Parkinson's disease (PD) is a disorder characterized by the degeneration of certain neuronal populations in the central and peripheral nervous system. One of the hallmarks of the disease is the toxic accumulation of proteins within susceptible neurons due to major impairment in the degradation/clearance protein systems.RTP801 is a pro-apoptotic protein that is sufficient and necessary to induce neuronal death in cellular and animal models of PD. RTP801 is also upregulated in sporadic and parkin mutant PD brains. Here, we report the role of NEDD4, an E3 ligase involved in α-synuclein degradation and PD pathogenesis, in the regulation of RTP801 protein levels and toxicity. NEDD4 polyubiquitinates RTP801 in a cell-free system and in cellular cultures, and they interact physically. NEDD4 conjugates K63-ubiquitin chains to RTP801 and targets it for degradation. NEDD4 regulates RTP801 protein levels in both cultured cells and in the brain tissue. NEDD4 levels are diminished in nigral neurons from human PD brains. Interestingly, neurotoxin 6-OHDA decreases dramatically NEDD4 protein expression but elevates RTP801 protein levels. Moreover, NEDD4 protects neuronal PC12 cells from both 6-OHDA and RTP801-induced toxicity. In primary cortical neurons, NEDD4 knockdown toxicity is mediated by RTP801 since the double knockdown of RTP801 and NEDD4 abrogates the loss of phospho Ser473-Akt and the appearance of caspase-cleaved spectrin fragments.Thus, NEDD4 ligase regulates RTP801 and is sensitive to PD-associated oxidative stress. This suggests that NEDD4 loss of function in PD could contribute importantly into neuronal death by elevating RTP801.

Drebrin is a major actin-filament-binding protein localized in mature dendritic spines. A recent in vivo immunoelectron microscopic study suggests that drebrin content at each dendritic spine is regulated by some unknown mechanisms. In the present in vitro study, we examined whether glutamate stimulation alters drebrin content in dendritic spines. Glutamate stimulation induced disappearance of drebrin immunostaining from dendritic spines but led to appearance of drebrin immunostaining in dendritic shafts and somata. The glutamate-induced shift of drebrin immunostaining was blocked by an NMDA receptor antagonist. Immunoblot analyses showed that both the total and the cytosolic drebrin remained unchanged and revealed that the drebrin shift was not due to drebrin degradation. These findings indicate that NMDA receptor activation induces a shift in subcellular distribution of drebrin associated with actin filaments, and that the shift might be a molecular basis for actin reorganization accompanied with synaptic plasticity.

Homeostatic synaptic plasticity (HSP) is important for maintaining neurons' excitability within the dynamic range and for protecting neurons from unconstrained long-term potentiation that can cause breakdown of synapse specificity (Turrigiano [2008] Cell 135:422-435). Knowledge of the molecular mechanism underlying this phenomenon remains incomplete, especially for the rapid form of HSP. To test whether HSP in adulthood depends on an F-actin binding protein, drebrin A, mice deleted of the adult isoform of drebrin (DAKO) but retaining the embryonic isoform (drebrin E) were generated. HSP was assayed by determining whether the NR2A subunit of N-methyl-D-aspartate receptors (NMDARs) can rise rapidly within spines following the application of an NMDAR antagonist, D-APV, onto the cortical surface. Electron microscopic immunocytochemistry revealed that, as expected, the D-APV treatment of wild-type (WT) mouse cortex increased the proportion of NR2A-immunolabeled spines within 30 minutes relative to basal levels in hemispheres treated with an inactive enantiomer, L-APV. This difference was significant at the postsynaptic membrane and postsynaptic density (i.e., synaptic junction) as well as at nonsynaptic sites within spines and was not accompanied by spine size changes. In contrast, the D-APV treatment of DAKO brains did not augment NR2A labeling within the spine cytoplasm or at the synaptic junction, even though basal levels of NR2A were not significantly different from those of WT cortices. These findings indicate that drebrin A is required for the rapid (<30 minutes) form of HSP at excitatory synapses of adult cortices, whereas drebrin E is sufficient for maintaining basal NR2A levels within spines.

