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Jeans AF, Oliver PL, Johnson R, Capogna M, Vikman J, Molnár Z, Babbs A, Partridge CJ, Salehi A, Bengtsson M, Eliasson L, Rorsman P, Davies KE. A dominant mutation in Snap25 causes impaired vesicle trafficking, sensorimotor gating, and ataxia in the blind-drunk mouse. Proc Natl Acad Sci U S A. 2007 Feb 13 ; 104(7):2431-6. Pubmed Abstract

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Primary Papers: A dominant mutation in Snap25 causes impaired vesicle trafficking, sensorimotor gating, and ataxia in the blind-drunk mouse.

Comment by:  William HonerAlasdair Barr
Submitted 19 February 2007
Posted 19 February 2007

A New SNP for SNAP-25—An Animal Model of Schizophrenia?
SNAP-25 is one of the three core presynaptic proteins that combine to form the SNARE ternary complex, and as such, it plays a critical role in calcium-triggered exocytic transmitter release. There are two isoforms, SNAP-25A and SNAP-25B, which are highly homologous proteins that differ by only nine amino acids (Bark and Wilson, 1994). SNAP-23 represents another member of this protein family, which shares an approximately 60 percent identity with SNAP-25 (Ravichandran et al., 1996). Current theories of vesicular membrane fusion emphasize the role of SNARE proteins. According to these models, syntaxin 1, synaptobrevin (VAMP) 2 and SNAP-25 regulate neuroexocytosis by forming a complex that forces vesicle and plasma membranes together, allowing the transmitter contained in the synaptic vesicle to enter the synaptic cleft (Jahn and Sudhof). The precise details of this mechanism continue to be elucidated, and the emerging picture represents a more complex mosaic than may initially have been expected. For example, recent evidence demonstrates that in the large, glutamatergic calyx of Held presynaptic terminal from rats, loss of SNAP-25 produces a graded loss of calcium-sensitive transmitter release, unlike loss of syntaxin or synaptobrevin, which generates an “all-or-nothing” block of release (Sakaba et al., 2005).

Data from in situ and immunohistochemical studies indicate that SNAP-25 displays a regional pattern of expression in the brain that changes developmentally. The cellular distribution of SNAP-25 also changes during development, as in the striatum, where SNAP-25 in the caudate nucleus is initially concentrated in axons, but subsequently localized in presynaptic regions of these axons (Oyler et al., 1992). During development, SNAP-25 plays a key role in neurite elongation and axonal growth. In adulthood, the primary function of SNAP-25 in the brain is the regulation of calcium-dependent exocytosis of neurotransmitter, although it is important to note that SNAP-25 also regulates exocytosis in non-neuronal systems, such as insulin secretion by pancreatic β cells (Gonelle-Gispert et al., 1999), and histamine release by gastric enterochromaffin-like cells (Hohne-Zell et al., 1997). The exocytic activity of SNAP-25 is regulated by protein kinase-dependent phosphorylation at a number of different sites (Nagy et al., 2004).

The report from Jeans et al. (Jeans et al., 2007) focuses on a new mutant mouse line, identified in a forward genetics mutagenesis screen. The authors determine that the mutant mouse line has an isoleucine-to-threonine amino acid switch in a region of SNAP-25 which is necessary for binding to the other partners in the SNARE complex. Interestingly, this mutation appeared to act to stabilize the SNARE complex. The authors identified abnormalities of synaptic transmission in neurons, which included reduced spontaneous mEPSCs and a depression of stimulated EPSCs at higher frequencies, consistent with a “functional depletion of synaptic vesicles due to defective synaptic vesicle mobilization and replenishment.” In vitro insulin release in pancreatic β cells was also impaired, although appeared normal in vivo. The mice also exhibited a number of behavioral abnormalities. These included impaired prepulse inhibition, anxiety-like behavior in one task, and decreased interest in a play object. It is posited that the current mouse line may be a useful model for investigating the role of SNAP-25 in schizophrenia.

