Layer 1 of somatosensory cortex: an important site for input to a tiny cortical compartment.

Neocortical layer 1 has been proposed to be at the center for top-down and bottom-up integration. It is a locus for interactions between long-range inputs, layer 1 interneurons, and apical tuft dendrites of pyramidal neurons. While input to layer 1 has been studied intensively, the level and effect of input to this layer has still not been completely characterized. Here we examined the input to layer 1 of mouse somatosensory cortex with retrograde tracing and optogenetics. Our assays reveal that local input to layer 1 is predominantly from layers 2/3 and 5 pyramidal neurons and interneurons, and that subtypes of local layers 5 and 6b neurons project to layer 1 with different probabilities. Long-range input from sensory-motor cortices to layer 1 of somatosensory cortex arose predominantly from layers 2/3 neurons. Our optogenetic experiments showed that intra-telencephalic layer 5 pyramidal neurons drive layer 1 interneurons but have no effect locally on layer 5 apical tuft dendrites. Dual retrograde tracing revealed that a fraction of local and long-range neurons was both presynaptic to layer 5 neurons and projected to layer 1. Our work highlights the prominent role of local inputs to layer 1 and shows the potential for complex interactions between long-range and local inputs, which are both in position to modify the output of somatosensory cortex.

Plasma membrane phospholipid phosphatase-related proteins as pleiotropic regulators of neuron growth and excitability.

Neuronal plasma membrane proteins are essential for integrating cell extrinsic and cell intrinsic signals to orchestrate neuronal differentiation, growth and plasticity in the developing and adult nervous system. Here, we shed light on the family of plasma membrane proteins phospholipid phosphatase-related proteins (PLPPRs) (alternative name, PRGs; plasticity-related genes) that fine-tune neuronal growth and synaptic transmission in the central nervous system. Several studies uncovered essential functions of PLPPRs in filopodia formation, axon guidance and branching during nervous system development and regeneration, as well as in the control of dendritic spine number and excitability. Loss of PLPPR expression in knockout mice increases susceptibility to seizures, and results in defects in sensory information processing, development of psychiatric disorders, stress-related behaviors and abnormal social interaction. However, the exact function of PLPPRs in the context of neurological diseases is largely unclear. Although initially described as active lysophosphatidic acid (LPA) ecto-phosphatases that regulate the levels of this extracellular bioactive lipid, PLPPRs lack catalytic activity against LPA. Nevertheless, they emerge as atypical LPA modulators, by regulating LPA mediated signaling processes. In this review, we summarize the effects of this protein family on cellular morphology, generation and maintenance of cellular protrusions as well as highlight their known neuronal functions and phenotypes of KO mice. We discuss the molecular mechanisms of PLPPRs including the deployment of phospholipids, actin-cytoskeleton and small GTPase signaling pathways, with a focus on identifying gaps in our knowledge to stimulate interest in this understudied protein family.

The impact of phosphorylated PTEN at threonine 366 on cortical connectivity and behaviour.

The lipid phosphatase PTEN (phosphatase and tensin homologue on chromosome 10) is a key tumour suppressor gene and an important regulator of neuronal signalling. PTEN mutations have been identified in patients with autism spectrum disorders, characterized by macrocephaly, impaired social interactions and communication, repetitive behaviour, intellectual disability, and epilepsy. PTEN enzymatic activity is regulated by a cluster of phosphorylation sites at the C-terminus of the protein. Here, we focused on the role of PTEN T366 phosphorylation and generated a knock-in mouse line in which Pten T366 was substituted with alanine (PtenT366A/T366A). We identify that phosphorylation of PTEN at T366 controls neuron size and connectivity of brain circuits involved in sensory processing. We show in behavioural tests that PtenT366/T366A mice exhibit cognitive deficits and selective sensory impairments, with significant differences in male individuals. We identify restricted cellular overgrowth of cortical neurons in PtenT366A/T366A brains, linked to increases in both dendritic arborization and soma size. In a combinatorial approach of anterograde and retrograde monosynaptic tracing using rabies virus, we characterize differences in connectivity to the primary somatosensory cortex of PtenT366A/T366A brains, with imbalances in long-range cortico-cortical input to neurons. We conclude that phosphorylation of PTEN at T366 controls neuron size and connectivity of brain circuits involved in sensory processing and propose that PTEN T366 signalling may account for a subset of autism-related functions of PTEN.

