Dendritic Protein Research Articles

Cyclin-Dependent Kinase 5 Regulates the Polarized Trafficking of Neuropeptide-Containing Dense-Core Vesicles in Caenorhabditis elegans Motor Neurons

The polarized trafficking of axonal and dendritic proteins is essential for the structure and function of neurons. Cyclin-dependent kinase 5 (CDK-5) and its activator CDKA-1/p35 regulate diverse aspects of nervous system development and function. Here, we show that CDK-5 and CDKA-1/p35 are required for the polarized distribution of neuropeptide-containing dense-core vesicles (DCVs) in Caenorhabditis elegans cholinergic motor neurons. In cdk-5 or cdka-1/p35 mutants, the predominantly axonal localization of DCVs containing INS-22 neuropeptides was disrupted and DCVs accumulated in dendrites. Time-lapse microscopy in DB class motor neurons revealed decreased trafficking of DCVs in axons and increased trafficking and accumulation of DCVs in cdk-5 mutant dendrites. The polarized distribution of several axonal and dendritic markers, including synaptic vesicles, was unaltered in cdk-5 mutant DB neurons. We found that microtubule polarity is plus-end out in axons and predominantly minus-end out in dendrites of DB neurons. Surprisingly, cdk-5 mutants had increased amounts of plus-end-out microtubules in dendrites, suggesting that CDK-5 regulates microtubule orientation. However, these changes in microtubule polarity are not responsible for the increased trafficking of DCVs into dendrites. Genetic analysis of cdk-5 and the plus-end-directed axonal DCV motor unc-104/KIF1A suggest that increased trafficking of UNC-104 into dendrites cannot explain the dendritic DCV accumulation. Instead, we found that mutations in the minus-end-directed motor cytoplasmic dynein, completely block the increased DCVs observed in cdk-5 mutant dendrites without affecting microtubule polarity. We propose a model in which CDK-5 regulates DCV polarity by both promoting DCV trafficking in axons and preventing dynein-dependent DCV trafficking into dendrites.

The Local Transcriptome in the Synaptic Neuropil Revealed by Deep Sequencing and High-Resolution Imaging

In neurons, dendritic protein synthesis is required for many forms of long-term synaptic plasticity. The population of mRNAs that are localized to dendrites, however, remains sparsely identified. Here, we use deep sequencing to identify the mRNAs resident in the synaptic neuropil in the hippocampus. Analysis of a neuropil data set yielded a list of 8,379 transcripts of which 2,550 are localized in dendrites and/or axons. Using a fluorescent barcode strategy to label individual mRNAs, we show that their relative abundance in the neuropil varies over 3 orders of magnitude. High-resolution in situ hybridization validated the presence of mRNAs in both cultured neurons and hippocampal slices. Among the many mRNAs identified, we observed a large fraction of known synaptic proteins including signaling molecules, scaffolds and receptors. These results reveal a previously unappreciated enormous potential for the local protein synthesis machinery to supply, maintain and modify the dendritic and synaptic proteome.

Adeno‐Associated Viral Vectors for Anterograde Axonal Tracing with Fluorescent Proteins in Nontransgenic and Cre Driver Mice

Harnessing the natural ability of viruses to infect post-mitotic cells such as neurons has provided an explosion of new methods to manipulate and reconstruct neural circuits in vivo. Here we describe the use of recombinant adeno-associated viral vectors (rAAV) for axonal tract tracing in nontransgenic and transgenic Cre driver mice. Two protocols are presented for stereotactic-guided placement of rAAV vectors into the live mouse brain using iontophoretic or nanoliter pressure injections. The methods discussed here will result in expression of fluorescent proteins in cell bodies, dendrites, and axons in targeted neurons, and can be easily adapted to address various experimental questions.

