Actin polymerization and longitudinal actin fibers in axon initial segment plasticity.

The location of the axon initial segment (AIS) at the junction between the soma and axon of neurons makes it instrumental in maintaining neural polarity and as the site for action potential generation. The AIS is also capable of large-scale relocation in an activity-dependent manner. This represents a form of homeostatic plasticity in which neurons regulate their own excitability by changing the size and/or position of the AIS. While AIS plasticity is important for proper functionality of AIS-containing neurons, the cellular and molecular mechanisms of AIS plasticity are poorly understood. Here, we analyzed changes in the AIS actin cytoskeleton during AIS plasticity using 3D structured illumination microscopy (3D-SIM). We showed that the number of longitudinal actin fibers increased transiently 3 h after plasticity induction. We further showed that actin polymerization, especially formin mediated actin polymerization, is required for AIS plasticity and formation of longitudinal actin fibers. From the formin family of proteins, Daam1 localized to the ends of longitudinal actin fibers. These results indicate that active re-organization of the actin cytoskeleton is required for proper AIS plasticity.

Gas7 Is a Novel Dendritic Spine Initiation Factor.

Brain stores new information by modifying connections between neurons. When new information is learnt, a group of neurons gets activated and they are connected to each other via synapses. Dendritic spines are protrusions along neuronal dendrites where excitatory synapses are located. Dendritic spines are the first structures to protrude out from the dendrite to reach out to other neurons and establish a new connection. Thus, it is expected that neuronal activity enhances spine initiation. However, the molecular mechanisms linking neuronal activity to spine initiation are poorly known. Membrane binding BAR domain proteins are involved in spine initiation, but it is not known whether neuronal activity affects BAR domain proteins. Here, we used bicuculline treatment to activate excitatory neurons in organotypic hippocampal slices. With this experimental setup, we identified F-BAR domain containing growth arrest-specific protein (Gas7) as a novel spine initiation factor responding to neuron activity. Upon bicuculline addition, Gas7 clustered to create spine initiation hotspots, thus increasing the probability to form new spines in activated neurons. Gas7 clustering and localization was dependent on PI3-kinase (PI3K) activity and intact F-BAR domain. Gas7 overexpression enhanced N-WASP localization to clusters as well as it increased the clustering of actin. Arp2/3 complex was required for normal Gas7-induced actin clustering. Gas7 overexpression increased and knock-down decreased spine density in hippocampal pyramidal neurons. Taken together, we suggest that Gas7 creates platforms under the dendritic plasma membrane which facilitate spine initiation. These platforms grow on neuronal activation, increasing the probability of making new spines and new connections between active neurons. As such, we identified a novel molecular mechanism to link neuronal activity to the formation of new connections between neurons.

Protrudin regulates FAK activation, endothelial cell migration and angiogenesis.

During angiogenesis, endothelial cells form protrusive sprouts and migrate towards the angiogenic stimulus. In this study, we investigate the role of the endoplasmic reticulum (ER)-anchored protein, Protrudin, in endothelial cell protrusion, migration and angiogenesis. Our results demonstrate that Protrudin regulates angiogenic tube formation in primary endothelial cells, Human umbilical vein endothelial cells (HUVECs). Analysis of RNA sequencing data and its experimental validation revealed cell migration as a prominent cellular function affected in HUVECs subjected to Protrudin knockdown. Further, our results demonstrate that knockdown of Protrudin inhibits focal adhesion kinase (FAK) activation in HUVECs and human aortic endothelial cells (HAECs). This is associated with a loss of polarized phospho-FAK distribution upon Protrudin knockdown as compared to Protrudin expressing HUVECs. Reduction of Protrudin also results in a perinuclear accumulation of mTOR and a decrease in VEGF-mediated S6K activation. However, further experiments suggest that the observed inhibition of angiogenesis in Protrudin knockdown cells is not affected by mTOR disturbance. Therefore, our findings suggest that defects in FAK activation and its abnormal subcellular distribution upon Protrudin knockdown are associated with a detrimental effect on endothelial cell migration and angiogenesis. Furthermore, mice with global Protrudin deletion demonstrate reduced retinal vascular progression. To conclude, our results provide evidence for a novel key role of Protrudin in endothelial cell migration and angiogenesis.