Precursor types predict the stability of neuronal branches.

Branches are critical for neuron function, generating the morphological complexity required for functional networks. They emerge from different, well-described, cytoskeletal precursor structures that elongate to branches. While branches are thought to be maintained by shared cytoskeletal regulators, our data from mouse hippocampal neurons indicate that the precursor structures trigger alternative branch maintenance mechanisms with differing stabilities. Whereas branches originating from lamellipodia or growth cone splitting events collapse soon after formation, branches emerging from filopodia persist. Furthermore, compared to other developing neurites, axons stabilise all branches and preferentially initiate branches from filopodia. These differences explain the altered stability of branches we observe in neurons lacking the plasma membrane protein phospholipid phosphatase-related protein 3 (PLPPR3, also known as PRG2) and in neurons treated with netrin-1. Rather than altering branch stability directly, PLPPR3 and netrin-1 boost a 'filopodia branch programme' on axons, thereby indirectly initiating more long-lived branches. In summary, we propose that studies on branching should distinguish overall stabilising effects from effects on precursor types, ideally using multifactorial statistical models, as exemplified in this study.

The actin binding protein drebrin helps to protect against the development of seizure-like events in the entorhinal cortex.

The actin binding protein drebrin plays a key role in dendritic spine formation and synaptic plasticity. Decreased drebrin protein levels have been observed in temporal lobe epilepsy, suggesting the involvement of drebrin in the disease. Here we investigated the effect of drebrin knockout on physiological and pathophysiological neuronal network activities in mice by inducing gamma oscillations, involved in higher cognitive functions, and by analyzing pathophysiological epileptiform activity. We found that loss of drebrin increased the emergence of spontaneous gamma oscillations suggesting an increase in neuronal excitability when drebrin is absent. Further analysis showed that although the kainate-induced hippocampal gamma oscillations were unchanged in drebrin deficient mice, seizure like events measured in the entorhinal cortex appeared earlier and more frequently. The results suggest that while drebrin is not essential for normal physiological network activity, it helps to protect against the formation of seizure like activities during pathological conditions. The data indicate that targeting drebrin function could potentially be a preventive or therapeutic strategy for epilepsy treatment.

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 ß1-integrin. Our work shows that DBN-mediated membrane trafficking in astrocytes is an important neuroprotective mechanism following traumatic brain injury in mice.

Harnessing PTEN's Growth Potential in Neuronal Development and Disease.

PTEN is a powerful regulator of neuronal growth. It globally suppresses axon extension and branching during both nervous system development and regeneration, by antagonizing growth-promoting PI3K/PI(3,4,5)P3 signaling. We recently identified that the transmembrane protein PRG2/LPPR3 functions as a modulator of PTEN function during axon morphogenesis. Our work demonstrates that through inhibition of PTEN activity, PRG2 stabilizes membrane PI(3,4,5)P3. In turn, PRG2 deficiency attenuates the formation of branches in a PTEN-dependent manner, albeit without affecting the overall growth capacity of extending axons. Thus, PRG2 is poised to temporally and locally relieve growth suppression mediated by PTEN in neurons and, in effect, to redirect growth specifically to axonal branches. In this commentary, we discuss potential implications and unresolved questions regarding the regulation of axonal PTEN in neurons. Given their widespread implication during neuronal development and regeneration, identification of mechanisms that confer spatiotemporal control of PTEN may unveil new approaches to reprogram PI3K signaling in neurodevelopmental disorders and regeneration research.

PI 3-kinase delta enhances axonal PIP3 to support axon regeneration in the adult CNS.