Association of microtubule associated protein-2, synaptophysin, and apolipoprotein E mRNA and protein levels with cognition and anxiety levels in aged female rhesus macaques

The dendritic protein microtubule associated protein 2 (MAP-2), the presynaptic marker synaptophysin (SYN), and apolipoprotein E (APOE), a protein which plays a role in lipid transport and metabolism and affects synaptic activity show changes with age. We analyzed post-mortem tissue from aged female rhesus macaques cognitively tested in a spatial maze and classified as good spatial performers (GSP) or poor spatial performers (PSP) and behaviorally tested in a playroom and classified as bold or reserved animals. MAP2, SYN, and APOE mRNA and protein levels in the prefrontal cortex (PFC), hippocampus, and amygdala, were assessed using qRT-PCR and western blot. In the amygdala, bold monkeys had higher levels of MAP2 and SYN mRNA than reserved monkeys. MAP2 mRNA correlated positively with amygdala size on the right, left, and combined left and right sides, while SYN mRNA levels correlated positively with the size of the right amygdala. In the hippocampus, SYN and APOE protein levels were higher in GSP than PSP animals. Thus, in aged nonhuman primates, classification of measures of anxiety is associated with differences in selected mRNA, but not protein, levels. In contrast, classification of cognitive performance is associated with differences in selected protein, but not mRNA, levels.

Dendritically targeted Bdnf mRNA is essential for energy balance and response to leptin

Mutations in the Bdnf gene, which produces transcripts with either short or long 3′ untranslated regions (3′UTRs), cause human obesity; however, the precise role of BDNF in the regulation of energy balance remains unknown. Here we show the relationship between long 3′UTR Bdnf mRNA, leptin, neuronal activation and body weight. We found that long 3′UTR Bdnf mRNA was enriched in dendrites of hypothalamic neurons and that insulin and leptin could stimulate its translation in dendrites. Furthermore, mice harboring a truncated long Bdnf 3′UTR developed severe hyperphagic obesity, which was completely rescued by viral expression of long 3′UTR Bdnf mRNA in the hypothalamus. In these animals the ability of leptin to activate hypothalamic neurons and to inhibit food intake was compromised despite normal activation of leptin receptors. These results reveal a novel mechanism linking leptin action to BDNF expression during hypothalamic-mediated regulation of body weight, while also implicating dendritic protein synthesis in this process.

Noradrenergic antidepressant responses to desipramine in vivo are reciprocally regulated by arrestin3 and spinophilin

Many antidepressant drugs, including the tricyclic antidepressant desipramine (DMI), are broadly understood to function by modulating central noradrenergic neurotransmission. α2 adrenergic receptors (α2ARs) are key regulators of the noradrenergic system, and previous work has implicated α2ARs in mediating the antidepressant activity of DMI in the rodent forced swim test (FST). However, little is known about intracellular regulators of antidepressant drug action. α2AR function is tightly regulated by its intracellular interacting partners arrestin and the dendritic protein spinophilin. We have previously established the competitive and reciprocal nature of these interacting proteins at the α2AR in the context of classic agonist effects, and have shown DMI to be a direct arrestin-biased ligand at the receptor. In the present study, we report that mice deficient in the α2AAR subtype lack DMI-induced antidepressant behavioral effects in the FST. As well, mice deficient in arrestin3 lack antidepressant response to DMI, while spinophilin-null mice have enhanced antidepressant response to DMI compared with wild-type controls, indicating that this α2AAR-mediated response is reciprocally regulated by arrestin and spinophilin. The characteristic of α2AAR-dependence and arrestin3 involvement was shared by the antidepressant effect of the classic α2AR agonist clonidine but not the non-tricyclic norepinephrine reuptake inhibitor reboxetine, supporting a model whereby DMI exerts its antidepressant effect through direct engagement of the α2AAR and arrestin3. Our results implicate arrestin- and spinophilin-mediated regulation of the α2AAR in the pharmacology of the noradrenergic antidepressant DMI, and suggest that manipulation of this mode of receptor regulation may represent a novel and viable therapeutic strategy.