The Cre-loxP system is invaluable for spatial and temporal control of gene knockout, knockin, and reporter expression in the mouse nervous system. However, we report varying probabilities of unexpected germline recombination in distinct Cre driver lines designed for nervous system-specific recombination. Selective maternal or paternal germline recombination is showcased with sample Cre lines. Collated data reveal germline recombination in over half of 64 commonly used Cre driver lines, in most cases with a parental sex bias related to Cre expression in sperm or oocytes. Slight differences among Cre driver lines utilizing common transcriptional control elements affect germline recombination rates. Specific target loci demonstrated differential recombination; thus, reporters are not reliable proxies for another locus of interest. Similar principles apply to other recombinase systems and other genetically targeted organisms. We hereby draw attention to the prevalence of germline recombination and provide guidelines to inform future research for the neuroscience and broader molecular genetics communities.

Hibernation or torpor is considered a possible tool to protect astronauts from the deleterious effects of space radiation that contains high-energy heavy ions. We induced synthetic torpor in rats by injecting adenosine 5'-monophosphate monohydrate (5'-AMP) i.p. and maintaining in low ambient temperature room (+ 16 °C) for 6 h immediately after total body irradiation (TBI) with accelerated carbon ions (C-ions). The 5'-AMP treatment in combination with low ambient temperature reduced skin temperature and increased survival following 8 Gy C-ion irradiation compared to saline-injected animals. Analysis of the histology of the brain, liver and lungs showed that 5'-AMP treatment following 2 Gy TBI reduced activated microglia, Iba1 positive cells in the brain, apoptotic cells in the liver, and damage to the lungs, suggesting that synthetic torpor spares tissues from energetic ion radiation. The application of 5'-AMP in combination with either hypoxia or low temperature environment for six hours following irradiation of rat retinal pigment epithelial cells delays DNA repair and suppresses the radiation-induced mitotic catastrophe compared to control cells. We conclude that synthetic torpor protects animals from cosmic ray-simulated radiation and the mechanism involves both hypothermia and hypoxia.

Cas9 targets genomic loci with high specificity. For knockin with double-strand break repair, however, Cas9 often leads to unintended on-target knockout rather than intended edits. This imprecision is a barrier for direct in vivo editing where clonal selection is not feasible. In this study, we demonstrate a high-throughput workflow to comparatively assess on-target efficiency and precision of editing outcomes. Using this workflow, we screened combinations of donor DNA and Cas9 variants, as well as fusions to DNA repair proteins. This yielded novel high-performance double-strand break repair editing agents and combinatorial optimizations, yielding increases in knockin efficiency and precision. Cas9-RC, a novel fusion Cas9 flanked by eRad18 and CtIP[HE], increased knockin performance in vitro and in vivo in the developing mouse brain. Continued comparative assessment of editing efficiency and precision with this framework will further the development of high-performance editing agents for in vivo knockin and future genome therapeutics.

Nedd4-2 is an E3 ubiquitin ligase in which missense mutation is related to familial epilepsy, indicating its critical role in regulating neuronal network activity. However, Nedd4-2 substrates involved in neuronal network function have yet to be identified. Using mouse lines lacking Nedd4-1 and Nedd4-2, we identified astrocytic channel proteins inwardly rectifying K+ channel 4.1 (Kir4.1) and Connexin43 as Nedd4-2 substrates. We found that the expression of Kir4.1 and Connexin43 is increased upon conditional deletion of Nedd4-2 in astrocytes, leading to an elevation of astrocytic membrane ion permeability and gap junction activity, with a consequent reduction of γ-oscillatory neuronal network activity. Interestingly, our biochemical data demonstrate that missense mutations found in familial epileptic patients produce gain-of-function of the Nedd4-2 gene product. Our data reveal a process of coordinated astrocytic ion channel proteostasis that controls astrocyte function and astrocyte-dependent neuronal network activity and elucidate a potential mechanism by which aberrant Nedd4-2 function leads to epilepsy.

