Dendritic Protrusions Research Articles

Direct Astrocytic Contacts Regulate Local Maturation of Dendritic Spines

Astrocytes contribute on both development and function of synapses, but it remains unclear whether direct astrocytic contacts regulate development of individual synapses. Two-photon time-lapse imaging of astrocytic and dendritic protrusive activity revealed the correlation of astrocytic contacts with both lifetime and morphological maturation of dendritic protrusions. Astrocytic motility was essential in maturation of spines, because its suppression by manipulating Rac1-dependent signaling in astrocytes resulted in induction of longer, filopodia-like dendritic protrusions. Manipulation of ephrin/Eph-dependent neuron-astrocyte signaling suggested involvement of this signaling pathway in astrocyte-dependent stabilization of newly generated dendritic protrusions. Our data support a model in which astrocytic protrusive activity in development acts as a key local regulator for stabilization of individual dendritic protrusions and subsequent maturation into spines.

NGL family PSD-95–interacting adhesion molecules regulate excitatory synapse formation

Synaptic cell adhesion molecules (CAMs) regulate synapse formation through their trans-synaptic and heterophilic adhesion. Here we show that postsynaptic netrin-G ligand (NGL) CAMs associate with netrin-G CAMs in an isoform-specific manner and, through their cytosolic tail, with the abundant postsynaptic scaffold postsynaptic density-95 (PSD-95). Overexpression of NGL-2 in cultured rat neurons increased the number of PSD-95-positive dendritic protrusions. NGL-2 located on heterologous cells or beads induced functional presynaptic differentiation in contacting neurites. Direct aggregation of NGL-2 on the surface membrane of dendrites induced the clustering of excitatory postsynaptic proteins. Competitive inhibition by soluble NGL-2 reduced the number of excitatory synapses. NGL-2 knockdown reduced excitatory, but not inhibitory, synapse numbers and currents. These results suggest that NGL regulates the formation of excitatory synapses.

PDGF regulates the actin cytoskeleton through hnRNP-K-mediated activation of the ubiquitin E3-ligase MIR

PDGF is a potent chemotactic mitogen and a strong inductor of fibroblast motility. In Swiss 3T3 fibroblasts, exposure to PDGF but not EGF or IGF-1 causes a rapid loss of actin stress fibers (SFs) and focal adhesions (FAs), which is followed by the development of retractile dendritic protrusions and induction of motility. The PDGF-specific actin reorganization was blocked by inhibition of Src-kinase and the 26S proteasome. PDGF induced Src-dependent association between the multifunctional transcription/translation regulator hnRNP-K and the mRNA-encoding myosin regulatory light-chain (MRLC)-interacting protein (MIR), a E(3)-ubiquitin ligase that is MRLC specific. This in turn rapidly increased MIR expression, and led to ubiquitination and proteasome-mediated degradation of MRLC. Downregulation of MIR by RNA muting prevented the reorganization of actin structures and severely reduced the migratory and wound-healing potential of PDGF-treated cells. The results show that activation of MIR and the resulting removal of diphosphorylated MRLC are essential for PDGF to instigate and maintain control over the actin-myosin-based contractile system in Swiss 3T3 fibroblasts. The PDGF induced protein destabilization through the regulation of hnRNP-K controlled ubiquitin -ligase translation identifies a novel pathway by which external stimuli can regulate phenotypic development through rapid, organelle-specific changes in the activity and stability of cytoskeletal regulators.

The polarity protein PAR-3 and TIAM1 cooperate in dendritic spine morphogenesis

PAR-3 (partitioning-defective gene 3) is essential for cell polarization in many contexts, including axon specification. However, polarity proteins have not been implicated in later steps of neuronal differentiation, such as dendritic spine morphogenesis. Here, we show that PAR-3 is necessary for normal spine development in primary hippocampal neurons. Depletion of PAR-3 causes the formation of multiple filopodia- and lamellipodia-like dendritic protrusions - a phenotype similar to neurons expressing activated Rac. PAR-3 regulates spine formation by binding the Rac guanine nucleotide-exchange factor (GEF) TIAM1, and spatially restricting it to dendritic spines. Thus, a balance of PAR-3 and TIAM1 is essential to modulate Rac-GTP levels and to allow spine morphogenesis.