Conditional RARα knockout mice reveal acute requirement for retinoic acid and RARα in homeostatic plasticity

All-trans retinoic acid (RA) plays important roles in brain development through regulating gene transcription. Recently, a novel post-developmental role of RA in mature brain was proposed. Specifically, RA rapidly enhanced excitatory synaptic transmission independent of transcriptional regulation. RA synthesis was induced when excitatory synaptic transmission was chronically blocked, and RA then activated dendritic protein synthesis and synaptic insertion of homomeric GluA1 AMPA receptors, thereby compensating for the loss of neuronal activity in a homeostatic fashion. This action of RA was suggested to be mediated by its canonical receptor RARα but no genetic evidence was available. Thus, we here tested the fundamental requirement of RARα in homeostatic plasticity using conditional RARα knockout (KO) mice, and additionally performed a structure-function analysis of RARα. We show that acutely deleting RARα in neurons eliminated RA's effect on excitatory synaptic transmission, and inhibited activity blockade-induced homeostatic synaptic plasticity. By expressing various RARα rescue constructs in RARα KO neurons, we found that the DNA-binding domain of RARα was dispensable for its role in regulating synaptic strength, further supporting the notion that RA and RARα act in a non-transcriptional manner in this context. By contrast, the ligand-binding domain (LBD) and the mRNA-binding domain (F-domain) are both necessary and sufficient for the function of RARα in homeostatic plasticity. Furthermore, we found that homeostatic regulation performed by the LBD/F-domains leads to insertion of calcium-permeable AMPA receptors. Our results confirm with unequivocal genetic approaches that RA and RARα perform essential non-transcriptional functions in regulating synaptic strength, and establish a functional link between the various domains of RARα and their involvement in regulating protein synthesis and excitatory synaptic transmission during homeostatic plasticity.

The clasp between NetrinG and NGL becomes crystal clear

The clasp between NetrinG and NGL becomes crystal clear

Lack of Pur-alpha alters postnatal brain development and causes megalencephaly

Pur-alpha (Purα) plays an important role in a variety of cellular processes including transcriptional regulation, cell proliferation and oncogenic transformation. To better understand the role of Purα in the developing and mature brain, we generated Purα-deficient mice, which we were able to raise to the age of six months. Purα(-/-) mice were born with no obvious pathological condition. We obtained convincing evidence that lack of Purα prolongs the postnatal proliferation of neuronal precursor cells both in the hippocampus and in the cerebellum, however, without affecting the overall number of postmitotic neurons. Independent of these findings, we observed alterations in the expression and distribution of the dendritic protein MAP2, the translation of which has been proposed previously to be Purα-dependent. At the age of 2 weeks, Purα(-/-) mice generated a continuous tremor which persisted throughout lifetime. Finally, adult Purα(-/-) mice displayed a megalencephaly and histopathological findings including axonal swellings and hyperphosphorylation of neurofilaments. Our studies underline the importance of Purα in the proliferation of neuronal precursor cells during postnatal brain development and suggest a role for Purα in the regulation of the expression and cellular distribution of dendritic and axonal proteins. Since recent studies implicate a link between Purα and the fragile X tremor/ataxia syndrome, our Purα(-/-) mouse model will provide new opportunities for understanding the mechanisms of neurodegeneration.

Barriers to Diffusion in Dendrites and Estimation of Calcium Spread Following Synaptic Inputs

The motion of ions, molecules or proteins in dendrites is restricted by cytoplasmic obstacles such as organelles, microtubules and actin network. To account for molecular crowding, we study the effect of diffusion barriers on local calcium spread in a dendrite. We first present a model based on a dimension reduction approach to approximate a three dimensional diffusion in a cylindrical dendrite by a one-dimensional effective diffusion process. By comparing uncaging experiments of an inert dye in a spiny dendrite and in a thin glass tube, we quantify the change in diffusion constants due to molecular crowding as Dcyto/Dwater = 1/20. We validate our approach by reconstructing the uncaging experiments using Brownian simulations in a realistic 3D model dendrite. Finally, we construct a reduced reaction-diffusion equation to model calcium spread in a dendrite under the presence of additional buffers, pumps and synaptic input. We find that for moderate crowding, calcium dynamics is mainly regulated by the buffer concentration, but not by the cytoplasmic crowding, dendritic spines or synaptic inputs. Following high frequency stimulations, we predict that calcium spread in dendrites is limited to small microdomains of the order of a few microns (<5 μm).