The establishment of axon-dendrite polarity is fundamental for radial migration of neurons during cortex development of mammals. We demonstrate that the E3 ubiquitin ligases WW-Containing Proteins 1 and 2 (Wwp1 and Wwp2) are indispensable for proper polarization of developing neurons. We show that knockout of Wwp1 and Wwp2 results in defects in axon-dendrite polarity in pyramidal neurons, and their aberrant laminar cortical distribution. Knockout of miR-140, encoded in Wwp2 intron, engenders phenotypic changes analogous to those upon Wwp1 and Wwp2 deletion. Intriguingly, transcription of the Wwp1 and Wwp2/miR-140 loci in neurons is induced by the transcription factor Sox9. Finally, we provide evidence that miR-140 supervises the establishment of axon-dendrite polarity through repression of Fyn kinase mRNA. Our data delineate a novel regulatory pathway that involves Sox9-[Wwp1/Wwp2/miR-140]-Fyn required for axon specification, acquisition of pyramidal morphology, and proper laminar distribution of cortical neurons.

Drebrin is an actin-binding protein that is preferentially expressed in the brain. It is highly localized in dendritic spines and regulates spine shapes. The embryonic-type (drebrin E) is expressed in the embryonic and early postnatal brain and is replaced by the adult-type (drebrin A) during development. In parallel, NMDA receptor (NMDAR)-dependent long-term depression (LTD) of synaptic transmission, induced by low-frequency stimulation (LFS), is dominant in the immature brain and decreases during development. Here, we report that drebrin regulates NMDAR-dependent and group 1 metabotropic glutamate receptor (mGluR)-dependent LTD induction in the hippocampus. While LFS induced NMDAR-dependent LTD in the developing hippocampus in wild-type (WT) mice, it did not induce LTD in developing drebrin E and A double knockout (DXKO) mice, indicating that drebrin is required for NMDAR-dependent LTD. On the other hand, LFS induced robust LTD dependent on mGluR5, one of group 1 mGluRs, in both developing and adult brains of drebrin A knockout (DAKO) mice, in which drebrin E is expressed throughout development and adulthood. Agonist-induced mGluR-dependent LTD was normal in WT and DXKO mice; however, it was enhanced in DAKO mice. Also, mGluR1, another group 1 mGluR, was involved in agonist-induced mGluR-dependent LTD in DAKO mice. These data suggest that abnormal drebrin E expression in adults promotes group 1 mGluR-dependent LTD induction. Therefore, while drebrin expression is critical for NMDAR-dependent LTD induction, developmental conversion from drebrin E to drebrin A prevents robust group 1 mGluR-dependent LTD.

Extracellular phosphorylation of proteins was suggested in the late 1800s when it was demonstrated that casein contains phosphate. More recently, extracellular kinases that phosphorylate extracellular serine, threonine, and tyrosine residues of numerous proteins have been identified. However, the functional significance of extracellular phosphorylation of specific residues in the nervous system is poorly understood. Here we show that synaptic accumulation of GluN2B-containing N-methyl-D-aspartate receptors (NMDARs) and pathological pain are controlled by ephrin-B-induced extracellular phosphorylation of a single tyrosine (p*Y504) in a highly conserved region of the fibronectin type III (FN3) domain of the receptor tyrosine kinase EphB2. Ligand-dependent Y504 phosphorylation modulates the EphB-NMDAR interaction in cortical and spinal cord neurons. Furthermore, Y504 phosphorylation enhances NMDAR localization and injury-induced pain behavior. By mediating inducible extracellular interactions that are capable of modulating animal behavior, extracellular tyrosine phosphorylation of EphBs may represent a previously unknown class of mechanism mediating protein interaction and function.

