Parkinson’s disease (PD) is a multisystemic disorder that manifests through motor and non-motor symptoms. Motor dysfunction is the most debilitating and it is caused by the degeneration of dopamine-producing neurons in the substantia nigra pars compacta (SNpc). A body of evidence indicates that synapse demise precedes by years neuronal death. Still, early synaptic dysfunctions in PD are poorly deciphered. Here we combined literature metanalysis, proteomics and phosphoproteomics with biochemical, imaging and electrophysiological measurements in neurons, brains and synaptosomes from knockout and knockin mouse models, as well as human iPSC-derived neurons associated with the PD-kinase LRRK2. We show that phosphorylation of LRRK2 at Ser935, which controls LRRK2 subcellular localization, rapidly increases upon brain-derived neurotrophic factor (BDNF) stimulation of differentiated SH-SY5Y cells and primary mouse neurons. Affinity-purification coupled with mass spectrometry (AP-MS/MS) analysis revealed that LRRK2 interactome is significantly reshaped upon BDNF stimulation, with an interconnected network of actin cytoskeleton-associated proteins increasing their binding to LRRK2. Accordingly, LRRK2 knockout neurons exhibit decreased TrkB signaling and fail to induce BDNF-dependent spinogenesis. In vivo , one-month old Lrrk2 knockout mice display defects in spine maturation, a phenotype that disappears with age. In human iPSC-derived cortical neurons, BDNF increases the frequency of miniature excitatory post-synaptic currents (mEPSC) in wild-type but not in the presence of LRRK2 knockout, functionally supporting a distinctive role of LRRK2 in BDNF-synaptic signaling. Finally, Lrrk2 G2019S PD mutant synaptosomes display differentially phosphorylated proteins enriched in categories related to postsynaptic structural organization. Taken together, our study discloses a critical function of LRRK2 in BDNF-dependent synaptic processes and identifies the postsynaptic actin cytoskeleton as a convergent site of LRRK2 pathophysiological activity.

Spinal cord injury (SCI) results in significant disruption of nerve fibers responsible for transmitting signals between the brain and body, often leading to partial or complete motor, sensory, and autonomic dysfunction below the injury site. Astrocytes are an important component in scar formation, crucial for suppression of injury propagation, effective wound healing, and the regulation of neuronal plasticity. Here, we identify the role of the actin-binding protein Drebrin (DBN) in reactive astrogliosis following SCI. SCI induces the upregulation of DBN in astrocytes, which controls immediate injury containment but also the long-term preservation of tissue integrity and healing in the spinal cord. DBN knockout results in enlarged spinal cord lesions, increased immune cell infiltration, and neurodegeneration. Mechanistically, DBN loss disrupts the polarization of scar border-forming astrocytes, leading to impaired encapsulation of the injury. In summary, DBN serves as a pivotal regulator of SCI outcome by modulating astrocytic polarity, which is essential for establishing a protective barrier confining the lesion site.

Phospholipid-phosphatase-related protein 3 (PLPPR3) belongs to a family of transmembrane proteins highly expressed in the nervous system where it regulates critical axonal growth processes during guidance, filopodia formation, and branching. However, little is known regarding its role in synapses and the signaling events regulating PLPPR3 function. Here, we identify 26 high-confidence phosphorylation sites in the intracellular domain of PLPPR3 using mass spectrometry. Biochemical characterization established one of these-S351-as a bona fide phosphorylation site of protein kinase A (PKA). PLPPR3 is enriched at presynaptic terminals, and deletion of PLPPR3 results in increased depolarization-induced synaptic vesicle release in hippocampal neurons. This tonic inhibitory signal toward depolarization-induced presynaptic activity is corrected by expression of PLPPR3 intracellular domain, but not a S351A phospho-dead mutant, in Plppr3 -/- hippocampal neurons. We propose that PLPPR3 phosphorylation under the control of PKA activity is a signaling integrator of presynaptic activity in hippocampal neurons.

Neurons, as long-lived non-dividing cells with complex morphology, depend on a highly elaborate secretory trafficking system which enables a constant turnover of proteins and membranes. Previously, it was shown that simplified, Golgi-related structures called Golgi satellites (GS) are present in the dendrites of primary hippocampal neurons. These organelles are distinct from the somatic Golgi complex and are involved in de novo glycosylation and local forward trafficking of membrane proteins. However, the question of whether GS are also targeted to the axons of principal neurons remained unanswered. In this study, we investigated the subcellular distribution of GS in adult hippocampal neurons. Our findings showed that GS are present all along the axon, extending to the distal tips of the growth cone. Similar to dendritic GS, the axonal organelles are labeled by the same GS markers and are capable of mature glycosylation. Live imaging experiments revealed the presence of both mobile and immobile GS in the axon, and that the switch between active transport and stalling of GS was modulated by neuronal firing. We found that GS frequently pause at en passant synapses and remain stationary for longer time periods at activated pre-synaptic boutons. This behavior is dependent on the actin cytoskeleton and the actin-based motor protein myosin VI. Overall, our study demonstrates that neuronal activity can dynamically regulate the positioning of GS in the axon, shedding light on the intricate mechanisms underlying organelle trafficking in neurons.

