Schizophrenia Research Forum - A Catalyst for Creative Thinking

Dendritic Spine Research—Putting Meat on the Bones

3 February 2006. Dendritic spines are rapidly becoming a darling of neuroscientists. They are the main site for excitatory synaptic transmission in the brain, and their waxing and waning underlie sophisticated modulations of neuronal circuits in development (Bock et al., 2005), not to mention mechanisms of learning and memory, such as long-term potentiation (see Matsuzaki et al., 2004) and long-term depression (see Zhou et al., 2004). All of the above make them particularly interesting to researchers studying schizophrenia.

Despite the budding interest, not much is known about where, how, or why these synaptic stems grow, but three independent basic science studies published January 19 shed some light on all these questions. And a study by David Lewis and colleagues at the University of Pittsburgh, published January 10, reports reduced expression of Cdc42 and Duo, important regulators of spine morphology, in schizophrenia.

Spirals of spines
First, the "where" of dendritic spines. John O’Brien and Nigel Unwin from the Medical Research Council Laboratory of Molecular Biology in Cambridge, England, reported in last week’s PNAS online that the distribution of dendritic spines is far from random, at least in the Purkinje neurons of the cerebellum. Rather, spines grow in elaborate and regular linear arrays, and they actually trace short-pitch helical paths around the dendrites.

O’Brien and Unwin coated Purkinje neurons from the mormyrid fish with a lipophilic fluorescent dye, and then examined tissue slices with a confocal microscope. Dendritic spines in the fish are less dense than in the mammalian brain, and the cerebellum is more regular. The authors found that in regions of low density, spines were evenly spaced along the dendrites, typically at about 0.54 μM apart. At high density this pattern was not easy to identify, but the authors detected a different pattern that also indicates regular spine spacing. In the fish, the Purkinje dendrites are long, straight, and parallel, forming a planar “palisade,” and the authors sometimes found lines of side-by-side spines in periodic groupings.

Using image analysis, O’Brien and Unwin generated a “diffraction pattern,” which revealed that spines along individual dendritic shafts trace a good approximation of a helix. The helix had a pitch of about 1.25 μM, which would fit about three spines per turn. “The helical ordering of spines gives rise to a set of similar surface lattices, the dimensions of which lead to approximately equal sampling of the surrounding space by the spineheads,” write the authors. In other words, it would seem the spines are arranged to maximize the probability that the dendritic arbor would interact with any afferent axon.

Extending these observations to mammals, the authors examined both the “weaver” mouse, a mutant that has fewer axons innervating the Purkinje cells, which have relatively straight dendritic shafts, and wild-type mice. The mammalian spines were more variable in shape, but even so, the authors detected a periodic spacing of about 0.58 μM. They also detected a helical pattern that was, again, slightly longer in pitch than in the fish. Because the periodicity and the pitch were very similar in all three cases, the authors conclude that this ordering of spines is an inherent property of dendrites that is not influenced by external factors, such as presynaptic activity. They also suggest that the periodicity indicates the involvement of a filamentous protein, such as actin. “Giant actin-binding proteins, such as nebulin, are more than long enough to span the interval between successive spines and could play a role in creating regularly spaced templates for the growth of filopodia, from which mature spines are thought to develop,” they write.

Myosin—not just for muscles anymore
Which brings this story straight to the “how” of dendritic spines. Morgan Sheng, at the Picower Institute for Learning and Memory at Massachusetts Institute of Technology, and colleagues there and elsewhere, report in the January 19 Neuron that myosin IIB plays a critical role in the formation of spines.

Myosin II was once dismissed as having no role in spine formation because experiments showed that the myosin inhibitor 2,3 butanedione-monoxime (BDM) had no effect on morphology or motility of dendritic spines. But BDM was subsequently shown to be a poor inhibitor of myosin IIB, one of the two isoforms found in non-muscle cells. That revelation, plus recent proteomic data suggesting the postsynaptic density is loaded with the protein (see Jordan et al., 2004; Peng et al., 2004) prompted Sheng and colleagues to reinvestigate a role for myosin IIB in spines.