Dendritic spines are the major transmitter reception compartments of glutamatergic synapses in most principal neurons of the mammalian brain and play a key role in the function of nerve cell circuits. The formation of functional spine synapses is thought to be critically dependent on presynaptic glutamatergic signaling. By analyzing CA1 pyramidal neurons in mutant hippocampal slice cultures that are essentially devoid of presynaptic transmitter release, we demonstrate that the formation and maintenance of dendrites and functional spines are independent of synaptic glutamate release.

Dendritic filopodia select synaptic partner axons by interviewing the cell surface of potential targets, but how filopodia decipher the complex pattern of adhesive and repulsive molecular cues to find appropriate contacts is unknown. Here, we demonstrate in cortical neurons that a single cue is sufficient for dendritic filopodia to reject or select specific axonal contacts for elaboration as synaptic sites. Super-resolution and live-cell imaging reveals that EphB2 is located in the tips of filopodia and at nascent synaptic sites. Surprisingly, a genetically encoded indicator of EphB kinase activity, unbiased classification, and a photoactivatable EphB2 reveal that simple differences in the kinetics of EphB kinase signaling at the tips of filopodia mediate the choice between retraction and synaptogenesis. This may enable individual filopodia to choose targets based on differences in the activation rate of a single tyrosine kinase, greatly simplifying the process of partner selection and suggesting a general principle.

Protein synthesis must be finely tuned in the developing nervous system as the final essential step of gene expression. This study investigates the architecture of ribosomes from the neocortex during neurogenesis, revealing Ebp1 as a high-occupancy 60S peptide tunnel exit (TE) factor during protein synthesis at near-atomic resolution by cryoelectron microscopy (cryo-EM). Ribosome profiling demonstrated Ebp1-60S binding is highest during start codon initiation and N-terminal peptide elongation, regulating ribosome occupancy of these codons. Membrane-targeting domains emerging from the 60S tunnel, which recruit SRP/Sec61 to the shared binding site, displace Ebp1. Ebp1 is particularly abundant in the early-born neural stem cell (NSC) lineage and regulates neuronal morphology. Ebp1 especially impacts the synthesis of membrane-targeted cell adhesion molecules (CAMs), measured by pulsed stable isotope labeling by amino acids in cell culture (pSILAC)/bioorthogonal noncanonical amino acid tagging (BONCAT) mass spectrometry (MS). Therefore, Ebp1 is a central component of protein synthesis, and the ribosome TE is a focal point of gene expression control in the molecular specification of neuronal morphology during development.

Abnormal metabotropic glutamate receptor (mGluR) activity could cause brain disorders; however, its regulation has not yet been fully understood. Here, we report that protein kinase N1 (PKN1), a protein kinase expressed predominantly in neurons in the brain, normalizes group 1 mGluR function by upregulating a neuronal glutamate transporter, excitatory amino acid transporter 3 (EAAT3), and supports silent synapse activation. Knocking out PKN1a, the dominant PKN1 subtype in the brain, unmasked abnormal input-nonspecific mGluR-dependent long-term depression (mGluR-LTD) and AMPA receptor (AMPAR) silencing in the developing hippocampus. mGluR-LTD was mimicked by inhibiting glutamate transporters in wild-type mice. Knocking out PKN1a decreased hippocampal EAAT3 expression and PKN1 inhibition reduced glutamate uptake through EAAT3. Also, synaptic transmission was immature; there were more silent synapses and fewer spines with shorter postsynaptic densities in PKN1a knockout mice than in wild-type mice. Thus, PKN1 plays a critical role in regulation of synaptic maturation by upregulating EAAT3 expression.