Remodeling of synaptic structures in the motor cortex following spinal cord injury

After spinal cord injury (SCI), structural reorganization occurs at multiple levels of the motor system including the motor cortex, and this remodeling may underlie recovery of motor function. The present study determined whether SCI leads to a remodeling of synaptic structures in the motor cortex. Dendritic spines in the rat motor cortex were visualized by confocal microscopy in fixed slices, and their density and morphology were analyzed after an overhemisection injury at C4 level. Spine density decreased at 7 days and partially recovered by 28 days. Spine head diameter significantly increased in a layer-specific manner. SCI led to a higher proportion of longer spines especially at 28 days, resulting in a roughly 10% increase in mean spine length. In addition, filopodium-like long dendritic protrusions were more frequently observed after SCI, suggesting an increase in synaptogenic events. This spine remodeling was accompanied by increased expression of polysialylated neural cell adhesion molecule, which attenuates adhesion between the pre- and postsynaptic membranes, in the motor cortex from as early as 3 days to 2 weeks after injury, suggesting a decrease in synaptic adhesion during the remodeling process. These results demonstrate time-dependent changes in spine density and morphology in the motor cortex following SCI. This synaptic remodeling seems to proceed with a time scale ranging from days to weeks. Elongation of dendritic spines may indicate a more immature and modifiable pattern of synaptic connectivity in the motor cortex being reorganized following SCI.

Morphological observation of tumor infiltrating immunocytes in human rectal cancer

To investigate the morphological characterization of tumor infiltrating dendritic cells (TIDCs) and tumor infiltrating lymphocytes (TILs) in human rectal cancer. Light and electron microscopy as well as immunohistochemistry were used to observe the distributive and morphological changes of TIDCs and TILs. TIDCs were mainly located in tumor-surrounding tissue. The number of TIDCs in the earlier stage was higher than that in the later stage (P<0.01). TILs were mainly seen in adjacent tissue of cancers and tumor-surrounding tissue. There were more TILs in the earlier stage than that in the later stage (P<0.01). Under electron microscope, TIDCs were irregular in shape and exhibited many dendritic protrusions. It is not obvious that cancer cells perforated the basement membrane and TILs were arranged along the basement membrane in the earlier stage. In the later stage, it is explicit that cancer cells perforated the basement membrane and surrounded by TILs. There were contacts among TIDCs, TILs and tumor cell. One TIDCs contacted one or several TILs which clustered around TIDCs. Glycogen granules were seen between TIDCs and TILs. The number of TIDCs and TILs is related with tumor progression There exist close relationships among TIDCs, TILs and tumor cell.

Familial Parkinson Mutant α-Synuclein Causes Dopamine Neuron Dysfunction in Transgenic Caenorhabditis elegans

Mutations in alpha-synuclein gene cause familial form of Parkinson disease, and deposition of wild-type alpha-synuclein as Lewy bodies occurs as a hallmark lesion of sporadic Parkinson disease and dementia with Lewy bodies, implicating alpha-synuclein in the pathogenesis of Parkinson disease and related neurodegenerative diseases. Dopamine neurons in substantia nigra are the major site of neurodegeneration associated with alpha-synuclein deposition in Parkinson disease. Here we establish transgenic Caenorhabditis elegans (TG worms) that overexpresses wild-type or familial Parkinson mutant human alpha-synuclein in dopamine neurons. The TG worms exhibit accumulation of alpha-synuclein in the cell bodies and neurites of dopamine neurons, and EGFP labeling of dendrites is often diminished in TG worms expressing familial Parkinson disease-linked A30P or A53T mutant alpha-synuclein, without overt loss of neuronal cell bodies. Notably, TG worms expressing A30P or A53T mutant alpha-synuclein show failure in modulation of locomotory rate in response to food, which has been attributed to the function of dopamine neurons. This behavioral abnormality was accompanied by a reduction in neuronal dopamine content and was treatable by administration of dopamine. These phenotypes were not seen upon expression of beta-synuclein. The present TG worms exhibit dopamine neuron-specific dysfunction caused by accumulation of alpha-synuclein, which would be relevant to the genetic and compound screenings aiming at the elucidation of pathological cascade and therapeutic strategies for Parkinson disease.

