Andrew Matus

Rapid Actin-Based Plasticity in Dendritic Spines

Dendritic spines have been proposed as primary sites of synaptic plasticity in the brain. Consistent with this hypothesis, spines contain high concentrations of actin, suggesting that they might be motile. To investigate this possibility, we made video recordings from hippocampal neurons expressing actin tagged with green fluorescent protein (GFP-actin). This reagent incorporates into actin-containing structures and allows the visualization of actin dynamics in living neurons. In mature neurons, recordings of GFP fluorescence revealed large actin-dependent changes in dendritic spine shape, similar to those inferred from previous studies using fixed tissues. Visible changes occurred within seconds, suggesting that anatomical plasticity at synapses can be extremely rapid. As well as providing a molecular basis for structural plasticity, the presence of motile actin in dendritic spines implicates the postsynaptic element as a primary site of this phenomenon.

Glutamate receptors regulate actin-based plasticity in dendritic spines.

Dendritic spines at excitatory synapses undergo rapid, actin-dependent shape changes which may contribute to plasticity in brain circuits. Here we show that actin dynamics in spines are potently inhibited by activation of either AMPA or NMDA subtype glutamate receptors. Activation of either receptor type inhibited actin-based protrusive activity from the spine head. This blockade of motility caused spines to round up so that spine morphology became both more stable and more regular. Inhibition of spine motility by AMPA receptors was dependent on postsynaptic membrane depolarization and influx of Ca2+ through voltage-activated channels. In combination with previous studies, our results suggest a two-step process in which spines initially formed in response to NMDA receptor activation are subsequently stabilized by AMPA receptors.

Activity-induced targeting of profilin and stabilization of dendritic spine morphology

Morphological changes in dendritic spines have been implicated in connective plasticity in brain circuitry, but the underlying pathway leading from synaptic transmission to structural change is unknown. Using primary neurons expressing GFP-tagged proteins, we found that profilin, a regulator of actin polymerization, is targeted to spine heads when postsynaptic NMDA receptors are activated and that actin-based changes in spine shape are concomitantly blocked. Profilin targeting was triggered by electrical stimulation patterns known to induce the long-term changes in synaptic responsiveness associated with memory formation. These results suggest that, in addition to electrophysiological changes, NMDA receptor activation initiates changes in the actin cytoskeleton of dendritic spines that stabilize synaptic structure.

Cytoskeletal Plasticity in Cells Expressing Neuronal Microtubule-Associated Proteins

MAP2 and tau are the two most prominent neuron-specific microtubule-associated proteins. They have been implicated in the stabilization of microtubules and consequently of neurite morphology. To investigate their influence on microtubule dynamics, we have tagged both proteins with green fluorescent protein and expressed them in non-neuronal cells. Time-lapse recordings of living cells showed that MAP2 and tau did not significantly affect the rates of microtubule growth and shrinkage. Longer recordings revealed the growth and disappearance of MAP-induced microtubule bundles coinciding with changes in cell shape. This supports the idea that microtubule dynamics are influenced by the cortical cytoskeleton. The dynamics-preserving stabilization of microtubules by MAP2 and tau thus provides a molecular basis for the morphological plasticity reported to exist in established neurites.

In situ localization of microtubule-associated protein mRNA in the developing and adult rat brain

We have used cDNA probes specific for three of the major brain microtubule-associated proteins (MAPs), MAP1, MAP2, and MAP5, to study the timing of appearance, relative abundance, and intracellular compartmentalization of MAP gene transcripts in developing rat brain. The MAP1 probe hybridizes throughout the brain, in both grey and white matter. MAP2 mRNA is detected only in grey matter and appears in cerebral neurons only after they have ceased dividing and have migrated to the cortical plate. The MAP5 cDNA hybridizes throughout the embryonic brain, but by P12, MAP5 mRNA distribution is restricted to relatively immature areas. MAP2 mRNA, found in dendrites in the developing brain, persists in some adult dendrites. MAP5 mRNA, like beta-tubulin mRNA, is found only in the cell bodies of developing neurons, indicating that the protein must be transported from the soma into processes. MAP1 mRNA is found only in the proximal regions of cortical pyramidal cell dendrites in both developing and adult brain. The diverse distributions of MAP gene transcripts emphasize the importance of these proteins in generating heterogeneity of microtubule function and indicate that MAP compartmentalization within neurons is regulated in part by differential mRNA transport.

Lithium reduces tau phosphorylation: effects in living cells and in neurons at therapeutic concentrations.