First author Jubin Ryu and colleagues first confirmed the proteomic data using immunohistochemistry to show that in cultured hippocampal neurons, the vast majority of neuronal myosin IIB congregates with postsynaptic density 95, a marker of synaptic sites. Then, to test if this myosin has functional significance, Ryu and colleagues dosed the neurons with blebbistatin, a molecule that specifically inhibits the ATPase activity of myosin II. Ryu and colleagues found that within minutes of adding the chemical, the mushroom shaped heads of the dendritic spines started to disappear, and by about half an hour, all that was left of most spines were long, thin filopodia. Knocking down myosin IIB by RNA interference had a similar effect.

Spine dynamics have long been linked to actin filaments, but these data indicate that growth and regression of actin filaments are not the only means to control spine morphology. Sheng and colleagues suggest that myosin, which helps maintain tension in actin filament networks in other scenarios, such as cytokinesis, may have the same effect in spines. By providing tangential force at the spine head membrane, myosin might counteract the outward push generated by growth of actin filaments, creating a mushroom head, much like the bud in yeast. In fact, without myosin, budding in yeast is disrupted. Data in Sheng’s paper supported this idea. Time-lapse confocal microscopy showed that the normal extension and retraction of the filopodia is reduced to just extension in the presence of blebbistatin, suggesting that myosin does rein in the filopodia.

Perhaps one of the most interesting aspects of myosin IIB is that its effects on morphology seem tightly linked to the spines’ function. When Ryu and colleagues measured excitatory postsynaptic potentials in blebbistatin-treated cells, they found that amplitudes were down by more than half. This may well be related to loss of AMPA receptors, because addition of the myosin II inhibitor led to depletion of these receptors.

A micro-chaperone for Limk1
Together, these two papers reveal new insights into where and how spines form, but they don’t address the question of why. That’s left up to Michael Greenberg and colleagues at Children’s Hospital and Harvard Medical School in Boston, and also at the Medical University of Vienna, Austria. First author Gerhard Schratt and colleagues reported in the January 19 Nature that spine volume is regulated by a microRNA.

Schratt and colleagues investigated the role of the brain microRNA-134 (miR-134), finding that it is localized near synaptic sites in dendrites on cultured hippocampal neurons. When overexpressed, it decreased the volume of dendritic spines. This result prompted the authors to search the genome for genes this small RNA might target. One of the sequences that partially matches the sequence of miR-134 is in the 3’ untranslated region of the gene for Lim domain-containing protein kinase (Limk1). The microRNA not only binds to this site, but it also inhibits translation of the kinase, Schratt and colleagues report.

This finding may be of particular interest to schizophrenia researchers. Ablation of Limk1 leads to cognitive defects in mice (see Meng et al., 2002), and the kinase has been linked to two molecules that are heavily implicated in synaptic plasticity, brain-derived neurotrophic factor (BDNF) and actin. The former induces expression of Limk1, while actin polymerization can be regulated by the Limk1 substrate, cofilin. Actin, of course, is involved in the extension of spine filopodia. In fact, Schratt and colleagues found that reporter genes only respond to BDNF if they carry the miR-134 binding site, indicating that the neurotrophin may somehow relieve repression of Limk1 translation.

The authors speculate that miR-134 may keep Limk1 mRNA in a dormant state while it is being transported to synaptic sites. “Upon synaptic stimulation, the release of BDNF may trigger activation of the TrkB/mTOR signaling pathways, which inactivates the miR-134-associated silencing complex by an as-yet-unknown mechanism, leading to enhanced Limk1 protein synthesis and spine growth,” suggest the authors.