Phosphatase and tensin homologue (PTEN) is the main antagonist of the phosphatidylinositol-3-kinase (PI3K)/AKT/ mTOR signalling pathway and mutated in 10%-20% of individuals with autism spectrum disorder (ASD) exhibiting macrocephaly. Hyperactive mTOR signalling is responsible for some aspects during PTEN-ASD progression, e.g. neuronal hypertrophy and-excitability, but PI3K/mTOR-independent processes have additionally been described. There is emerging evidence that PTEN regulates gene transcription, spliceosome formation and pre-mRNA splicing independently of PI3K/mTOR. Altered splicing is a hallmark of brains from individuals with idiopathic and PTEN-ASD, however, molecular mechanisms are yet to be identified. We performed RNA-sequencing (RNA-Seq), followed by analysis of altered transcript splicing in Pten-deficient primary cortical mouse neurons, which we compared with published data from PTEN-deficient human neuronal stem cells. This analysis identified that transcripts were globally mis-spliced in a developmentally regulated fashion and cluster in synaptic and gene expression regulatory processes. Strikingly, splicing defects following Pten-deficiency represent a significant number of other known ASD-susceptibility genes. Furthermore, we show that exons with strong 3 ' splice sites are more frequently mis-spliced under Pten-deficient conditions. Our study indicates that PTEN-ASD is a multifactorial condition involving the dysregulation of other known ASD-susceptibility genes.

Neurons are long-lived postmitotic cells that capitalize on autophagy to remove toxic or defective proteins and organelles to maintain neurotransmission and the integrity of their functional proteome. Mutations in autophagy genes cause congenital diseases, sharing prominent brain dysfunctions including epilepsy, intellectual disability, and neurodegeneration. Ablation of core autophagy genes in neurons or glia disrupts normal behavior, leading to motor deficits, memory impairment, altered sociability, and epilepsy, which are associated with defects in synapse maturation, plasticity, and neurotransmitter release. In spite of the importance of autophagy for brain physiology, the substrates of neuronal autophagy and the mechanisms by which defects in autophagy affect synaptic function in health and disease remain controversial. Here, we summarize the current state of knowledge on neuronal autophagy, address the existing controversies and inconsistencies in the field, and provide a roadmap for future research on the role of autophagy in the control of synaptic function.

Microglial functions rely on their morphodynamic versatility and inflammatory response, yet the molecular determinants, particularly ion channels and receptors, remain poorly understood. Here, we identify chloride intracellular channel 1 (CLIC1), a protein known to exist in both soluble and membrane-associated forms, as highly enriched in human and murine microglia, with minimal expression in other brain cells. Acute blockade or genetic deletion of CLIC1 markedly attenuates microglial surveillance by reducing ramification and motility, without affecting chemotaxis. This phenotype is recapitulated in xenografted human microglia and human brain tissue. Mechanistically, CLIC1 effects involve interactions with actin-binding ezrin, radixin, and moesin (ERM) proteins, suggesting a role in linking the plasma membrane to the cytoskeleton. Contrary to its name, CLIC1 functions are chloride-independent and thus unlikely to reflect ion channel activity. This is supported by patch-clamp electrophysiology revealing lack of chloride conductance in surveillant microglia. Following ATP-evoked activation, CLIC1 blockade strongly suppresses NLRP3-dependent interleukin-1β release, suggesting therapeutic potential against neuroinflammation.

>Lysophosphatidic acid(LPA) is a pleiotropic lipid agonist essential for functions of the central nervous system(CNS).It is abundant in the developing and adult brain while its concentration in biological fluids,including cerebrospinal fluid,varies significantly(Figure 1A;Yung et al.,2014).LPA actually corresponds to a variety of lipid species that include different stereoisomers with either saturated or unsaturated fatty acids bearing likely differentiated biological activities(Figure 1A;Yung et al.,2014;Hernández-Araiza et al.,

The phosphatase and tensin homolog (PTEN) is a vital protein that maintains an inhibitory brake for cellular proliferation and growth. Accordingly, PTEN loss-of-function mutations are associated with a broad spectrum of human pathologies. Despite its importance, there is currently no method to directly monitor PTEN activity with cellular specificity within intact biological systems. Here we describe the development of a FRET-based biosensor using PTEN conformation as a proxy for the PTEN activity state, for two-photon fluorescence lifetime imaging microscopy. We identify a point mutation that allows the monitoring of PTEN activity with minimal interference to endogenous PTEN signaling. We demonstrate imaging of PTEN activity in cell lines, intact Caenorhabditis elegans and in the mouse brain. Finally, we develop a red-shifted sensor variant that allows us to identify cell-type-specific PTEN activity in excitatory and inhibitory cortical cells. In summary, our approach enables dynamic imaging of PTEN activity in vivo with unprecedented spatial and temporal resolution.