Expression of Semaphorin 4F in neurons and brain oligodendrocytes and the regulation of oligodendrocyte precursor migration in the optic nerve

Semaphorins are secreted or membrane-anchored proteins that play critical roles in neural development and adult brain plasticity. Sema4F is a transmembrane semaphorin found on glutamatergic synapses, in which it is attached to the PSD-95-scaffolding protein. Here we further examined the expression of Sema4F by raising specific antibodies. We show that Sema4F protein is widely expressed by neurons during neural development and in the adult brain. We also demonstrate a preferential localization of this protein in postsynaptic dendrites. Moreover, Sema4F is expressed not only by neurons but also by oligodendrocyte precursors in the optic nerve and along the migratory pathways of oligodendroglial cells, and also by subsets of postnatal oligodendroglial cells in the brain. Finally, in vitro experiments demonstrate that endogenous Sema4F expressed by brain cells of oligodendroglial lineage regulates the outgrowth migration of oligodendrocyte precursors and promotes their differentiation. The present data extend our knowledge about the expression of Sema4F and uncover a novel function in the control of oligodendrocyte precursor migration in the developing brain.

A stochastic model describing transport of PSD-95 molecules in spiny dendrites provides the basis for synaptic plasticity

Molecules in neurons are in a state far from equilibrium. Therefore their transport properties are strongly affected by fluctuations. We present a model of stochastic molecular transport in neurons which have their synapses located in the spines of a dendrite. In this model we assume that the molecules perform a random walk between the spines that trap the walkers. If the molecules are assumed to interact with each other inside the spines, the trapping time in each spine depends on the number of molecules in the respective trap. The corresponding mathematical problem has non-trivial solutions even in the absence of external disorder due to self-organization phenomena. We obtain the stationary distributions of the number of walkers in the traps for different kinds of on-site interactions between the walkers and furthermore analyze how birth and death processes of the random walkers affect these distributions. We apply this model to describe the dynamics of the PSD-95 proteins in spiny dendrites. PSD-95 is the most abundant molecule in the post-synaptic density (PSD) located in the spines. It is observed that these molecules have high turnover rates and that neighboring spines are constantly exchanging individual molecules. Thus we predict the distribution of PSD-95 cluster sizes that determine the size of the synapse and thus the synaptic strength. Finally, we show that in the model non-equilibrium inter-spine dynamics of PSD-95 molecules can provide the basis for locally controlled synaptic plasticity through activity-dependent ubiquitinization of PSD-95 molecules.

Proteomics, Ultrastructure, and Physiology of Hippocampal Synapses in a Fragile X Syndrome Mouse Model Reveal Presynaptic Phenotype

Fragile X syndrome (FXS), the most common form of hereditary mental retardation, is caused by a loss-of-function mutation of the Fmr1 gene, which encodes fragile X mental retardation protein (FMRP). FMRP affects dendritic protein synthesis, thereby causing synaptic abnormalities. Here, we used a quantitative proteomics approach in an FXS mouse model to reveal changes in levels of hippocampal synapse proteins. Sixteen independent pools of Fmr1 knock-out mice and wild type mice were analyzed using two sets of 8-plex iTRAQ experiments. Of 205 proteins quantified with at least three distinct peptides in both iTRAQ series, the abundance of 23 proteins differed between Fmr1 knock-out and wild type synapses with a false discovery rate (q-value) <5%. Significant differences were confirmed by quantitative immunoblotting. A group of proteins that are known to be involved in cell differentiation and neurite outgrowth was regulated; they included Basp1 and Gap43, known PKC substrates, and Cend1. Basp1 and Gap43 are predominantly expressed in growth cones and presynaptic terminals. In line with this, ultrastructural analysis in developing hippocampal FXS synapses revealed smaller active zones with corresponding postsynaptic densities and smaller pools of clustered vesicles, indicative of immature presynaptic maturation. A second group of proteins involved in synaptic vesicle release was up-regulated in the FXS mouse model. In accordance, paired-pulse and short-term facilitation were significantly affected in these hippocampal synapses. Together, the altered regulation of presynaptically expressed proteins, immature synaptic ultrastructure, and compromised short-term plasticity points to presynaptic changes underlying glutamatergic transmission in FXS at this stage of development.