Dendritic Spine Initiation in Brain Development, Learning and Diseases and Impact of BAR-Domain Proteins.

Dendritic spines are small, bulbous protrusions along neuronal dendrites where most of the excitatory synapses are located. Dendritic spine density in normal human brain increases rapidly before and after birth achieving the highest density around 2-8 years. Density decreases during adolescence, reaching a stable level in adulthood. The changes in dendritic spines are considered structural correlates for synaptic plasticity as well as the basis of experience-dependent remodeling of neuronal circuits. Alterations in spine density correspond to aberrant brain function observed in various neurodevelopmental and neuropsychiatric disorders. Dendritic spine initiation affects spine density. In this review, we discuss the importance of spine initiation in brain development, learning, and potential complications resulting from altered spine initiation in neurological diseases. Current literature shows that two Bin Amphiphysin Rvs (BAR) domain-containing proteins, MIM/Mtss1 and SrGAP3, are involved in spine initiation. We review existing literature and open databases to discuss whether other BAR-domain proteins could also take part in spine initiation. Finally, we discuss the potential molecular mechanisms on how BAR-domain proteins could regulate spine initiation.

A look into the future of the COVID-19 pandemic in Europe: an expert consultation.

How will the coronavirus disease 2019 (COVID-19) pandemic develop in the coming months and years? Based on an expert survey, we examine key aspects that are likely to influence the COVID-19 pandemic in Europe. The challenges and developments will strongly depend on the progress of national and global vaccination programs, the emergence and spread of variants of concern (VOCs), and public responses to non-pharmaceutical interventions (NPIs). In the short term, many people remain unvaccinated, VOCs continue to emerge and spread, and mobility and population mixing are expected to increase. Therefore, lifting restrictions too much and too early risk another damaging wave. This challenge remains despite the reduced opportunities for transmission given vaccination progress and reduced indoor mixing in summer 2021. In autumn 2021, increased indoor activity might accelerate the spread again, whilst a necessary reintroduction of NPIs might be too slow. The incidence may strongly rise again, possibly filling intensive care units, if vaccination levels are not high enough. A moderate, adaptive level of NPIs will thus remain necessary. These epidemiological aspects combined with economic, social, and health-related consequences provide a more holistic perspective on the future of the COVID-19 pandemic.

The axonal radial contractility: Structural basis underlying a new form of neural plasticity.

Axons are the longest cellular structure reaching over a meter in the case of human motor axons. They have a relatively small diameter and contain several cytoskeletal elements that mediate both material and information exchange within neurons. Recently, a novel type of axonal plasticity, termed axonal radial contractility, has been unveiled. It is represented by dynamic and transient diameter changes of the axon shaft to accommodate the passages of large organelles. Mechanisms underpinning this plasticity are not fully understood. Here, we first summarised recent evidence of the functional relevance for axon radial contractility, then discussed the underlying structural basis, reviewing nanoscopic evidence of the subtle changes. Two models are proposed to explain how actomyosin rings are organised. Possible roles of non-muscle myosin II (NM-II) in axon degeneration are discussed. Finally, we discuss the concept of periodic functional nanodomains, which could sense extracellular cues and coordinate the axonal responses. Also see the video abstract here: https://youtu.be/ojCnrJ8RCRc.

Carbonic anhydrase seven bundles filamentous actin and regulates dendritic spine morphology and density.

Intracellular pH is a potent modulator of neuronal functions. By catalyzing (de)hydration of CO2 , intracellular carbonic anhydrase (CAi ) isoforms CA2 and CA7 contribute to neuronal pH buffering and dynamics. The presence of two highly active isoforms in neurons suggests that they may serve isozyme-specific functions unrelated to CO2 -(de)hydration. Here, we show that CA7, unlike CA2, binds to filamentous actin, and its overexpression induces formation of thick actin bundles and membrane protrusions in fibroblasts. In CA7-overexpressing neurons, CA7 is enriched in dendritic spines, which leads to aberrant spine morphology. We identified amino acids unique to CA7 that are required for direct actin interactions, promoting actin filament bundling and spine targeting. Disruption of CA7 expression in neocortical neurons leads to higher spine density due to increased proportion of small spines. Thus, our work demonstrates highly distinct subcellular expression patterns of CA7 and CA2, and a novel, structural role of CA7.