Activity-Dependent Dendritic Spine Structural Plasticity Is Regulated by Small GTPase Rap1 and Its Target AF-6

Activity-dependent remodeling of dendritic spines is essential for neural circuit development and synaptic plasticity, but the mechanisms that coordinate synaptic structural and functional plasticity are not well understood. Here we investigate the signaling pathways that enable excitatory synapses to undergo activity-dependent structural modifications. We report that activation of NMDA receptors in cultured cortical neurons induces spine morphogenesis and activation of the small GTPase Rap1. Rap1 bimodally regulates spine morphology: activated Rap1 recruits the PDZ domain-containing protein AF-6 to the plasma membrane and induces spine neck elongation, while inactive Rap1 dissociates AF-6 from the membrane and induces spine enlargement. Rap1 also regulates spine content of AMPA receptors: thin spines induced by Rap1 activation have reduced GluR1-containing AMPA receptor content, while large spines induced by Rap1 inactivation are rich in AMPA receptors. These results identify a signaling pathway that regulates activity-dependent synaptic structural plasticity and coordinates it with functional plasticity.

Overexpression of drebrin A in immature neurons induces the accumulation of F-actin and PSD-95 into dendritic filopodia, and the formation of large abnormal protrusions

Drebrin A is a neuron-specific F-actin binding protein, and plays a pivotal role in the spine formation. In this study, we expressed drebrin A tagged with green fluorescent protein (GFP-DA) in hippocampal neurons at 7–9 days in vitro when presynaptic terminals are not fully maturated. GFP-DA was accumulated in dendritic protrusions and formed large abnormal structures. Since these structures were similar to filopodia in terms of lack of MAP2 immunostaining, we named them “megapodia” meaning large dendritic filopodia. F-actin and PSD-95 were also accumulated in megapodia, and their amounts were significantly correlated with that of GFP-DA. However, the expression of GFP-DA did not result in the promotion of the morphological change from filopodia into spines. These results demonstrate that drebrin A accumulates spine-resident proteins via protein–protein interaction in filopodia, and suggest that the spine formation requires the concurrence of the increase of drebrin-A expression and the functional presynaptic contact.

Spines reach out

B ored dendritic spines look for new challenges, say David Richards, R. Anne McKinney (in work done at the University of Zurich, Switzerland), and colleagues. Spines are small dendritic protrusions on which excitatory synapses connect to axons. Recent evidence suggested that spines are mobile even in adults. Richards et al. now suggest that this mobility allows for post-developmental synaptic rewiring. The group found that mobile spines formed filopodium-like protrusions extending toward neighboring axons. The protrusions are a response to glutamate in situations when the spine is receiving little from its own axonal partner. Small amounts of glutamate caused the protrusions to extend toward the glutamate source. A large dose, however, repressed protrusion formation for up to 20 min. This may be due to the strong influx of calcium in excited synapses, which is known to inhibit cytoskeletal changes. The prevalence of protrusions in inactive spines might allow them to seek out more active presynaptic partners. “If activity is very low, a spine gets restless,” says Richards. “But if it is close enough to another presynaptic terminal, some of [that terminal's] glutamate can diffuse and weakly activate the spine. Now it's found a [new] potential source of glutamate, so it heads in that direction.” This rewiring might explain how stroke sufferers are able to recover certain neurological functions. Reference: Richards, D.A., et al. 2005. Proc. Natl. Acad. Sci. USA. doi:.10.1073/pnas.0501881102 [PubMed] [Cross Ref]

