Doctor of Philosophy, University of California Irvine (2009)
Thomas Sudhof, Postdoctoral Research Mentor
Brain-derived neurotrophic factor (BDNF) has emerged as a possible broad-spectrum treatment for the plasticity losses found in rodent models of human conditions associated with memory and cognitive deficits. We have tested this strategy in the particular case of ovariectomy. The actin polymerization in spines normally found after patterned afferent stimulation was greatly reduced, along with the stabilization of long-term potentiation, in hippocampal slices prepared from middle-aged ovariectomized rats. Both effects were fully restored by a 60-minute infusion of 2 nM BDNF. Comparable rescue results were obtained after elevating endogenous BDNF protein levels in hippocampus with 4 daily injections of a short half-life ampakine (positive modulator of ?-amino-3-hydroxy-5-methyl-4-isoxazolepropionate [AMPA]-type glutamate receptors). These results provide the first evidence that minimally invasive, mechanism-based drug treatments can ameliorate defects in spine plasticity caused by depressed estrogen levels.
View details for DOI 10.1016/j.neurobiolaging.2010.06.008
View details for Web of Science ID 000301506800008
View details for PubMedID 20674095
Learning-induced neurotrophic signaling at synapses is widely held to be critical for neuronal viability in adult brain. A previous study provided evidence that unsupervised learning of a novel environment is accompanied by activation of the TrkB receptor for brain-derived neurotrophic factor (BDNF) in hippocampal field CA1b of adult rats. Here we report that this effect is regionally differentiated, in accord with "engram" type memory encoding. A 30 min exposure to a novel, complex environment caused a marked, NMDA receptor-dependent increase in postsynaptic densities associated with activated (phosphorylated) Trk receptors in rostral hippocampus. Increases were pronounced in field CA3a, moderate in the dentate gyrus, and absent in field CA1a. Synapses with Trk activation were significantly larger than their neighbors. Surprisingly, unsupervised learning had no effect on Trk phosphorylation in more temporal sections of hippocampus. It thus appears that commonplace forms of learning interact with regional predispositions to produce spatially differentiated effects on BDNF signaling.
View details for DOI 10.1523/JNEUROSCI.3549-10.2010
View details for Web of Science ID 000284096300025
View details for PubMedID 21068315
Reorganization of the actin cytoskeleton is essential for synaptic plasticity and memory formation. Presently, the mechanisms that trigger actin dynamics during these brain processes are poorly understood. In this study, we show that myosin II motor activity is downstream of LTP induction and is necessary for the emergence of specialized actin structures that stabilize an early phase of LTP. We also demonstrate that myosin II activity contributes importantly to an actin-dependent process that underlies memory consolidation. Pharmacological treatments that promote actin polymerization reversed the effects of a myosin II inhibitor on LTP and memory. We conclude that myosin II motors regulate plasticity by imparting mechanical forces onto the spine actin cytoskeleton in response to synaptic stimulation. These cytoskeletal forces trigger the emergence of actin structures that stabilize synaptic plasticity. Our studies provide a mechanical framework for understanding cytoskeletal dynamics associated with synaptic plasticity and memory formation.
View details for DOI 10.1016/j.neuron.2010.07.016
View details for Web of Science ID 000281534600010
View details for PubMedID 20797537
The abnormal spine morphology found in fragile X syndrome (FXS) is suggestive of an error in the signaling cascades that organize the actin cytoskeleton. We report here that physiological activation of the small GTPase Rac1 and its effector p-21 activated kinase (PAK), two enzymes critically involved in actin management and functional synaptic plasticity, is impaired at hippocampal synapses in the Fmr1-knock-out (KO) mouse model of FXS. Theta burst afferent stimulation (TBS) caused a marked increase in the number of synapses associated with phosphorylated PAK in adult hippocampal slices from wild-type, but not Fmr1-KO, mice. Stimulation-induced activation of synaptic Rac1 was also absent in the mutants. The polymerization of spine actin that occurs immediately after theta stimulation appeared normal in mutant slices but the newly formed polymers did not properly stabilize, as evidenced by a prolonged vulnerability to a toxin (latrunculin) that disrupts dynamic actin filaments. Latrunculin also reversed long-term potentiation when applied at 10 min post-TBS, a time point at which the potentiation effect is resistant to interference in wild-type slices. We propose that a Rac>PAK signaling pathway needed for rapid stabilization of activity-induced actin filaments, and thus for normal spine morphology and lasting synaptic changes, is defective in FXS.
