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Schizophrenia-Related Changes Emerge in Kalirin-knockout Mice

1 September 2009. Genetically engineered mice without the brain protein kalirin show sparse dendritic spines and decreased glutamatergic signaling in the frontal cortex, according to a study in the August 4 PNAS. The mice also show changes in cognition and behavior that are reminiscent of those seen in some people with schizophrenia. Furthermore, some of the deficits emerged only as the knockout mice matured, echoing developmental aspects of the disease. The work, from Peter Penzes of Northwestern University in Chicago, Illinois, and colleagues, fits with the known role of kalirin in dendritic spine formation, and raises the intriguing possibility that lower kalirin levels seen in brain tissue of people with schizophrenia could play a part in the disease.

In the brain, most excitatory nerve impulses land on dendritic spines, postsynaptic linchpins of learning and memory (see SRF related news story; SRF news story; and SRF news story). Research on neurons, and more recently, on mice, suggests that dendritic spine formation depends on kalirin (for reviews, see Penzes and Jones, 2008; Rabiner et al., 2005; Sommer and Budreck, 2009). The protein, a guanine-nucleotide exchange factor, prompts Rac1, a GTPase from the Rho family, to bind GTP. This triggers remodeling of actin, which forms a framework for the spines.

The schizophrenia connection unfolds from observations that the prefrontal cortex of subjects with the disease contains too few dendritic spines (see, e.g., Glantz and Lewis, 2000). This may go hand-in-hand with their shortage of kalirin-7, the most plentiful kalirin isoform in normal adult brains (see SRF related news story).

To shed light on how kalirin might affect phenotypes tied to neuropsychiatric disease, first author Michael Cahill and his coauthors developed mice without the gene KALRN, which encodes kalirin. “Surprisingly, we found that the knockout of KALRN produces striking, yet selective, age-dependent, anatomical, functional, and behavioral deficits, which resemble disease-related phenotypes,” they write.

To the naked eye, the mice looked normal, despite the absence of kalirin. However, their prefrontal cortex showed less Rac1 activity than that of wild-type mice. Since Rac1 activity in the hippocampus remained normal, the researchers think that Rac guanine-nucleotide exchange factors other than kalirin may keep communication channels in the hippocampus open. The cortex, on the other hand, may need kalirin to keep neurons talking.

In contrast to the Rac1 findings, the cortex and hippocampus of the knockout mice showed normal levels of Cdc42, a Rho-like GTPase not controlled by kalirin. “This further indicates that kalirin loss produces a specific deficit in cortical Rac1-GTP levels,” write Cahill and colleagues.

Quiet gene stymies signaling
Next, Cahill and colleagues explored how silencing KALRN affects the dendrites. In the frontal cortex, but not the hippocampus, they found only scattered dendritic spines in the knockout mice. This low density of dendritic spines appeared age-dependent: The researchers saw it only at age 12 weeks and not at three weeks, suggesting a parallel between the development of dysfunctional spine formation and the course of schizophrenia, which typically manifests in adolescence or later.

In mature cortical neurons in culture, overexpression of kalirin-7 reversed the deficits, further implicating kalirin in spine formation. The protein may also help slip the AMPA type of glutamate receptors into spines and control neural signaling involving these receptors (Xie et al., 2007). In fact, when Cahill and colleagues examined layer 5 pyramidal neurons from the frontal cortex, they found reduced neurotransmission mediated by AMPA receptors in the knockout mice.

Fleshing out the phenotype
When the researchers turned to cognition and behavior, they found changes in the knockout mice that echo some of those found in subjects with schizophrenia. For instance, at age 12 weeks, the kalirin-deficient mice performed poorly on tests of working memory, despite having tested normal at three weeks. In contrast, their reference memory, which involves associations across trials, seemed to match that of their wild-type peers. To explain these findings, Cahill and colleagues note that working memory taps connections between the prefrontal cortex and the hippocampus, whereas reference memory stems from the hippocampus and does not rely on the prefrontal cortex.

Besides working memory impairments, the knockout mice also showed flawed sensory-motor gating in a prepulse inhibition paradigm. In addition, they lacked a healthy interest in socializing. At age 12 weeks, they walked excessively in their cages, which they had not done when they were younger. The atypical antipsychotic clozapine curbed their hyperactivity but did little for their memory.

Four-legged schizophrenia models?
Clearly, this study sheds light on kalirin’s in vivo effects. It says much about the protein’s ability to promote Rac1 activity, spine building, and glutamatergic signaling, with consequences for cognition and behavior. In addition, Cahill and colleagues present some evidence that it influences only selective outcomes, bolstering the case that it may play a unique role in cortical and behavioral deficits of interest in schizophrenia.

“The age-dependent delayed functional and behavioral impairments in schizophrenia have been difficult to explain in humans and to model in animals,” write the researchers. Time will tell whether kalirin-knockout mice can help with those tasks.—Victoria L. Wilcox.

Reference:
Cahill ME, Xie Z, Day M, Barbolina MV, Miller CA, Weiss C, Radulovic J, Sweatt JD, Disterhoft JF, Surmeier J, Penzes P. Kalirin regulates cortical spine morphogenesis and disease-related behavioral phenotypes. PNAS. 2009 Aug 4;106(31):13058-13063. Abstract

Comments on Related News


Related News: Dendritic Spine Research—Putting Meat on the Bones

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

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