A GIT1/PIX/Rac/PAK Signaling Module Regulates Spine Morphogenesis and Synapse Formation through MLC

Three of seven recently identified genes mutated in nonsyndromic mental retardation are involved in Rho family signaling. Two of the gene products, alpha-p-21-activated kinase (PAK) interacting exchange factor (alphaPIX) and PAK3, form a complex with the synaptic adaptor protein G-protein-coupled receptor kinase-interacting protein 1 (GIT1). Using an RNA interference approach, we show that GIT1 is critical for spine and synapse formation. We also show that Rac is locally activated in dendritic spines using fluorescence resonance energy transfer. This local activation of Rac is regulated by PIX, a Rac guanine nucleotide exchange factor. PAK1 and PAK3 serve as downstream effectors of Rac in regulating spine and synapse formation. Active PAK promotes the formation of spines and dendritic protrusions, which correlates with an increase in the number of excitatory synapses. These effects are dependent on the kinase activity of PAK, and PAK functions through phosphorylating myosin II regulatory light chain (MLC). Activated MLC causes an increase in dendritic spine and synapse formation, whereas inhibiting myosin ATPase activity results in decreased spine and synapse formation. Finally, both activated PAK and activated MLC can rescue the defects of GIT1 knockdown, suggesting that PAK and MLC are downstream of GIT1 in regulating spine and synapse formation. Our results point to a signaling complex, consisting of GIT1, PIX, Rac, and PAK, that plays an essential role in the regulation of dendritic spine and synapse formation and provides a potential mechanism by which alphaPIX and PAK3 mutations affect cognitive functions in mental retardation.

Syntenin is involved in the developmental regulation of neuronal membrane architecture

Syntenin is a ∼33 kDa scaffolding protein that we have shown previously to bind to kainate receptor subunits via a PDZ interaction. Here we show that syntenin has a tightly regulated developmental profile in neurons and is most abundant in the period of intense growth and synapse formation and stabilization. There is extensive colocalization of syntenin and kainate receptors with particularly intense labeling for both proteins at growth cones. Overexpression of GFP-syntenin in both young and mature neurons evokes marked changes in neuronal morphology by increasing the number of dendritic protrusions. These results are consistent with the involvement of syntenin in controlling membrane organization and suggest that by interaction with kainate receptors it may play a role in determining the formation and maturation of synapses.

Tetraspanin CD82 Attenuates Cellular Morphogenesis through Down-regulating Integrin α6-Mediated Cell Adhesion

Tetraspanin CD82 has been implicated in integrin-mediated functions such as cell motility and invasiveness. Although tetraspanins associate with integrins, it is unknown if and how CD82 regulates the functionality of integrins. In this study, we found that Du145 prostate cancer cells underwent morphogenesis on the reconstituted basement membrane Matrigel to form an anastomosing network of multicellular structures. This process entirely depends on integrin alpha6, a receptor for laminin. After CD82 is expressed in Du145 cells, this cellular morphogenesis was abolished, indicating a functional cross-talk between CD82 and alpha6 integrins. Interestingly, antibodies against other tetraspanins expressed in Du145 cells such as CD9, CD81, and CD151 did not block this integrin alpha6-dependent morphogenesis. We further found that CD82 significantly inhibited cell adhesion on laminin 1. Notably, the level of alpha6 integrins on the cell surface was down-regulated upon CD82 expression, although total cellular alpha6 protein levels remained unchanged in CD82-expressing cells. This down-regulation indicates that the diminished cell adhesiveness of CD82-expressing Du145 cells on laminin likely resulted from less cell surface expression of alpha6 integrins. As expected, CD82 physically associated with the integrin alpha6 in Du145-CD82 transfectant cells, suggesting that the formation of the CD82-integrin alpha6 complex reduces alpha6 integrin cell surface expression. Finally, the internalization of cell surface integrin alpha6 is significantly enhanced upon CD82 expression. In conclusion, our results indicate that 1) CD82 attenuates integrin alpha6 signaling during a cellular morphogenic process; 2) the decreased surface expression of alpha6 integrins in CD82-expressing cells is likely responsible for the diminished adhesiveness on laminin and, subsequently, results in the attenuation of alpha6 integrin-mediated cellular morphogenesis; and 3) the accelerated internalization of integrin alpha6 upon CD82 expression correlates with the down-regulation of cell surface integrin alpha6.