Tropomyosin Tpm3.1 Is Required to Maintain the Structure and Function of the Axon Initial Segment.

The axon initial segment (AIS) is the site of action potential initiation and serves as a cargo transport filter and diffusion barrier that helps maintain neuronal polarity. The AIS actin cytoskeleton comprises actin patches and periodic sub-membranous actin rings. We demonstrate that tropomyosin isoform Tpm3.1 co-localizes with actin patches and that the inhibition of Tpm3.1 led to a reduction in the density of actin patches. Furthermore, Tpm3.1 showed a periodic distribution similar to sub-membranous actin rings but Tpm3.1 was only partially congruent with sub-membranous actin rings. Nevertheless, the inhibition of Tpm3.1 affected the uniformity of the periodicity of actin rings. Furthermore, Tpm3.1 inhibition led to reduced accumulation of AIS structural and functional proteins, disruption in sorting somatodendritic and axonal proteins, and a reduction in firing frequency. These results show that Tpm3.1 is necessary for the structural and functional maintenance of the AIS.

NuMA1 facilitates the assembly of the axon initial segment by promoting the retention of neurofascin-186.

Axon initial segment (AIS) functionality relies on a specific organization of the AIS with high enrichment of structural and functional proteins. In this issue, Torii et al. (2019. J. Cell. Biol.https://doi.org/10.1083/jcb.201907048) describe a mechanism for achieving a high density of proteins in the nascent AIS.

MIM-Deficient Mice Exhibit Anatomical Changes in Dendritic Spines, Cortex Volume and Brain Ventricles, and Functional Changes in Motor Coordination and Learning.

In this study, we performed a comprehensive behavioral and anatomical analysis of the Missing in Metastasis (Mtss1/MIM) knockout (KO) mouse brain. We also analyzed the expression of MIM in different brain regions at different ages. MIM is an I-BAR containing membrane curving protein, shown to be involved in dendritic spine initiation and dendritic branching in Purkinje cells in the cerebellum. Behavioral analysis of MIM KO mice revealed defects in both learning and reverse-learning, alterations in anxiety levels and reduced dominant behavior, and confirmed the previously described deficiency in motor coordination and pre-pulse inhibition. Anatomically, we observed enlarged brain ventricles and decreased cortical volume. Although MIM expression was relatively low in hippocampus after early development, hippocampal pyramidal neurons exhibited reduced density of thin and stubby dendritic spines. Learning deficiencies can be connected to all detected anatomical changes. Both behavioral and anatomical findings are typical for schizophrenia mouse models.

Chemical LTD, but not LTP, induces transient accumulation of gelsolin in dendritic spines.

Synaptic plasticity underlies central brain functions, such as learning. Ca2+ signaling is involved in both strengthening and weakening of synapses, but it is still unclear how one signal molecule can induce two opposite outcomes. By identifying molecules, which can distinguish between signaling leading to weakening or strengthening, we can improve our understanding of how synaptic plasticity is regulated. Here, we tested gelsolin's response to the induction of chemical long-term potentiation (cLTP) or long-term depression (cLTD) in cultured rat hippocampal neurons. We show that gelsolin relocates from the dendritic shaft to dendritic spines upon cLTD induction while it did not show any relocalization upon cLTP induction. Dendritic spines are small actin-rich protrusions on dendrites, where LTD/LTP-responsive excitatory synapses are located. We propose that the LTD-induced modest - but relatively long-lasting - elevation of Ca2+ concentration increases the affinity of gelsolin to F-actin. As F-actin is enriched in dendritic spines, it is probable that increased affinity to F-actin induces the relocalization of gelsolin.