Cytoskeletal proteins perturbed in schizophrenia
Dendritic spines have also caught the attention of some schizophrenia researchers (see, e.g., Law et al. 2004), as has synaptic dysfunction in general (for review, see Harrison and Weinberger, 2005; Owen et al., 2005). Previously, David Lewis and colleagues demonstrated layer-specific reductions in spine density in the dorsolateral prefrontal cortex in people with schizophrenia (Kolluri et al., 2005; Glantz and Lewis, 2000). In the present paper, first author Justin Hill, Takanori Hashimoto, and Lewis put a finer focus on these abnormalities, exploring whether they correlate with changes in expression of any of five proteins known to be involved in the regulation of spine morphology.

Hill and colleagues report reductions of mRNA for two proteins, Cdc42 and Duo, in schizophrenia versus control postmortem tissue. The changes were detected in gray matter, but not white matter, and correlated with spine density in individual study subjects. The RhoGTPase Cdc42 is known to be critical for the formation of filopodia, and Duo, a GDP-GTP exchange factor, is thought to be involved in maintaining mature spines and the apposition between pre- and postsynaptic sites. Curiously, the researchers did not detect corresponding changes in proteins closely related to these two, such as RhoA, Rac1, or drebrin. In an effort to control for possible effects of antipsychotic medications, the researchers administered drugs to macaques but found no changes in mRNAs of the proteins of interest from either first- or second-generation antipsychotics (haloperidol and olanzapine). "Taken together, a decrease in the expression of both Cdc42 and Duo mRNAs would be expected to result in a reduction in the formation of new spines and an impairment in the maintenance of existing, mature spines," they write. It might be surprising that the other cytoskeletal mRNAs assayed were unchanged, given their close relationships to Cdc42 and Duo in regulating spines. But, as the authors point out, changes in levels of Duo, for example, could affect activation of its downstream target Rac1, which helps maintain spine stability, without necessarily changing levels of the target protein.

Because their earlier work had found lower spine density in dendrites of the pyramidal cells of layer 3, but not those of layer 6, Hill and colleagues guessed that they might find corresponding differences in the distribution of Cdc42 and Duo mRNAs. This did not turn out to be the case—a finding that the authors interpret to support the idea that changes in the expression of the two cytoskeletal proteins are causal, and not a consequence of changes of spine density. If Cdc42 and Duo expression had been found to be reduced only in layer 3, they reason, this would have left open the possibility that spine density reductions precede changes in expression of the proteins. But what, then, causes the selective reduction in these proteins, and of spines, in layer 3? Differential activation could be the explanation: The pyramidal cells of layers 3 and 6 have quite different sources of input, in part due to radically different dendritic arbor patterns. As excitatory input is required for spine formation, the authors speculate that a reduction of excitatory input to only layer 3 might underlie reduced spine density in its pyramidal cells.—Tom Fagan and Hakon Heimer.

References:
Hill JJ, Hashimoto T, Lewis DA. Molecular mechanisms contributing to dendritic spine alterations in the prefrontal cortex of subjects with schizophrenia. Mol Psychiatry . 2006 Jan 10 ; Abstract

O’Brien J, Unwin N. Organization of spines on the dendrites of Purkinje cells. PNAS early edition. 19 January, 2006. Abstract

Ryu J, Liu L, Wong TP, Wu DC, Burette A, Weinberg R, Wang YT, Sheng M. A critical role for myosin IIB in dendritic spine morphology and synaptic function. Neuron. 2006 Jan 19;49(2):175-82. Abstract

Schratt GM, Tuebing F, Nigh EA, Kane CG, Sabatini ME, Kiebler M, Greenberg ME. A brain-specific microRNA regulates dendritic spine development. Nature. January 19, 2006;439:283-289. Abstract

Comments on News and Primary Papers
Comment by:  Amanda Jayne Law, SRF Advisor
Submitted 13 February 2006
Posted 13 February 2006

The formation of dendritic spines during development and their structural plasticity in the adult brain are critical aspects of synaptogenesis and synaptic plasticity. Actin is the major cytoskeletal source of dendritic spines, and polymerization/depolymerization of actin is the primary determinant of spine motility and morphogenesis. Some, but not all, postmortem studies in schizophrenia have identified reduced dendritic spine density in neurons of the hippocampal formation and dorsolateral prefrontal cortex (for review, see Honer et al., 2000); however, little is known about the underlying pathogenic mechanisms affecting synaptic function in the disease.