The Importance of Dendritic Mitochondria in the Morphogenesis and Plasticity of Spines and Synapses

The proper intracellular distribution of mitochondria is assumed to be critical for normal physiology of neuronal cells, but direct evidence for this idea is lacking. Extension or movement of mitochondria into dendritic protrusions correlates with the development and morphological plasticity of spines. Molecular manipulations of dynamin-like GTPases Drp1 and OPA1 that reduce dendritic mitochondria content lead to loss of synapses and dendritic spines, whereas increasing dendritic mitochondrial content or mitochondrial activity enhances the number and plasticity of spines and synapses. Thus, the dendritic distribution of mitochondria is essential and limiting for the support of synapses. Reciprocally, synaptic activity modulates the motility and fusion/fission balance of mitochondria and controls mitochondrial distribution in dendrites.

Imaging the motility of dendritic protrusions and axon terminals: roles in axon sampling and synaptic competition

Dendritic spines and filopodia display actin-based morphological plasticity. The function of this rapid motility is unknown. Its ubiquitous expression during development has led to the hypothesis that motility plays a role in synaptogenesis. We investigated this by simultaneously imaging presynaptic boutons and dendritic protrusions in acute hippocampal slices from GFP-M transgenic mice loaded with FM 1-43 followed by immunostaining. Postsynaptic motility was inversely correlated with the presence of stable synaptic contacts. Filopodia were highly motile and made transient interactions, whereas spines were less motile and had stable contacts, although they could still move together with a synaptic terminal. "Head morphing" of spines was associated with interactions with more than one presynaptic terminal. Our data indicate that filopodia motility could serve to transiently sample the surrounding neuropil, while the motility of established spines could mediate interactions with two axonal terminals. Spine "morphing" could therefore be the morphological signature for synaptic input competition in central synapses.

The dynamic turnover and functional roles of α-actinin in dendritic spines

Excitatory synapses are located on actin-rich protrusions known as dendritic spines. α-Actinin is an actin binding protein enriched in the postsynaptic density (PSD) of excitatory synapses. Because it also binds to NMDA receptors and other PSD components, α-actinin has been proposed to link NMDA receptors and the PSD to the underlying actin cytoskeleton of the dendritic spine. Although α-actinin has been implicated in modulation of NMDA receptor activity, the cell biological function of α-actinin in neurons is unknown. We report here that α-actinin is concentrated in spines. Both the actin binding domain and the spectrin repeat region (which interacts with NMDA receptors) of α-actinin2 are required for spine targeting. In live imaging experiments, Venus-tagged α-actinin2 in dendritic spines showed faster turnover than PSD-95, as determined by fluorescent recovery after photobleaching (FRAP), and individual spines often showed marked fluctuations in α-actinin content over a time-scale of minutes. Overexpression of α-actinin2 increased the length and density of dendritic protrusions in cultured hippocampal neurons, an effect that requires the actin binding domain and the spectrin repeats of α-actinin. These results suggest that α-actinin regulates spine morphology and density.