Lysophosphatidic acid (LPA) is a bioactive phospholipid that participates in critical processes in neural development and adult brain function and is implicated in various pathophysiological conditions. Along with its six well-characterized receptors, atypical regulators of LPA signaling have also been suggested, including phospholipid phosphatase-related proteins (PLPPRs). PLPPRs have been mostly studied in the developing brain where they control LPA-dependent axon guidance, cortical network hyperexcitability, and glutamatergic neurotransmission. PLPPR4 and PLPPR3 represent two closely related proteins reported to localize predominantly in dendrites and axons, respectively, and differ in their developmental expression patterns. Herein, we have revised the expression patterns of PLPPRs in the cerebellum, dorsal and ventral hippocampus, prefrontal cortex (PFC), nucleus accumbens, and striatum during development and in the adult using quantitative PCR. Expression patterns of Plppr2,4 and 5 were consistent with previous studies, whereas Plppr3 and Plppr1 exhibited a unique expression profile in nucleus accumbens (NAc) and striatum in later developmental and adult stages, which we verified at the protein level for PLPPR3. To investigate neuron type-specific expression at the single cell level, we developed a bioinformatic tool to analyze recent single-cell RNA-sequencing data in the cerebral cortex and hippocampus of adult mice. Our analysis revealed a widespread but also selective adult neuron-type expression with higher expression levels of Plppr3, Plppr1, and Plppr5 in GABAergic and Plppr4 and Plppr2 in glutamatergic neurons. PLPPR4 has been identified as a post-synaptic modulator of LPA levels in glutamatergic synapses operating via an uptake mechanism, to control LPA-dependent cortical network hyperexcitability. Using subcellular fractionation experiments, we found that both PLPPR4 and PLPPR3 are co-expressed in adult synaptosomal membranes. Furthermore, flow cytometry experiments in HEK293 cells showed comparable LPA uptake by PLPPR4 and PLPPR3, whereas PLPRR3, but not PLPPR4, induced also uptake of monoacylglycerol, the dephosphorylation product of LPA. We propose that synaptic LPA may be subject to both pre-synaptic and post-synaptic mechanisms of regulation by PLPPRs in addition to LPARs in developing and adult synapses.

Cristina Kroon and Britta Eickholt discuss current barriers to effective science communication and propose strategies to overcome them.

Brain function emerges from a highly complex network of specialized cells that are interlinked by billions of synapses. The synaptic connectivity between neurons is established between the elongated processes of their axons and dendrites or, together, neurites. To establish these connections, cellular neurites have to grow in highly specialized, cell-type dependent patterns covering extensive distances and connecting with thousands of other neurons. The outgrowth and branching of neurites are tightly controlled during development and are a commonly used functional readout of imaging in the neurosciences. Manual analysis of neuronal morphology from microscopy images, however, is very time intensive and prone to bias. Most automated analyses of neurons rely on reconstruction of the neuron as a whole without a semantic analysis of each neurite. A fully-automated classification of all neurites still remains unavailable in open-source software. Here we present a standalone, GUI-based software for batch-quantification of neuronal morphology in two-dimensional fluorescence micrographs of cultured neurons with minimal requirements for user interaction. Single neurons are first reconstructed into binarized images using a Hessian-based segmentation algorithm to detect thin neurite structures combined with intensity- and shape-based reconstruction of the cell body. Neurites are then classified into axon, dendrites and their branches of increasing order using a geodesic distance transform of the cell skeleton. The software was benchmarked against a published dataset and reproduced the phenotype observed after manual annotation. Our tool promises accelerated and improved morphometric studies of neuronal morphology by allowing for consistent and automated analysis of large datasets.

Phospholipid-phosphatase related protein 3 (PLPPR3, previously known as Plasticity Related Gene 2 or PRG2) belongs to a family of transmembrane proteins, highly expressed in neuronal development, which regulate critical growth processes in neurons. Prior work established crucial functions of PLPPR3 in axon guidance, filopodia formation and axon branching. However, little is known regarding the signaling events regulating PLPPR3 function. We identify here 26 high-confidence phosphorylation sites in the intracellular domain of PLPPR3 using mass spectrometry. Biochemical characterization established one of these – S351 – as a bona fide phosphorylation site of PKA. Experiments in neuronal cell lines suggest that phosphorylation of S351 does not regulate filopodia formation. Instead, it regulates binding to BASP1, a signaling molecule previously implicated in axonal growth and regeneration. Interestingly, both PLPPR3 intracellular domain and BASP1 enrich in presynapses in primary neurons. We propose that the presynaptic PLPPR3-BASP1 complex may function as novel signaling integrator at neuronal synapses.### Competing Interest StatementThe authors have declared no competing interest.