Many different factors and proteins are known to control dendritic spine development and remodeling (see Ethell and Pasquale, 2005). Comprehensive investigation of the effectors and signaling pathways involved in regulating actin dynamics may provide insight into the molecular mechanisms mediating altered cortical microcircuitry in the disease.

David Lewis and colleagues have previously reported reduced spine density in the basilar dendrites of pyramidal neurons in laminar III of the DLPFC (though this is not clearly a laminar-specific finding). In their current study, Hill et al. extended these investigations to examine gene expression levels for members of the RhoGTPase family of intracellular signaling molecules (e.g., Cdc42, Rac1, RhoA, Duo), and Debrin, an F-actin binding protein, all of which are critical signal transduction molecules involved in spine formation and maintenance. Their aim was to determine whether alterations in the expression of one of more molecules may underlie the reduced spine density seen in the disorder. Hill et al. report that reductions in Cdc42 and Duo mRNA are observed in the DLPFC in schizophrenia and correlate with spine density on deep layer III pyramidal neurons. This paper provides preliminary evidence that "gene expression levels of certain mRNAs encoding proteins known to be key regulators of dendritic spines are reduced in the DLPFC in schizophrenia." However, the paper also reports that these two mRNAs are reduced in lamina where significant reductions in spine density are not observed in schizophrenia. These results may suggest, as the authors discuss, that reduced expression of Cdc42 and Duo might contribute to, but is not sufficient to cause reduced, spine density.

Synaptic dysfunction has received increasing attention as a key feature of schizophrenia’s neuropathology and possibly its genetic etiology (Law et al., 2004). Neuregulin 1 (NRG1), a lead schizophrenia susceptibility gene, is known to be a critical upstream regulator of signal transduction pathways modulating cytoskeletal dynamics, playing pivotal roles in synapse formation and function. We have previously reported that isoform-specific alterations of the NRG1 gene and its primary receptor, ErbB4, are apparent in the brain in schizophrenia and related to genetic risk for the disease (Law et al, 2005a, Law et al, 2005b). Altered NRG1/ErbB4 signaling in schizophrenia may be a pathway to aberrant cortical neurodevelopment and synaptic function via dysregulation of specific intracellular signaling pathways linked to actin. The lack of significant alterations in gene expression levels for proteins such as Rac1 and RhoA in the DLPFC (gray matter, as reported by Hill and colleagues) in schizophrenia might be because the primary defect may not lie with the expression of these molecules but with the upstream modulation of their function and activity. Therefore, investigation of the proteins themselves, their phosphorylation status and activity, will be useful in understanding how genes effect molecular pathways that mediate biological risk for schizophrenia. The study of intracellular signaling cascades may be a route to a closer understanding of the biological mechanisms underpinning the association of genes such as NRG1 and ErbB4 with schizophrenia and their relationship to its neuropathology.

References:

Ethell IM, Pasquale EB. Molecular mechanisms of dendritic spine development and remodeling. Prog Neurobiol. 2005 Feb;75(3):161-205. Epub 2005 Apr 2. Review. Abstract

Honer G, Young C, and Falkai P, 2000. Synaptic Pathology in the Neuropathology of Schizophrenia, Progress and interpretation. Oxford University Press, edited by Paul J Harrison and Gareth W. Roberts, pp105-136.