There is a large body of evidence that points to a link between SNAP-25 and schizophrenia (we recently reviewed this in Barr et al., 2006). As the authors of the study mention, there is strong genetic and postmortem data which support a role for this presynaptic protein in the etiology of the disorder. We previously examined SNAP-25, syntaxin, and synaptobrevin in the anterior frontal cortex sample from four groups of subjects: controls, schizophrenia with natural causes of death, schizophrenia with suicide as a cause of death, and depression with suicide (Honer et al., 2002). SNAP-25 levels were low, but only in the schizophrenia natural death group. However, we also developed an in vitro assay to look at formation of the SNARE complex. This assay showed increased likelihood of SNARE complex formation in the samples from both groups with suicide as a cause of death. More recently, we reported that levels of SNAP-25 were decreased in the hippocampus in schizophrenia, and that in subregions of the hippocampus, lower levels of SNAP-25 were associated with premorbid cognitive impairment (Barr et al., 2006). Interestingly, we also reported in a separate study on rats that the typical antipsychotic drugs haloperidol and chlorpromazine act to increase levels of SNAP-25 in the hippocampal trisynaptic pathway (Barr et al., 2006).

The question then arises: How useful will the current mutant SNAP-25 line of mice be in uncovering a role for this presynaptic protein in schizophrenia? The present study represents an important step in that the authors have developed a fertile, viable line of mice with altered SNAP-25 function (synaptobrevin 2 and SNAP-25 homozygous knockout mice are perinatally lethal). The authors are perhaps premature, though, to discount the importance of two alternate models of reduced SNAP-25 function, which include the hemizygous SNAP-25 knockout mouse and the coloboma mutant mouse line. Jeans and colleagues state that the SNAP-25 “heterozygotes show no apparent phenotype,” while the “spontaneous mouse mutant coloboma carries a heterozygous deletion spanning four genes including Snap25, but its locomotor hyperactivity phenotype is likely to depend on the additional loss of one or more of the other genes within the deleted region” (Jeans et al., 2007). These statements represent the truth, but not necessarily the whole truth. To our knowledge, the behavioral phenotype of the hemizygous SNAP-25 knockout mouse has never been reported in detail. The original manuscript by Washbourne et al. does not mention that complex behavioral tasks, such as prepulse inhibition or anxiety paradigms, were ever conducted, leaving open the question as to whether similar effects would be observed as in the I67T mutant mice (Washbourne et al., 2002).

The coloboma mutant mouse line has a contiguous gene defect that results in altered monoamine neurotransmission and locomotor hyperactivity, the latter being reduced by treatment with d-amphetamine (Wilson, 2000). The coloboma line has been put forward as an animal of attention-deficit hyperactivity disorder (ADHD). While additional developmental defects in this mouse, such as ocular dysmorphology, may result from loss of gene information other than SNAP-25, the behavioral hyperactivity was rescued by a SNAP-25 transgene (Steffensen et al., 1999). Although schizophrenia and ADHD are distinct clinical disorders, they may share some common neurocognitive impairments, such as attentional and executive deficits (Banaschewski et al., 2005). Thus, it is too early yet to bury the SNAP-25 hemizygous knockout and coloboma mutant mice as research models for understanding SNAP-25 in schizophrenia, especially as the issue of pleiotropic developmental deficits in the I67T mutant mice remains unresolved. More detailed behavioral phenotyping of these alternate strains of mice is required, and especially in tasks that are homologous to those where deficits are observed in schizophrenia.

The behavioral changes that Jeans and colleagues describe include several that provide intriguing preliminary evidence for schizophrenia-like abnormalities. The I67T mutant mice display reduced prepulse inhibition of the acoustic startle reflex at a single prepulse intensity of 90 dB. As schizophrenia patients typically show robust deficits in prepulse inhibition across a range of prepulse intensities, and in both acoustic as well as multimodal acoustic/tactile gating paradigms (Braff et al., 2001), which can be modeled in rodents (Geyer et al., 2002; Geyer et al., 2001), it will be necessary to evaluate how extensive are the sensorimotor gating deficits in these mice. Clearly, it will be necessary to determine if the prepulse inhibition deficits can be reversed by treatment with both first- and second-generation antipsychotic drugs, as loss of prepulse inhibition is restored by these drugs in many animal models of schizophrenia. We have also recently demonstrated that mutant mice with alterations in the reelin signaling pathway, which is implicated in schizophrenia, were more sensitive to the disruptive effects of the NMDA-receptor antagonist phencyclidine on prepulse inhibition (Barr et al., 2007), consistent with the greater sensitivity of schizophrenia patients to the effects of this class of drugs (Krystal et al., 2003). Similar studies, using both NMDA-receptor antagonists and dopamine agonists, could be conducted in prepulse inhibition and other behavioral tasks to determine if the current line of mice exhibits any evidence for pre-existing neurochemical “sensitization.”