View details for DOI 10.1523/JNEUROSCI.1077-10.2010
View details for Web of Science ID 000281197900003
View details for PubMedID 20720104
Learning-induced trophic activity is thought to be critical for maintaining health of the aging brain. We report here that learning, acting through an unexpected pathway, activates synaptic receptors for one of the brain's primary trophic factors. Unsupervised learning, but not exploratory activity alone, robustly increased the number of postsynaptic densities associated with activated (phosphorylated) forms of BDNF's TrkB receptor in adult rat hippocampus; these increases were blocked by an NMDA receptor antagonist. Similarly, stimulation of hippocampal slices at the learning-related theta frequency increased synaptic TrkB phosphorylation in an NMDA receptor-dependent fashion. Theta burst stimulation, which was more effective in this regard than other stimulation patterns, preferentially engaged NMDA receptors that, in turn, activated Src kinases. Blocking the latter, or scavenging extracellular TrkB ligands, prevented theta-induced TrkB phosphorylation. Thus, synaptic TrkB activation was dependent upon both ligand presentation and postsynaptic signaling cascades. These results show that afferent activity patterns and cellular events involved in memory encoding initiate BDNF signaling through synaptic TrkB, thereby ensuring that learning will trigger neurotrophic support.
View details for DOI 10.1073/pnas.0912973107
View details for Web of Science ID 000276642100084
View details for PubMedID 20356829
Estrogen, in addition to its genomic effects in brain, causes rapid and reversible changes to synaptic operations. We report here that these acute actions are due to selective activation of an actin-signaling cascade normally used in the production of long-term potentiation (LTP). Estrogen, or a selective agonist of the steroid's beta-receptor, caused a modest increase in fast glutamatergic transmission and a pronounced facilitation of LTP in adult hippocampal slices; both effects were completely eliminated by latrunculin, a toxin that prevents actin filament assembly. Estrogen also increased spine concentrations of filamentous actin and strongly enhanced its polymerization in association with LTP. A search for the origins of these effects showed that estrogen activates the small GTPase RhoA and phosphorylates (inactivates) the actin severing protein cofilin, a downstream target of RhoA. Moreover, an antagonist of RhoA kinase (ROCK) blocked estrogen's synaptic effects. Estrogen thus emerges as a positive modulator of a RhoA>ROCK>LIM kinase>cofilin pathway that regulates the subsynaptic cytoskeleton. It does not, however, strongly affect a second LTP-related pathway, involving the GTPases Rac and Cdc42 and their effector p21-activated kinase, which may explain why its acute effects are reversible. Finally, ovariectomy depressed RhoA activity, spine cytoskeletal plasticity, and LTP, whereas brief infusions of estrogen rescued plasticity, suggesting that the deficits in plasticity arise from acute, as well as genomic, consequences of hormone loss.
View details for DOI 10.1523/JNEUROSCI.3059-09.2009
View details for Web of Science ID 000270845000033
View details for PubMedID 19828812
The releasable factor adenosine blocks the formation of long-term potentiation (LTP). These experiments used this observation to uncover the synaptic processes that stabilize the potentiation effect. Brief adenosine infusion blocked stimulation-induced actin polymerization within dendritic spines along with LTP itself in control rat hippocampal slices but not in those pretreated with the actin filament stabilizer jasplakinolide. Adenosine also blocked activity-driven phosphorylation of synaptic cofilin but not of synaptic p21-activated kinase (PAK). A search for the upstream origins of these effects showed that adenosine suppressed RhoA activity but only modestly affected Rac and Cdc42. A RhoA kinase (ROCK) inhibitor reproduced adenosine's effects on cofilin phosphorylation, spine actin polymerization, and LTP, whereas a Rac inhibitor did not. However, inhibitors of Rac or PAK did prolong LTP's vulnerability to reversal by latrunculin, a toxin which blocks actin filament assembly. Thus, LTP induction initiates two synaptic signaling cascades: one (RhoA-ROCK-cofilin) leads to actin polymerization, whereas the other (Rac-PAK) stabilizes the newly formed filaments.