Law AJ, Weickert CS, Hyde TM, Kleinman JE, Harrison PJ. Reduced spinophilin but not microtubule-associated protein 2 expression in the hippocampal formation in schizophrenia and mood disorders: molecular evidence for a pathology of dendritic spines. Am J Psychiatry. 2004 Oct;161(10):1848-55. Abstract

Law, 2005a. Soc Neurosci Abstract, SFN Annual Meeting, Washington DSC, 2005. Neuregulin1 and schizophrenia: A pathway to altered cortical circuits. Also See SfN 2005 research news: Cortical Deficits in Schizophrenia: Have Genes, Will Hypothesize.

Law 2005b ACNP Abstract, Neuropsychopharmacology, vol. 30, Supplement 1. SNPing away at NRG1 and ErbB4 gene expression in schizophrenia.

View all comments by Amanda Jayne Law

Primary Papers: Molecular mechanisms contributing to dendritic spine alterations in the prefrontal cortex of subjects with schizophrenia.

Comment by:  Patricia Estani
Submitted 14 February 2006
Posted 14 February 2006
  I recommend this paper

Comments on Related News


Related News: Architect of Synaptic Plasticity Links Spine Form and Function

Comment by:  Akira Sawa, SRF Advisor
Submitted 29 December 2007
Posted 29 December 2007

Synaptic disturbance in the pathology of schizophrenia is a well-established idea. Lewis’s lab has reported decreased synaptic spine density in brains from patients with schizophrenia (Glantz and Lewis, 2000). Although it is unclear whether this is primary or secondary, expression of kalirin-7-associated molecules is decreased (Hill et al., 2006). Thus, kalirin-7-associated cellular signaling in synaptic spines may have implication for the pathology of schizophrenia. In this sense, I regard the recent publication from Penzes’s lab as very interesting in schizophrenia research.

It is still unclear whether kalirin-7 may interact with genetic susceptibility factors for schizophrenia, such as ErbB4 and DISC1. Until the protein interactions are tested by co-immunoprecipitation at endogenous protein levels, as well as validated by cell staining, we cannot tell whether or not such factors are really associated with the kalirin-7 pathway. This putative protein interaction of kalirin-7 with DISC1 or ErbB4 will be an important issue to address in the future.

In Penzes’s neuronal cultures, he has focused on spine formation in pyramidal neurons, but not in interneurons. Thus, the mechanism proposed in his study will be useful to consider possible pathology in pyramidal neurons in brains of patients with schizophrenia.

View all comments by Akira Sawa

Related News: DISC1 and SNAP23 Emerge In NMDA Receptor Signaling

Comment by:  Jacqueline Rose
Submitted 2 March 2010
Posted 2 March 2010
  I recommend the Primary Papers

The newly published paper by Katherine Roche and Paul Roche reports SNAP-23 expression in neuron dendrites and examines the possible role of this neuronal SNAP-23 protein. To this point, SNAP-23 has traditionally been discussed in reference to vesicle trafficking in epithelial cells (see Rodriguez-Boulan et al., 2005 for review), so it is of interest to determine the function of SNAP-23 in neurons. Suh et al. report that surface NMDA receptor expression and NMDA-mediated currents are inhibited following SNAP-23 knockdown. Further, SNAP-23 knockdown results in a specific decrease in NR2B subunit insertion; previously, the NR2B subunit has been reported to preferentially localize to recycling endosomes compared to NR2A (Lavezzari et al., 2004). Given these findings, it is reasonable to conclude that SNAP-23 may be involved in maintaining NMDA receptor surface expression possibly by binding to NMDA-specific recycling endosomes.

Interestingly, there is recent evidence that PKC-induced NMDA receptor insertion is mediated by another neuronal SNARE protein; postsynaptic SNAP-25 (Lau et al., 2010). It is possible that activity-induced NMDA receptor trafficking is mediated by SNAP-25, while baseline maintenance of NMDA receptor levels relies on SNAP-23. Other evidence to suggest a strictly regulatory role for SNAP-23 in neuronal NMDA insertion is the finding that activity-dependent receptor insertion from early endosomes has previously been reported to be restricted to AMPA-type glutamate receptors (Park et al., 2004). However, it is possible that activity-induced insertion of AMPA receptors occurs via a distinct endosome pool than NMDA receptors; AMPA and NMDA receptor trafficking has been reported to proceed by distinct vesicle trafficking pathways (Jeyifous et al., 2009).