Sub-membranous actin rings in the axon initial segment are resistant to the action of latrunculin.

The axon initial segment (AIS) comprises a sub-membranous lattice containing periodic actin rings. The overall AIS structure is insensitive to actin-disrupting drugs, but the effects of actin-disrupting drugs on actin rings lack consensus. We examined the effect of latrunculin A and B on the actin cytoskeleton of neurons in culture and actin rings in the AIS. Both latrunculin A and B markedly reduced the overall amount of F-actin in treated neurons in a dose-dependent manner, but the periodicity of actin rings remained unaffected. The insensitivity of AIS actin rings to latrunculin suggests they are relatively stable.

ASD-Associated De Novo Mutations in Five Actin Regulators Show Both Shared and Distinct Defects in Dendritic Spines and Inhibitory Synapses in Cultured Hippocampal Neurons.

Many actin cytoskeleton-regulating proteins control dendritic spine morphology and density, which are cellular features often altered in autism spectrum disorder (ASD). Recent studies using animal models show that autism-related behavior can be rescued by either manipulating actin regulators or by reversing dendritic spine density or morphology. Based on these studies, the actin cytoskeleton is a potential target pathway for developing new ASD treatments. Thus, it is important to understand how different ASD-associated actin regulators contribute to the regulation of dendritic spines and how ASD-associated mutations modulate this regulation. For this study, we selected five genes encoding different actin-regulating proteins and induced ASD-associated de novo missense mutations in these proteins. We assessed the functionality of the wild-type and mutated proteins by analyzing their subcellular localization, and by analyzing the dendritic spine phenotypes induced by the expression of these proteins. As the imbalance between excitation and inhibition has been suggested to have a central role in ASD, we additionally evaluated the density, size and subcellular localization of inhibitory synapses. Common for all the proteins studied was the enrichment in dendritic spines. ASD-associated mutations induced changes in the localization of α-actinin-4, which localized less to dendritic spines, and for SWAP-70 and SrGAP3, which localized more to dendritic spines. Among the wild-type proteins studied, only α-actinin-4 expression caused a significant change in dendritic spine morphology by increasing the mushroom spine density and decreasing thin spine density. We hypothesized that mutations associated with ASD shift dendritic spine morphology from mushroom to thin spines. An M554V mutation in α-actinin-4 (ACTN4) resulted in the expected shift in dendritic spine morphology by increasing the density of thin spines. In addition, we observed a trend toward higher thin spine density with mutations in myosin IXb and SWAP-70. Myosin IIb and myosin IXb expression increased the proportion of inhibitory synapses in spines. The expression of mutated myosin IIb (Y265C), SrGAP3 (E469K), and SWAP-70 (L544F) induced variable changes in inhibitory synapses.

Longitudinal two-photon imaging in somatosensory cortex of behaving mice reveals dendritic spine formation enhancement by subchronic administration of low-dose ketamine.

Ketamine, a well-known anesthetic, has recently attracted renewed attention as a fast-acting antidepressant. A single dose of ketamine induces rapid synaptogenesis, which may underlie its antidepressant effect. To test whether repeated exposure to ketamine triggers sustained synaptogenesis, we administered a sub-anesthetic dose of ketamine (10 mg/kg i.p.) once-daily for 5 days, and repeatedly imaged dendritic spines of the YFP-expressing pyramidal neurons in somatosensory cortex of awake female mice using in vivo two-photon microscopy. We found that the spine formation rate became significantly higher at 72-132 h after the first ketamine injection (but not at 6-24 h), while the rate of elimination of pre-existing spines remained unchanged. In contrast to the net gain of spines observed in ketamine-treated mice, the vehicle-injected control mice exhibited a net loss typical for young-adult animals undergoing synapse pruning. Ketamine-induced spinogenesis was correlated with increased PSD-95 and phosphorylated actin, consistent with formation of new synapses. Moreover, structural synaptic plasticity caused by ketamine was paralleled by a significant improvement in the nest building behavioral assay. Taken together, our data show that subchronic low-dose ketamine induces a sustained shift towards spine formation.