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.

The deepest layer of the cortex (layer 6b [L6b]) contains relatively few neurons, but it is the only cortical layer responsive to the potent wake-promoting neuropeptide orexin/hypocretin. Can these few neurons significantly influence brain state? Here, we show that L6b-photoactivation causes a surprisingly robust enhancement of attention-associated high-gamma oscillations and population spiking while abolishing slow waves in sleep-deprived mice. To explain this powerful impact on brain state, we investigated L6b's synaptic output using optogenetics, electrophysiology, and monoCaTChR ex vivo. We found powerful output in the higher-order thalamus and apical dendrites of L5 pyramidal neurons, via L1a and L5a, as well as in superior colliculus and L6 interneurons. L6b subpopulations with distinct morphologies and short- and long-term plasticities project to these diverse targets. The L1a-targeting subpopulation triggered powerful NMDA-receptor-dependent spikes that elicited burst firing in L5. We conclude that orexin/hypocretin-activated cortical neurons form a multifaceted, fine-tuned circuit for the sustained control of the higher-order thalamocortical system.

Preclinical cardiovascular research relies heavily on non-invasive in-vivo echocardiography in mice and rats to assess cardiac function and morphology, since the complex interaction of heart, circulation, and peripheral organs are challenging to mimic ex-vivo. While n-numbers of annually used laboratory animals worldwide approach 200 million, increasing efforts are made by basic scientists aiming to reduce animal numbers in cardiovascular research according to the 3R’s principle. The chicken egg is well-established as a physiological correlate and model for angiogenesis research but has barely been used to assess cardiac (patho-) physiology. Here, we tested whether the established in-ovo system of incubated chicken eggs interfaced with commercially available small animal echocardiography would be a suitable alternative test system in experimental cardiology. To this end, we defined a workflow to assess cardiac function in 8–13-day-old chicken embryos using a commercially available high resolution ultrasound system for small animals (Vevo 3100, Fujifilm Visualsonics Inc.) equipped with a high frequency probe (MX700; centre transmit: 50 MHz). We provide detailed standard operating procedures for sample preparation, image acquisition, data analysis, reference values for left and right ventricular function and dimensions, and inter-observer variabilities. Finally, we challenged incubated chicken eggs with two interventions well-known to affect cardiac physiology—metoprolol treatment and hypoxic exposure—to demonstrate the sensitivity of in-ovo echocardiography. In conclusion, in-ovo echocardiography is a feasible alternative tool for basic cardiovascular research, which can easily be implemented into the small animal research environment using existing infrastructure to replace mice and rat experiments, and thus, reduce use of laboratory animals according to the 3R principle.

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 specifically focussed on the role of Pten T366 phosphorylation and generated a knock-in mouse line in which Pten T366 was substituted with alanine ( Pten T366A/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 Pten T366/T366A mice exhibit cognitive deficits and selective sensory impairments, with significant differences in male individuals. We identify restricted cellular overgrowth of cortical neurons in Pten T366A/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 Pten T366A/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.

Brain function emerges from a highly complex network of specialized cells that are interlinked by billions of synapses. The synaptic connectivity between neurons is established between the elongated processes of their axons and dendrites or, together, neurites. To establish these billions of often far-reaching connections, cellular neurites have to grow in highly specialized, cell-type dependent patterns covering often mm distances and connecting with thousands of other neurons. The outgrowth and branching of neurites are tightly controlled during development and are a commonly used functional readout of imaging in the neurosciences. Manual analysis of neuronal morphology from microscopy images, however, is very time intensive and error prone. Especially fully automated segmentation and classification of all neurites remain unavailable in open-source software. Here we present a standalone, GUI-based software for batch-quantification of neuronal morphology in fluorescence micrographs with minimal requirements for user interaction. Neurons are segmented using a Hessian-based algorithm to detect thin neurite structures combined with intensity- and shape-based detection of the cell body. To measure the number of branches in a neuron accurately, rather than just determining branch points, neurites are classified into axon, dendrites and their branches of increasing order by their length using a geodesic distance transform of the cell skeleton. The software was benchmarked against a large, published dataset and reproduced the phenotype observed after manual annotation before. Our tool promises greatly accelerated and improved morphometric studies of neuronal morphology by allowing for consistent and automated analysis of large datasets.

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.