View details for DOI 10.1083/jcb.200901084
View details for Web of Science ID 000267939500009
View details for PubMedID 19596849
Recent demonstrations that positive modulators of AMPA-type glutamate receptors (ampakines) increase neuronal brain-derived neurotrophic factor (BDNF) expression have suggested a novel strategy for treating neurodegenerative diseases. However, reports that AMPA and BDNF receptors are down-regulated by prolonged activation raise concerns about the extent to which activity-induced increases in BDNF levels can be sustained without compromising glutamate receptor function. The present study constitutes an initial test of whether ampakines can cause enduring increases in BDNF content and signaling without affecting AMPA receptor (AMPAR) expression. Prolonged (12-24 h) treatment with the ampakine CX614 reduced AMPAR subunit (glutamate receptor subunit (GluR) 1-3) mRNA and protein levels in cultured rat hippocampal slices whereas treatment with AMPAR antagonists had the opposite effects. The cholinergic agonist carbachol also depressed GluR1-3 mRNA levels, suggesting that AMPAR down-regulation is a global response to extended periods of elevated neuronal activity. Analyses of time courses and thresholds indicated that BDNF expression is influenced by lower doses of, and shorter treatments with, the ampakine than is AMPAR expression. Accordingly, daily 3 h infusions of CX614 chronically elevated BDNF content with no effect on GluR1-3 concentrations. Restorative deconvolution microscopy provided the first evidence that chronic up-regulation of BDNF is accompanied by increased activation of the neurotrophin's TrkB-Fc receptor at spine synapses. These results show that changes in BDNF and AMPAR expression are dissociable and that up-regulation of the former leads to enhanced trophic signaling at excitatory synapses. These findings are encouraging with regard to the feasibility of using ampakines to tonically enhance BDNF-dependent functions in adult brain.
View details for DOI 10.1016/j.neuroscience.2008.12.018
View details for Web of Science ID 000263863500029
View details for PubMedID 19141314
Estrogen, in addition to its genomic effects, triggers rapid synaptic changes in hippocampus and cortex. Here we summarize evidence that the acute actions of the steroid arise from actin signaling cascades centrally involved in long-term potentiation (LTP). A 10-min infusion of E2 reversibly increased fast EPSPs and promoted theta burst-induced LTP within adult hippocampal slices. The latter effect reflected a lowered threshold and an elevated ceiling for the potentiation effect. E2's actions on transmission and plasticity were completely blocked by latrunculin, a toxin that prevents actin polymerization. E2 also caused a reversible increase in spine concentrations of filamentous (F-) actin and markedly enhanced polymerization caused by theta burst stimulation (TBS). Estrogen activated the small GTPase RhoA, but not the related GTPase Rac, and phosphorylated (inactivated) synaptic cofilin, an actin severing protein targeted by RhoA. An inhibitor of RhoA kinase (ROCK) thoroughly suppressed the synaptic effects of E2. Collectively, these results indicate that E2 engages a RhoA >ROCK> cofilin> actin pathway also used by brain-derived neurotrophic factor and adenosine, and therefore belongs to a family of 'synaptic modulators' that regulate plasticity. Finally, we describe evidence that the acute signaling cascade is critical to the depression of LTP produced by ovariectomy.
View details for PubMedID 20419049
Recent work has added strong support to the long-standing hypothesis that the stabilization of both long-term potentiation and memory requires rapid reorganization of the spine actin cytoskeleton. This development has led to new insights into the origins of cognitive disorders, and raised the possibility that a diverse array of memory problems, including those associated with diabetes, reflect disturbances to various components of the same mechanism. In accord with this argument, impairments to long-term potentiation in mouse models of Huntington's disease and in middle-aged rats have both been linked to problems with modulatory factors that control actin polymerization in spine heads. Complementary to the common mechanism hypothesis is the idea of a single treatment for addressing seemingly unrelated memory diseases. First tests of the point were positive: Brain-Derived Neurotrophic Factor (BDNF), a potent activator of actin signaling cascades in adult spines, rescued potentiation in Huntington's disease mutant mice, middle-aged rats, and a mouse model of Fragile-X syndrome. A similar reversal of impairments to long-term potentiation was obtained in middle-aged rats by up-regulating BDNF production with brief exposures to ampakines, a class of drugs that positively modulate AMPA-type glutamate receptors. Work now in progress will test if chronic elevation of BDNF enhances memory in normal animals.