Dendritic spine actin cytoskeleton in autism spectrum disorder.

Dendritic spines are small actin-rich protrusions from neuronal dendrites that form the postsynaptic part of most excitatory synapses. Changes in the shape and size of dendritic spines correlate with the functional changes in excitatory synapses and are heavily dependent on the remodeling of the underlying actin cytoskeleton. Recent evidence implicates synapses at dendritic spines as important substrates of pathogenesis in neuropsychiatric disorders, including autism spectrum disorder (ASD). Although synaptic perturbations are not the only alterations relevant for these diseases, understanding the molecular underpinnings of the spine and synapse pathology may provide insight into their etiologies and could reveal new drug targets. In this review, we will discuss recent findings of defective actin regulation in dendritic spines associated with ASD.

DHCR24 exerts neuroprotection upon inflammation-induced neuronal death.

BACKGROUND: DHCR24, involved in the de novo synthesis of cholesterol and protection of neuronal cells against different stress conditions, has been shown to be selectively downregulated in neurons of the affected brain areas in Alzheimer's disease. METHODS: Here, we investigated whether the overexpression of DHCR24 protects neurons against inflammation-induced neuronal death using co-cultures of mouse embryonic primary cortical neurons and BV2 microglial cells upon acute neuroinflammation. Moreover, the effects of DHCR24 overexpression on dendritic spine density and morphology in cultured mature mouse hippocampal neurons and on the outcome measures of ischemia-induced brain damage in vivo in mice were assessed. RESULTS: Overexpression of DHCR24 reduced the loss of neurons under inflammation elicited by LPS and IFN-γ treatment in co-cultures of mouse neurons and BV2 microglial cells but did not affect the production of neuroinflammatory mediators, total cellular cholesterol levels, or the activity of proteins linked with neuroprotective signaling. Conversely, the levels of post-synaptic cell adhesion protein neuroligin-1 were significantly increased upon the overexpression of DHCR24 in basal growth conditions. Augmentation of DHCR24 also increased the total number of dendritic spines and the proportion of mushroom spines in mature mouse hippocampal neurons. In vivo, overexpression of DHCR24 in striatum reduced the lesion size measured by MRI in a mouse model of transient focal ischemia. CONCLUSIONS: These results suggest that the augmentation of DHCR24 levels provides neuroprotection in acute stress conditions, which lead to neuronal loss in vitro and in vivo.

New waves in dendritic spine actin cytoskeleton: From branches and bundles to rings, from actin binding proteins to post-translational modifications.

Dendritic spines are small actin-rich protrusions from neuronal dendrites that form the postsynaptic part of most excitatory synapses. Changes in the number or strength of synapses are physiological mechanisms behind learning. The growth and maturation of dendritic spines and the activity-induced changes to their morphology are all based on changes to the actin cytoskeleton. In this review, we will discuss the regulation of the actin cytoskeleton in dendritic spine formation and maturation, as well as in synaptic strengthening. Concerning spine formation, we will focus on spine initiation, which has received less attention in the literature. We will also examine the recently revealed regulation of the actin cytoskeleton through post-translational modifications of actin monomers, in addition to the conventional regulation of actin via actin-binding proteins.

The initiation of post-synaptic protrusions.

The post-synaptic spines of neuronal dendrites are highly elaborate membrane protrusions. Their anatomy, stability and density are intimately linked to cognitive performance. The morphological transitions of spines are powered by coordinated polymerization of actin filaments against the plasma membrane, but how the membrane-associated polymerization is spatially and temporally regulated has remained ill defined. Here, we discuss our recent findings showing that dendritic spines can be initiated by direct membrane bending by the I-BAR protein MIM/Mtss1. This lipid phosphatidylinositol (PI(4,5)P2) signaling-activated membrane bending coordinated spatial actin assembly and promoted spine formation. From recent advances, we formulate a general model to discuss how spatially concentrated protein-lipid microdomains formed by multivalent interactions between lipids and actin/membrane regulatory proteins might launch cell protrusions.