Peripheral nervous system (PNS) neurons support axon regeneration into adulthood, whereas central nervous system (CNS) neurons lose regenerative ability after development. To better understand this decline whilst aiming to improve regeneration, we focused on phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol (3,4,5)-trisphosphate (PIP3 ). We demonstrate that adult PNS neurons utilise two catalytic subunits of PI3K for axon regeneration: p110α and p110δ. However, in the CNS, axonal PIP3 decreases with development at the time when axon transport declines and regenerative competence is lost. Overexpressing p110α in CNS neurons had no effect; however, expression of p110δ restored axonal PIP3 and increased regenerative axon transport. p110δ expression enhanced CNS regeneration in both rat and human neurons and in transgenic mice, functioning in the same way as the hyperactivating H1047R mutation of p110α. Furthermore, viral delivery of p110δ promoted robust regeneration after optic nerve injury. These findings establish a deficit of axonal PIP3 as a key reason for intrinsic regeneration failure and demonstrate that native p110δ facilitates axon regeneration by functioning in a hyperactive fashion.

Layer 6b Is Driven by Intracortical Long-Range Projection Neurons.

Layer 6b (L6b), the deepest neocortical layer, projects to cortical targets and higher-order thalamus and is the only layer responsive to the wake-promoting neuropeptide orexin/hypocretin. These characteristics suggest that L6b can strongly modulate brain state, but projections to L6b and their influence remain unknown. Here, we examine the inputs to L6b ex vivo in the mouse primary somatosensory cortex with rabies-based retrograde tracing and channelrhodopsin-assisted circuit mapping in brain slices. We find that L6b receives its strongest excitatory input from intracortical long-range projection neurons, including those in the contralateral hemisphere. In contrast, local intracortical input and thalamocortical input were significantly weaker. Moreover, our data suggest that L6b receives far less thalamocortical input than other cortical layers. L6b was most strongly inhibited by PV and SST interneurons. This study shows that L6b integrates long-range intracortical information and is not part of the traditional thalamocortical loop.

The Axonal Membrane Protein PRG2 Inhibits PTEN and Directs Growth to Branches.

In developing neurons, phosphoinositide 3-kinases (PI3Ks) control axon growth and branching by positively regulating PI3K/PI(3,4,5)P3, but how neurons are able to generate sufficient PI(3,4,5)P3 in the presence of high levels of the antagonizing phosphatase PTEN is difficult to reconcile. We find that normal axon morphogenesis involves homeostasis of elongation and branch growth controlled by accumulation of PI(3,4,5)P3 through PTEN inhibition. We identify a plasma membrane-localized protein-protein interaction of PTEN with plasticity-related gene 2 (PRG2). PRG2 stabilizes membrane PI(3,4,5)P3 by inhibiting PTEN and localizes in nanoclusters along axon membranes when neurons initiate their complex branching behavior. We demonstrate that PRG2 is both sufficient and necessary to account for the ability of neurons to generate axon filopodia and branches in dependence on PI3K/PI(3,4,5)P3 and PTEN. Our data indicate that PRG2 is part of a neuronal growth program that induces collateral branch growth in axons by conferring local inhibition of PTEN.

PTEN in Autism and Neurodevelopmental Disorders.

Phosphatase and tensin homolog (PTEN) is a classical tumor suppressor that antagonizes phosphatidylinositol 3-phosphate kinase (PI3K)/AKT signaling. Although there is a strong association of PTEN germline mutations with cancer syndromes, they have also been described in a subset of patients with autism spectrum disorders with macrocephaly characterized by impairments in social interactions and communication, repetitive behavior and, occasionally, epilepsy. To investigate PTEN's role during neurodevelopment and its implication for autism, several conditional Pten knockout mouse models have been generated. These models are valuable tools to understand PTEN's spatiotemporal roles during neurodevelopment. In this review, we will highlight the anatomical and phenotypic results from animal studies and link them to cellular and molecular findings.

Optically Induced Calcium-Dependent Gene Activation and Labeling of Active Neurons Using CaMPARI and Cal-Light.