View details for DOI 10.1016/j.ejphar.2007.11.082
View details for Web of Science ID 000256149300002
View details for PubMedID 18374328
Mice lacking expression of the fragile X mental retardation 1 (Fmr1) gene have deficits in types of learning that are dependent on the hippocampus. Here, we report that long-term potentiation (LTP) elicited by threshold levels of theta burst afferent stimulation (TBS) is severely impaired in hippocampal field CA1 of young adult Fmr1 knock-out mice. The deficit was not associated with changes in postsynaptic responses to TBS, NMDA receptor activation, or levels of punctate glutamic acid decarboxylase-65/67 immunoreactivity. TBS-induced actin polymerization within dendritic spines was also normal. The LTP impairment was evident within 5 min of induction and, thus, may not be secondary to defects in activity-initiated protein synthesis. Protein levels for both brain-derived neurotrophic factor (BDNF), a neurotrophin that activates pathways involved in spine cytoskeletal reorganization, and its TrkB receptor were comparable between genotypes. BDNF infusion had no effect on baseline transmission or on postsynaptic responses to theta burst stimulation, but nonetheless fully restored LTP in slices from fragile X mice. These results indicate that the fragile X mutation produces a highly selective impairment to LTP, possibly at a step downstream of actin filament assembly, and suggest a means for overcoming this deficit. The possibility of a pharmacological therapy based on these results is discussed.
View details for DOI 10.1523/JNEUROSCI.2624-07.2007
View details for Web of Science ID 000249981400006
View details for PubMedID 17913902
Stabilization of long-term potentiation (LTP) is commonly proposed to involve changes in synaptic morphology and reorganization of the spine cytoskeleton. Here we tested whether, as predicted from this hypothesis, induction of LTP by theta-burst stimulation activates an actin regulatory pathway and alters synapse morphology within the same dendritic spines. TBS increased severalfold the numbers of spines containing phosphorylated (p) p21-activated kinase (PAK) or its downstream target cofilin; the latter regulates actin filament assembly. The PAK/cofilin phosphoproteins were increased at 2 min but not 30 s post-TBS, peaked at 7 min, and then declined. Double immunostaining for the postsynaptic density protein PSD95 revealed that spines with high pPAK or pCofilin levels had larger synapses (+60-70%) with a more normal size frequency distribution than did neighboring spines. Based on these results and simulations of shape changes to synapse-like objects, we propose that theta stimulation markedly increases the probability that a spine will enter a state characterized by a large, ovoid synapse and that this morphology is important for expression and later stabilization of LTP.
View details for DOI 10.1523/JNEUROSCI.0164-07.2007
View details for Web of Science ID 000246720600011
View details for PubMedID 17507558
Brain-derived neurotrophic factor (BDNF) is an extremely potent, positive modulator of theta burst induced long-term potentiation (LTP) in the adult hippocampus. The present studies tested whether the neurotrophin exerts its effects by facilitating cytoskeletal changes in dendritic spines. BDNF caused no changes in phalloidin labeling of filamentous actin (F-actin) when applied alone to rat hippocampal slices but markedly enhanced the number of densely labeled spines produced by a threshold level of theta burst stimulation. Conversely, the BDNF scavenger TrkB-Fc completely blocked increases in spine F-actin produced by suprathreshold levels of theta stimulation. TrkB-Fc also blocked LTP consolidation when applied 1-2 min, but not 10 min, after theta trains. Additional experiments confirmed that p21 activated kinase and cofilin, two actin-regulatory proteins implicated in spine morphogenesis, are concentrated in spines in mature hippocampus and further showed that both undergo rapid, dose-dependent phosphorylation after infusion of BDNF. These results demonstrate that the influence of BDNF on the actin cytoskeleton is retained into adulthood in which it serves to positively modulate the time-dependent LTP consolidation process.
View details for DOI 10.1523/JNEUROSCI.4037-06.2007
View details for Web of Science ID 000245103600032
View details for PubMedID 17360925