Although SNAP-23 may not be involved in activity-dependent early endosome receptor trafficking, it is possible that SNAP-23 operates in other pathways linked to activity-induced NMDA receptor trafficking. For instance, SNAP-23 may be the SNARE protein by which lipid raft shuttling of NMDA receptors occurs. SNAP-23 has been found to preferentially associate with lipid rafts over SNAP-25 in PC12 cells (Salaün et al., 2005). As well, NMDA receptors have been found to associate with lipid raft associated proteins flotilin-1 and -2 in neurons (Swanwick et al., 2009). Lipid raft trafficking of NMDA receptors to post-synaptic densities has been reported to follow global ischemia (Besshoh et al., 2005), and the possibility remains that under certain circumstances, NMDA trafficking occurs by lipid raft association to SNAP-23.

Taken together, the discovery of post-synaptic SNARE proteins offers several avenues of research to determine their roles and functions in glutamatergic synapse organization. Further, investigating disruption of synaptic receptor organization presents several possibilities for potential etiologies of disorders linked to compromised glutamate signaling like schizophrenia.

References:

Besshoh, S., Bawa, D., Teves, L., Wallace, M.C. and Gurd, J.W. (2005). Increased phosphorylation and redistribution of NMDA receptors between synaptic lipid rafts and post-synaptic densities following transient global ischemia in the rat brain. Journal of Neurochemistry, 93: 186-194. Abstract

Jeyifous, O., Waites, C.L., Specht, C.G., Fujisawa, S., Schubert, M., Lin, E.I., Marshall, J., Aoki, C., de Silva, T., Montgomery, J.M., Garner, C.C. and Green, W.N. (2009). SAP97 and CASK mediate sorting of NMDA receptors through a previously unknown secretory pathway. Nature Neuroscience, 12: 1011-1019. Abstract

Lau, C.G., Takayasu, Y., Rodenas-Ruano, A., Paternain, A.V., Lerma, J., Bennet, M.V.L. and Zukin, R.S. (2010). SNAP-25 is a target of protein kinase C phosphorylation critical to NMDA receptor trafficking. Journal of Neuroscience, 30: 242-254. Abstract

Lavezzari, G., McCallum, J., Dewey, C.M. and Roche, K.W. (2004). Subunit-specific regulation of NMDA receptor endocytosis. Journal of Neuroscience, 24: 6383-6391. Abstract

Park, M., Penick, E.C., Edward, J.G., Kauer, J.A. and Ehlers, M.D. (2004). Recycling endosomes supply AMPA receptors for LTP. Science, 305: 1972-1975. Abstract

Rodriguez-Boulan, E., Kreitzer, G. and Müsch, A. (2005) Organization of vesicular trafficking in epithelia. Nature Reviews: Molecular Cell Biology, 6: 233-247. Abstract

Salaün, C., Gould, G.W. and Chamberlain, L.H. (2005). The SNARE proteins SNAP-25 and SNAP-23 display different affinities for lipid rafts in PC12 cells. Journal of Biological Chemistry, 280: 1236-1240. Abstract

Suh, Y.H., Terashima, A., Petralia, R.S., Wenthold, R.J., Isaac, J.T.R., Roche, K.W. and Roche, P.A. (2010). A neuronal role for SNAP-23 in postsynaptic glutamate receptor trafficking. Nat Neurosci. 2010 Mar;13(3):338-43. Abstract

Swanwick, C.C., Shapiro, M.E., Chang, Y.Z. and Wenthold, R.J. (2009). NMDA receptors interact with flotillin-1 and -2, lipid raft-associated proteins. FEBS Letters, 583: 1226-1230. Abstract

View all comments by Jacqueline Rose