albeit with distinct binding affinities. PFN4, on the other hand, did not interact with actin or polyproline in vitro. Rather, it seemed to be specialized for phospholipid binding, possibly providing cellular functions which are distinct from actin dynamics regulation.</p