The advent of optogenetic methods has made it possible to use endogeneously produced molecules to image and manipulate cellular, subcellular, and synaptic activity. It has also led to the development of photoactivatable calcium-dependent indicators that mark active synapses, neurons, and circuits. Furthermore, calcium-dependent photoactivation can be used to trigger gene expression in active neurons. Here we describe two sets of protocols, one using CaMPARI and a second one using Cal-Light. CaMPARI, a calcium-modulated photoactivatable ratiometric integrator, enables rapid network-wide, tunable, all-optical functional circuit mapping. Cal-Light, a photoactivatable calcium sensor, while slower to respond than CaMPARI, has the capacity to trigger the expression of genes, including effectors, activators, indicators, or other constructs. Here we describe the rationale and provide procedures for using these two calcium-dependent constructs (1) in vitro in dissociated primary neuronal cell cultures (CaMPARI & Cal-Light); (2) in vitro in acute brain slices for circuit mapping (CaMPARI); (3) in vivo for triggering photoconversion or gene expression (CaMPARI & Cal-Light); and finally, (4) for recovering photoconverted neurons post-fixation with immunocytochemistry (CaMPARI). The approaches and protocols we describe are examples of the potential uses of both CaMPARI & Cal-Light. The ability to mark and manipulate neurons that are active during specific epochs of behavior has a vast unexplored experimental potential.

Unique properties of PTEN-L contribute to neuroprotection in response to ischemic-like stress.

Phosphatase and tensin homolog (PTEN) signalling might influence neuronal survival after brain ischemia. However, the influence of the less studied longer variant termed PTEN-L (or PTENα) has not been studied to date. Therefore, we examined the translational variant PTEN-L in the context of neuronal survival. We identified PTEN-L by proteomics in murine neuronal cultures and brain lysates and established a novel model to analyse PTEN or PTEN-L variants independently in vitro while avoiding overexpression. We found that PTEN-L, unlike PTEN, localises predominantly in the cytosol and translocates to the nucleus 10-20 minutes after glutamate stress. Genomic ablation of PTEN and PTEN-L increased neuronal susceptibility to oxygen-glucose deprivation. This effect was rescued by expression of either PTEN-L indicating that both PTEN isoforms might contribute to a neuroprotective response. However, in direct comparison, PTEN-L replaced neurons were protected against ischemic-like stress compared to neurons expressing PTEN. Neurons expressing strictly nuclear PTEN-L NLS showed increased vulnerability, indicating that nuclear PTEN-L alone is not sufficient in protecting against stress. We identified mutually exclusive binding partners of PTEN-L or PTEN in cytosolic or nuclear fractions, which were regulated after ischemic-like stress. GRB2-associated-binding protein 2, which is known to interact with phosphoinositol-3-kinase, was enriched specifically with PTEN-L in the cytosol in proximity to the plasma membrane and their interaction was lost after glutamate exposure. The present study revealed that PTEN and PTEN-L have distinct functions in response to stress and might be involved in different mechanisms of neuroprotection.

ATM phosphorylation of the actin-binding protein drebrin controls oxidation stress-resistance in mammalian neurons and C. elegans.

Drebrin (DBN) regulates cytoskeletal functions during neuronal development, and is thought to contribute to structural and functional synaptic changes associated with aging and Alzheimer's disease. Here we show that DBN coordinates stress signalling with cytoskeletal dynamics, via a mechanism involving kinase ataxia-telangiectasia mutated (ATM). An excess of reactive oxygen species (ROS) stimulates ATM-dependent phosphorylation of DBN at serine-647, which enhances protein stability and accounts for improved stress resilience in dendritic spines. We generated a humanized DBN Caenorhabditis elegans model and show that a phospho-DBN mutant disrupts the protective ATM effect on lifespan under sustained oxidative stress. Our data indicate a master regulatory function of ATM-DBN in integrating cytosolic stress-induced signalling with the dynamics of actin remodelling to provide protection from synapse dysfunction and ROS-triggered reduced lifespan. They further suggest that DBN protein abundance governs actin filament stability to contribute to the consequences of oxidative stress in physiological and pathological conditions.

Importin α5 Regulates Anxiety through MeCP2 and Sphingosine Kinase 1.