Reversible Inhibition of PSD-95 mRNA Translation by miR-125a, FMRP Phosphorylation, and mGluR Signaling

The molecular mechanism for how RISC and microRNAs selectively and reversibly regulate mRNA translation in response to receptor signaling is unknown but could provide a means for temporal and spatial control of translation. Here we show that miR-125a targeting PSD-95 mRNA allows reversible inhibition of translation and regulation by gp1 mGluR signaling. Inhibition of miR-125a increased PSD-95 levels in dendrites and altered dendritic spine morphology. Bidirectional control of PSD-95 expression depends on miR-125a and FMRP phosphorylation status. miR-125a levels at synapses and its association with AGO2 are reduced in Fmr1 KO. FMRP phosphorylation promotes the formation of an AGO2-miR-125a inhibitory complex on PSD-95 mRNA, whereas mGluR signaling of translation requires FMRP dephosphorylation and release of AGO2 from the mRNA. These findings reveal a mechanism whereby FMRP phosphorylation provides a reversible switch for AGO2 and microRNA to selectively regulate mRNA translation at synapses in response to receptor activation.

A Role for Myosin VI in the Localization of Axonal Proteins

In neurons polarized trafficking of vesicle-bound membrane proteins gives rise to the distinct molecular composition and functional properties of axons and dendrites. Despite their central role in shaping neuronal form and function, surprisingly little is known about the molecular processes that mediate polarized targeting of neuronal proteins. Recently, the plus-end-directed motor Myosin Va was shown to play a critical role in targeting of transmembrane proteins to dendrites; however, the role of myosin motors in axonal targeting is unknown. Here we show that Myosin VI, a minus-end-directed motor, plays a vital role in the enrichment of proteins on the surface of axons. Engineering non-neuronal proteins to interact with Myosin VI causes them to become highly concentrated at the axonal surface in dissociated rat cortical neurons. Furthermore, disruption of either Myosin VI function or expression leads to aberrant dendritic localization of axonal proteins. Myosin VI mediates the enrichment of proteins on the axonal surface at least in part by stimulating dendrite-specific endocytosis, a mechanism that has been shown to underlie the localization of many axonal proteins. In addition, a version of Channelrhodopsin 2 that was engineered to bind to Myosin VI is concentrated at the surface of the axon of cortical neurons in mice in vivo, suggesting that it could be a useful tool for probing circuit structure and function. Together, our results indicate that myosins help shape the polarized distributions of both axonal and dendritic proteins.

Insulin Signaling and Autism

ORIGINAL RESEARCH article Front. Endocrinol., 14 October 2011Sec. Cellular Endocrinology https://doi.org/10.3389/fendo.2011.00054

Fragile X Protein FMRP Is Required for Homeostatic Plasticity and Regulation of Synaptic Strength by Retinoic Acid

Homeostatic synaptic plasticity adjusts the strength of synapses during global changes in neural activity, thereby stabilizing the overall activity of neural networks. Suppression of synaptic activity increases synaptic strength by inducing synthesis of retinoic acid (RA), which activates postsynaptic synthesis of AMPA-type glutamate receptors (AMPARs) in dendrites and promotes synaptic insertion of newly synthesized AMPARs. Here, we show that fragile X mental retardation protein (FMRP), an RNA-binding protein that regulates dendritic protein synthesis, is essential for increases in synaptic strength induced by RA or by blockade of neural activity in the mouse hippocampus. Although activity-dependent RA synthesis is maintained in Fmr1 knock-out neurons, RA-dependent dendritic translation of GluR1-type AMPA receptors is impaired. Intriguingly, FMRP is only required for the form of homeostatic plasticity that is dependent on both RA signaling and local protein synthesis. Postsynaptic expression of wild-type or mutant FMRP(I304N) in knock-out neurons reduced the total, surface, and synaptic levels of AMPARs, implying a role for FMRP in regulating AMPAR abundance. Expression of FMRP lacking the RGG box RNA-binding domain had no effect on AMPAR levels. Importantly, postsynaptic expression of wild-type FMRP, but not FMRP(I304N) or FMRPΔRGG, restored synaptic scaling when expressed in knock-out neurons. Together, these findings identify an unanticipated role for FMRP in regulating homeostatic synaptic plasticity downstream of RA. Our results raise the possibility that at least some of the symptoms of fragile X syndrome reflect impaired homeostatic plasticity and impaired RA signaling.