Split reporter protein-based genetic section systems are widely used to identify and characterize protein-protein interactions (PPI). The assembly of split markers that antagonize toxins, rather than required for synthesis of missing metabolites, facilitates the seeding of high density of cells and selective growth. Here we present a newly developed split chloramphenicol acetyltransferase (split-CAT) -based genetic selection system. The N terminus fragment of CAT is fused downstream of the protein of interest and the C terminus fragment is tethered upstream to its postulated partner. We demonstrate the system's advantages for the study of PPIs. Moreover, we show that co-expression of a functional ubiquitylation cascade where the target and ubiquitin are tethered to the split-CAT fragments results in ubiquitylation-dependent selective growth. Since proteins do not have to be purified from the bacteria and due to the high sensitivity of the split-CAT reporter, detection of challenging protein cascades and post-translation modifications is enabled. In addition, we demonstrate that the split-CAT system responds to small molecule inhibitors and molecular glues (GLUTACs). The absence of ubiquitylation-dependent degradation and deubiquitylation in E. coli significantly simplify the interpretation of the results. We harnessed the developed system to demonstrate that like NEDD4, UBE3B also undergoes self-ubiquitylation-dependent inactivation. We show that self-ubiquitylation of UBE3B on K665 induces oligomerization and inactivation in yeast and mammalian cells respectively. Finally, we showcase the advantages of split-CAT in the study of human diseases by demonstrating that mutations in UBE3B that cause Kaufman oculocerebrofacial syndrome exhibit clear E. coli growth phenotypes.

Munc13 catalyzes the transit of syntaxin from a closed complex with Munc18 into the ternary SNARE complex. Here we report a new function of Munc13, independent of Munc18: it promotes the proper syntaxin/synaptobrevin subconfiguration during assembly of the ternary SNARE complex. In cooperation with Munc18, Munc13 additionally ensures the proper syntaxin/SNAP-25 subconfiguration. In a reconstituted fusion assay with SNAREs, complexin, and synaptotagmin, inclusion of both Munc13 and Munc18 quadruples the Ca2+-triggered amplitude and achieves Ca2+ sensitivity at near-physiological concentrations. In Munc13-1/2 double-knockout neurons, expression of a constitutively open mutant of syntaxin could only minimally restore neurotransmitter release relative to Munc13-1 rescue. Together, the physiological functions of Munc13 may be related to regulation of proper SNARE complex assembly.

Beyond its role in parturition and lactation, oxytocin influences higher brain processes that control social behavior of mammals, and perturbed oxytocin signaling has been linked to the pathogenesis of several psychiatric disorders. However, it is still largely unknown how oxytocin exactly regulates neuronal function. We show that early, transient oxytocin exposure in vitro inhibits the development of hippocampal glutamatergic neurons, leading to reduced dendrite complexity, synapse density, and excitatory transmission, while sparing GABAergic neurons. Conversely, genetic elimination of oxytocin receptors increases the expression of protein components of excitatory synapses and excitatory synaptic transmission in vitro. In vivo, oxytocin-receptor-deficient hippocampal pyramidal neurons develop more complex dendrites, which leads to increased spine number and reduced γ-oscillations. These results indicate that oxytocin controls the development of hippocampal excitatory neurons and contributes to the maintenance of a physiological excitation/inhibition balance, whose disruption can cause neurobehavioral disturbances.