Importins mediate transport from synapse to soma and from cytoplasm to nucleus, suggesting that perturbation of importin-dependent pathways should have significant neuronal consequences. A behavioral screen on five importin α knockout lines revealed that reduced expression of importin α5 (KPNA1) in hippocampal neurons specifically decreases anxiety in mice. Re-expression of importin α5 in ventral hippocampus of knockout animals increased anxiety behaviors to wild-type levels. Hippocampal neurons lacking importin α5 reveal changes in presynaptic plasticity and modified expression of MeCP2-regulated genes, including sphingosine kinase 1 (Sphk1). Knockout of importin α5, but not importin α3 or α4, reduces MeCP2 nuclear localization in hippocampal neurons. A Sphk1 blocker reverses anxiolysis in the importin α5 knockout mouse, while pharmacological activation of sphingosine signaling has robust anxiolytic effects in wild-type animals. Thus, importin α5 influences sphingosine-sensitive anxiety pathways by regulating MeCP2 nuclear import in hippocampal neurons.

Improved methods for marking active neuron populations.

Marking functionally distinct neuronal ensembles with high spatiotemporal resolution is a key challenge in systems neuroscience. We recently introduced CaMPARI, an engineered fluorescent protein whose green-to-red photoconversion depends on simultaneous light exposure and elevated calcium, which enabled marking active neuronal populations with single-cell and subsecond resolution. However, CaMPARI (CaMPARI1) has several drawbacks, including background photoconversion in low calcium, slow kinetics and reduced fluorescence after chemical fixation. In this work, we develop CaMPARI2, an improved sensor with brighter green and red fluorescence, faster calcium unbinding kinetics and decreased photoconversion in low calcium conditions. We demonstrate the improved performance of CaMPARI2 in mammalian neurons and in vivo in larval zebrafish brain and mouse visual cortex. Additionally, we herein develop an immunohistochemical detection method for specific labeling of the photoconverted red form of CaMPARI. The anti-CaMPARI-red antibody provides strong labeling that is selective for photoconverted CaMPARI in activated neurons in rodent brain tissue.

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.

Investigation of hippocampal synaptic transmission and plasticity in mice deficient in the actin-binding protein Drebrin.

The dynamic regulation of the actin cytoskeleton plays a key role in controlling the structure and function of synapses. It is vital for activity-dependent modulation of synaptic transmission and long-term changes in synaptic morphology associated with memory consolidation. Several regulators of actin dynamics at the synapse have been identified, of which a salient one is the postsynaptic actin stabilising protein Drebrin (DBN). It has been suggested that DBN modulates neurotransmission and changes in dendritic spine morphology associated with synaptic plasticity. Given that a decrease in DBN levels is correlated with cognitive deficits associated with ageing and dementia, it was hypothesised that DBN protein abundance instructs the integrity and function of synapses. We created a novel DBN deficient mouse line. Analysis of gross brain and neuronal morphology revealed no phenotype in the absence of DBN. Electrophysiological recordings in acute hippocampal slices and primary hippocampal neuronal cultures showed that basal synaptic transmission, and both long-term and homeostatic synaptic plasticity were unchanged, suggesting that loss of DBN is not sufficient in inducing synapse dysfunction. We propose that the overall lack of changes in synaptic function and plasticity in DBN deficient mice may indicate robust compensatory mechanisms that safeguard cytoskeleton dynamics at the synapse.

RIM-binding protein 2 regulates release probability by fine-tuning calcium channel localization at murine hippocampal synapses.

The tight spatial coupling of synaptic vesicles and voltage-gated Ca2+ channels (CaVs) ensures efficient action potential-triggered neurotransmitter release from presynaptic active zones (AZs). Rab-interacting molecule-binding proteins (RIM-BPs) interact with Ca2+ channels and via RIM with other components of the release machinery. Although human RIM-BPs have been implicated in autism spectrum disorders, little is known about the role of mammalian RIM-BPs in synaptic transmission. We investigated RIM-BP2-deficient murine hippocampal neurons in cultures and slices. Short-term facilitation is significantly enhanced in both model systems. Detailed analysis in culture revealed a reduction in initial release probability, which presumably underlies the increased short-term facilitation. Superresolution microscopy revealed an impairment in CaV2.1 clustering at AZs, which likely alters Ca2+ nanodomains at release sites and thereby affects release probability. Additional deletion of RIM-BP1 does not exacerbate the phenotype, indicating that RIM-BP2 is the dominating RIM-BP isoform at these synapses.