BACKGROUND The mechanism of action of lithium remains to be determined satisfactorily. Recent studies suggested a possible role in inhibiting glycogen synthase kinase-3 (GSK-3), previously shown to phosphorylate the protein tau. Tau is expressed mainly in neurons, where it functions to stabilize microtubules in a phosphorylation-dependent manner. METHODS Neurons and transfected non-neuronal cells were treated with lithium and the phosphorylation of tau at multiple epitopes examined by western blotting and by immunocytochemistry. Using green fluorescent protein as a tag we examined the effects of lithium on phosphorylated tau in living cells. RESULTS Lithium reversibly reduced tau phosphorylation at therapeutic concentrations, and even at high concentrations did not alter neuronal morphology. Green fluorescent protein tagged-tau when phosphorylated by GSK-3 was diffusely distributed; treatment with lithium resulted in association with microtubules and then bundle formation. Removing lithium allowed observation of the dissolution of bundles and gradual dissociation of tau from microtubules in living cells. CONCLUSIONS Lithium may have multiple effects in brain, but at least one action is demonstrated to be a relative inhibition of GSK-3-induced tau phosphorylation. These results carry implications for future studies of the actions of mood-stabilizing drugs and indeed of the molecular mechanisms of affective disorders.

Attenuation of microtubule-associated protein 1B expression by antisense oligodeoxynucleotides inhibits initiation of neurite outgrowth

Microtubule-associated protein 1B, formerly also known as microtubule-associated protein 5, is the first structural microtubule accessory protein to appear in outgrowing axons. In PC12 pheochromocytoma cells microtubule-associated protein 1B levels increase several-fold after the addition of nerve growth factor and this increase is correlated with the initiation of process formation. To determine whether microtubule-associated protein 1B is essential for neurite outgrowth, we used antisense oligodeoxynucleotides to inhibit its expression in nerve growth factor-treated PC12 cells in the rat. The application of several different antisense oligodeoxynucleotides to the microtubule-associated protein 1B mRNA sequence inhibited both microtubule-associated protein 1B expression and neurite extension. Specificity was shown by the lack of effect of control sense oligonucleotides and by the lack of effect of the microtubule-associated protein 1B antisense oligodeoxynucleotides on the expression of either tubulin or microtubule-associated protein 3, another microtubule-associated protein whose synthesis is stimulated by nerve growth factor treatment of PC12 cells. After removal of the antisense oligodeoxynucleotides, microtubule-associated protein 1B expression recovered to normal levels and the cells grew normal neurites with the timing and morphological characteristics of normal nerve growth factor-induced outgrowth, indicating that the blockade was not because of non-specific toxic effects. These results indicate that microtubule-associated protein 1B is an essential component of the molecular mechanism underlying the formation of neuronal processes.

Molecular Cloning of Microtubule-Associated Protein 1 (MAP1A) and Microtubule-Associated Protein 5 (MAP1B): Identification of Distinct Genes and Their Differential Expression in Developing Brain

Abstract: cDNA clones encoding microtubule‐associated proteins 1 (MAP1/MAP1A) and 5 (MAP5/MAP1B) were isolated and have been used to study their structural relationship as well as their regulated expression in developing rat brain. cDNA clones specific for MAP1 hybridized to a single 10‐kb rat brain mRNA, and analysis of genomic DNA by Southern blotting indicated the existence of a single MAP1 gene. A second set of cDNAs specific for MAP5 hybridized to a single 11‐kb mRNA in rat brain and also detected a single gene. By analysis of hybrid mouse‐hamster cell lines, the MAP1 gene was located to mouse chromosome 2, designated Mtap‐1, and the MAP5 gene to chromosome 13, designated Mtap‐5 MAP1 and MAP5 mRNAs were expressed with different temporal patterns during rat brain development that mirrored the appearance of their protein products, suggesting that expression of these proteins is under transcriptional control. These results taken together demonstrate that although MAP1 and MAP5 have some properties that are similar, they are structurally distinct proteins whose transcription is differently regulated from separate genes.

GFP illuminates the cytoskeleton.

Until recently, cytoskeleton research has relied primarily on immunofluorescence microscopy techniques, requiring fixation and hence killing of the specimen before the analysis. The sole method for visualizing cytoskeletal dynamics in living cells has been the microinjection of purified and fluorescently labelled protein, but technical difficulties have precluded its widespread use. The recent introduction of green fluorescent protein (GFP) has enabled visualization of proteins and cytoskeletal dynamics with only minimal perturbations of the living cell and has opened new horizons for studying the cytoskeleton.

Growth of dendritic spines: a continuing story

Dendritic spines, which are present at the vast majority of excitatory synapses in the central nervous system, have a specialized cytoskeleton of dynamic actin filaments that makes them capable of rapid morphological plasticity. During development, structural remodeling of nascent spines is an important factor in experience-dependent shaping of neuronal circuits, whereas in the adult brain spines maintain a balance between morphological stability and plasticity.