Local Presynaptic Activity Gates Homeostatic Changes in Presynaptic Function Driven by Dendritic BDNF Synthesis

Homeostatic synaptic plasticity is important for maintaining stability of neuronal function, but heterogeneous expression mechanisms suggest that distinct facets of neuronal activity may shape the manner in which compensatory synaptic changes are implemented. Here, we demonstrate that local presynaptic activity gates a retrograde form of homeostatic plasticity induced by blockade of AMPA receptors (AMPARs) in cultured hippocampal neurons. We show that AMPAR blockade produces rapid (<3 hr) protein synthesis-dependent increases in both presynaptic and postsynaptic function and that the induction of presynaptic, but not postsynaptic, changes requires coincident local activity in presynaptic terminals. This "state-dependent" modulation of presynaptic function requires postsynaptic release of brain-derived neurotrophic factor (BDNF) as a retrograde messenger, which is locally synthesized in dendrites in response to AMPAR blockade. Taken together, our results reveal a local crosstalk between active presynaptic terminals and postsynaptic signaling that dictates the manner by which homeostatic plasticity is implemented at synapses.

Dendritic Sodium Channels Regulate Network Integration in Globus Pallidus Neurons: A Modeling Study

The globus pallidus (GP) predominantly contains GABAergic projection neurons that occupy a central position in the indirect pathway of the basal ganglia. They have long dendrites that can extend through one-half the diameter of the GP in rats, potentially enabling convergence and interaction between segregated basal ganglia circuits. Because of the length and fine diameter of GP dendrites, however, it is unclear how much influence distal synapses have on spiking activity. Dendritic expression of fast voltage-dependent Na(+) channels (NaF channels) can enhance the importance of distal excitatory synapses by allowing for dendritic spike initiation and by subthreshold boosting of EPSPs. Antibody labeling has demonstrated the presence of NaF channel proteins in GP dendrites, but the quantitative expression density of the channels remains unknown. We built a series of nine GP neuron models that differed only in their dendritic NaF channel expression level to assess the functional impact of this parameter. The models were all similar in their basic electrophysiological features; however, higher expression levels of dendritic NaF channels increased the relative effectiveness of distal inputs for both excitatory and inhibitory synapses, broadening the effective extent of the dendritic tree. Higher dendritic NaF channel expression also made the neurons more resistant to tonic inhibition and highly sensitive to clustered synchronous excitation. The dendritic NaF channel expression pattern may therefore be a critical determinant of convergence for both the striatopallidal and subthalamopallidal projections, while also dictating which spatiotemporal input patterns are most effective at driving GP neuron output.

Neuroligin Trafficking Deficiencies Arising from Mutations in the α/β-Hydrolase Fold Protein Family

Despite great functional diversity, characterization of the alpha/beta-hydrolase fold proteins that encompass a superfamily of hydrolases, heterophilic adhesion proteins, and chaperone domains reveals a common structural motif. By incorporating the R451C mutation found in neuroligin (NLGN) and associated with autism and the thyroglobulin G2320R (G221R in NLGN) mutation responsible for congenital hypothyroidism into NLGN3, we show that mutations in the alpha/beta-hydrolase fold domain influence folding and biosynthetic processing of neuroligin3 as determined by in vitro susceptibility to proteases, glycosylation processing, turnover, and processing rates. We also show altered interactions of the mutant proteins with chaperones in the endoplasmic reticulum and arrest of transport along the secretory pathway with diversion to the proteasome. Time-controlled expression of a fluorescently tagged neuroligin in hippocampal neurons shows that these mutations compromise neuronal trafficking of the protein, with the R451C mutation reducing and the G221R mutation virtually abolishing the export of NLGN3 from the soma to the dendritic spines. Although the R451C mutation causes a local folding defect, the G221R mutation appears responsible for more global misfolding of the protein, reflecting their sequence positions in the structure of the protein. Our results suggest that disease-related mutations in the alpha/beta-hydrolase fold domain share common trafficking deficiencies yet lead to discrete congenital disorders of differing severity in the endocrine and nervous systems.