Age-dependence in the homeostatic upregulation of hippocampal dendritic spine number during blocked synaptic transmission

Homeostatic regulation of spine number in mature hippocampal neurons results in more dendritic spines when synaptic transmission is blocked, providing a mechanism to compensate for diminished synaptic input. It is unsettled whether blockade of synaptic transmission also elevates spine number during development. To address this question, synaptic transmission was blocked in rat hippocampal slices during critical developmental stages of spine formation at postnatal days (P) 6–P22 and compared to adults. CA1 pyramidal cells were labeled with DiI and maintained for 5 h in one of three conditions, control artificial cerebrospinal fluid (ACSF), block media containing synaptic transmission antagonists in ACSF, or block media containing synaptic transmission antagonists in a nominally calcium-free ACSF with high magnesium. Slices were fixed in mixed aldehydes, sectioned, and the lateral dendrites were imaged in stratum radiatum with confocal microscopy. Dendritic spine density was quantified per unit length of dendrite. At P6–7 there were only a few protrusions emerging from the dendrites, which were predominantly filopodia-like in appearance. At both P11–12 and P15–16 there was a mixture of dendritic spines and filopodia-like structures. By P20–22 dendritic spines predominated and spine density was about 82% of the adult level. Dendritic spine density increased during blocked synaptic transmission at P20–22 as in adults, but was unchanged during blockade at younger ages. When extracellular calcium was nominally zero, dendritic spine density further increased on P20–22 dendrites as in adults. In contrast, spine density decreased along P11–12 dendrites under the nominally zero calcium condition. Under control conditions, dendritic protrusions were longer at P6–7 than at all other ages, which did not differ from one another. When synaptic transmission was blocked, dendritic protrusions further elongated at P6–7 only. Under the nominally zero calcium condition with blocked synaptic transmission, dendritic protrusions shortened at P11–12 only. These findings reveal age-dependent changes in the manifestation of homeostatic control of dendritic spines that could be mediated by maturational changes in mechanisms regulating postsynaptic calcium.

Activated natural killer cells adhere to cultured hippocampal neurons and affect the dendritic morphology

To examine the manner of interactions between immune cells and central nervous system (CNS) neurons, mouse hippocampal neurons were co-cultured with lymphokine (IL-2)-activated killer (LAK) cells. Immunocytochemical and time-lapse observations indicated that LAK cells migrated along neuronal processes and made adhesive contacts with them. In addition to the direct physical effects, LAK cells released glutamate, induced the formation of beads-like structure in the dendrites of about 14% of hippocampal neurons and caused the reduction of dendritic protrusions. These results suggest that infiltrating immune cells can form direct adhesive connections with CNS neurons and affect their dendritic morphology.

Temporal dynamics of NMDA receptor-induced changes in spine morphology and AMPA receptor recruitment to spines

Recent studies have shown that the activation of NMDA receptors can induce rapid changes in dendritic morphology and synaptic recruitment of AMPA receptors in dendritic spines. Here, we analyze the time course of NMDA receptor-induced changes in dendrite morphology and recruitment of AMPA receptors to synapses in cultured neurons. Activation of NMDA receptors causes a rapid transient increase in the size of preexisting spines and then the gradual formation of new dendritic protrusions and spines. NMDA receptor activation also induced GFP-tagged AMPA receptors to cluster in dendrites and to be inserted into the surface of dendritic spines. These results indicate that NMDA receptor activation induces several phases of dendritic plasticity, initial expansion of dendritic spines, followed by the de novo formation of spines and AMPA receptor dendritic clustering and surface expression on spines. Each of these forms of plasticity may have significant effects on the efficacy of synaptic transmission.

Vortex microavalanches in superconducting Pb thin films

Local magnetization measurements on 100 nm type-II superconducting Pb thin films show that flux penetration changes qualitatively with temperature. Small flux jumps at the lowest temperatures gradually increase in size, then disappear near T = 0.7Tc. Comparison with other experiments suggests that the avalanches correspond to dendritic flux protrusions. Reproducibility of the first flux jumps in a decreasing magnetic field indicates a role for defect structure in determining avalanches. We also find a temperature-independent final magnetization after flux jumps, analogous to the angle of repose of a sandpile.