Soybean isoflavones, such as genistein, daidzein, and its metabolite, S-equol, are widely known as phytoestrogens. Their biological actions are thought to be exerted via the estrogen signal transduction pathway. Estrogens, such as 17β-estradiol (E2), play a crucial role in the development and functional maintenance of the central nervous system. E2 bind to the nuclear estrogen receptor (ER) and regulates morphogenesis, migration, functional maturation, and intracellular metabolism of neurons and glial cells. In addition to binding to nuclear ER, E2 also binds to the G-protein-coupled estrogen receptor (GPER) and activates the nongenomic estrogen signaling pathway. Soybean isoflavones also bind to the ER and GPER. However, the effect of soybean isoflavone on brain development, particularly glial cell function, remains unclear. We examined the effects of soybean isoflavones using an astrocyte-enriched culture and astrocyte-derived C6 clonal cells. Isoflavones increased glial cell migration. This augmentation was suppressed by co-exposure with G15, a selective GPER antagonist, or knockdown of GPER expression using RNA interference. Isoflavones also activated actin cytoskeleton arrangement via increased actin polymerization and cortical actin, resulting in an increased number and length of filopodia. Isoflavones exposure increased the phosphorylation levels of FAK (Tyr397 and Tyr576/577), ERK1/2 (Thr202/Tyr204), Akt (Ser473), and Rac1/cdc42 (Ser71), and the expression levels of cortactin, paxillin and ERα. These effects were suppressed by knockdown of the GPER. Co-exposure of isoflavones to the selective RhoA inhibitor, rhosin, selective Cdc42 inhibitor, casin, or Rac1/Cdc42 inhibitor, ML-141, decreased the effects of isoflavones on cell migration. These findings indicate that soybean isoflavones exert their action via the GPER to activate the PI3K/FAK/Akt/RhoA/Rac1/Cdc42 signaling pathway, resulting in increased glial cell migration. Furthermore, in silico molecular docking studies to examine the binding mode of isoflavones to the GPER revealed the possibility that isoflavones bind directly to the GPER at the same position as E2, further confirming that the effects of the isoflavones are at least in part exerted via the GPER signal transduction pathway. The findings of the present study indicate that isoflavones may be an effective supplement to promote astrocyte migration in developing and/or injured adult brains.

The seat of higher-order cognitive abilities in mammals, the neocortex, is a complex structure, organized in several layers. The different subtypes of principal neurons are distributed in precise ratios and at specific positions in these layers and are generated by the same neural progenitor cells (NPCs), steered by a spatially and temporally specified combination of molecular cues that are incompletely understood. Recently, we discovered that an alternatively spliced isoform of the TrkC receptor lacking the kinase domain, TrkC-T1, is a determinant of the corticofugal projection neuron (CFuPN) fate. Here, we show that the finely tuned balance between TrkC-T1 and the better known, kinase domain-containing isoform, TrkC-TK+, is cell type-specific in the developing cortex and established through the antagonistic actions of two RNA-binding proteins, Srsf1 and Elavl1. Moreover, our data show that Srsf1 promotes the CFuPN fate and Elavl1 promotes the callosal projection neuron (CPN) fate in vivo via regulating the distinct ratios of TrkC-T1 to TrkC-TK+. Taken together, we connect spatio-temporal expression of Srsf1 and Elavl1 in the developing neocortex with the regulation of TrkC alternative splicing and transcript stability and neuronal fate choice, thus adding to the mechanistic and functional understanding of alternative splicing in vivo.

PCSK9 induces lysosomal degradation of the low-density lipoprotein (LDL) receptor (LDLR) in the liver, hereby preventing removal of LDL cholesterol from the circulation. Accordingly, PCSK9 inhibitory antibodies and siRNA potently reduce LDL cholesterol to unprecedented low levels and are approved for treatment of hypercholesterolemia. In addition, PCSK9 inactivation alters the levels of several other circulating lipid classes and species. Brain function is critically influenced by cholesterol and lipid composition. However, it remains unclear how the brain is affected long-term by the reduction in circulating lipids as achieved with potent lipid lowering therapeutics such as PCSK9 inhibitors. Furthermore, it is unknown if locally expressed PCSK9 affects neuronal circuits through regulation of receptor levels. We have studied the effect of lifelong low peripheral cholesterol levels on brain lipid composition and behavior in adult PCSK9 KO mice. In addition, we studied the effect of PCSK9 on neurons in culture and in vivo in the developing cerebral cortex. We found that PCSK9 reduced LDLR and neurite complexity in cultured neurons, but neither PCSK9 KO nor overexpression affected cortical development in vivo. Interestingly, PCSK9 deficiency resulted in changes of several lipid classes in the adult cortex and cerebellum. Despite the observed changes, PCSK9 KO mice had unchanged behavior compared to WT controls. In conclusion, our findings demonstrate that altered PCSK9 levels do not compromise brain development or function in mice, and are in line with clinical trials showing that PCSK9 inhibitors have no adverse effects on cognitive function.