Little should be read into the elusive anxiety-like behavioral effects in the I67T mutant mice, whereby anxiety is observed in one paradigm, but not another. Anxiety is a complicated psychological construct, and different aspects of anxiety are measured by different tasks in animals, which may or may not be affected by the degree of stressfulness of the conditions (Holmes et al., 2002). In humans, self-report of anxiety presumably provides a global measure. Although anxiety is a commonly occurring comorbid condition with schizophrenia, it is not a requirement for an animal model of schizophrenia.

The 20 percent reduction in time spent playing with a “toy” by I67T mutant mice is suggested to model aspects of the “negative symptomatology” of schizophrenia. This is in apparent contrast to the significant increase in time spent interacting with a conspecific on the first trial of a social recognition task (although this issue is not raised in the manuscript). It is clear that withdrawal from the surrounding environment and reduced interest in normally rewarding stimuli are features of schizophrenia (Wolf, 2006), and thus the reduced time with the toy may be an important behavioral index of abnormal motivation. However, social withdrawal is equally, if not more so, a negative symptom of schizophrenia (Horan et al., 2007), and so it will be important in future studies with these mutant mice to determine why initial social interest is increased, whereas it is decreased for inanimate objects. It will also be possible to evaluate whether the reduced time spent playing with the toy can be reversed by treatment with antipsychotic drugs, although negative symptoms are largely unresponsive to the effects of most pharmacotherapies (Erhart et al., 2006). An additional caveat regarding the novel mutant mice is the presence of an ataxic gait, which is not characteristically seen in schizophrenia or alternate presynaptic protein knockout mouse models of the disorder, such as complexin II mutant mice (Glynn et al., 2003), and should be controlled for in sensitive behavioral tests that may be affected by motor movement.

Future behavioral studies with this line of mutant mice should emphasize current priorities for research in schizophrenia, such as elucidating the physiological basis of neurocognitive deficits in the disorder. These have been espoused in detail by research groups such as the MATRICS/TURNS initiative (Marder, 2006). There are now numerous, well-validated murine paradigms for measurement of neuropsychological constructs such as memory, attention, and executive function, and a better understanding of the molecular basis of deficits in these cognitive domains is desperately needed, which may well provide the basis for novel pharmacotherapies. There are currently few compounds with known capacity to alter the physiological function of SNAP-25, but given that the protein has multiple phosphorylation sites, and displays direct interactions with a number of ion channels and transporters (Barr et al., 2006), there is reason for optimism.

Finally, other forms of genetic variation in SNAP-25 related to some aspects of the phenotype of schizophrenia are worthy of consideration. For example, we previously observed a relationship between genetic variation in SNAP-25 and treatment response in schizophrenia (Muller et al., 2005). In the context of the pancreatic β cell abnormalities in the study by Jeans and colleagues, it is of further interest that the same genetic variation in SNAP-25 which predicted antipsychotic treatment response also showed a suggestive relationship with weight gain. In conclusion, we congratulate the authors of the present study for a valuable contribution to the growing body of data which support a link between SNAP-25 and schizophrenia, and we hope that they will continue to expand on their interesting behavioral and physiological findings.

References
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Jeans AF, Oliver PL, Johnson R, Capogna M, Vikman J, Molnar Z, Babbs A, Partridge CJ, Salehi A, Bengtsson M, Eliasson L, Rorsman P, Davies KE. A dominant mutation in Snap25 causes impaired vesicle trafficking, sensorimotor gating, and ataxia in the blind-drunk mouse. Proc Natl Acad Sci U S A. 2007 Feb 13;104(7):2431-6. Epub 2007 Feb 5. Abstract

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View all comments by William Honer
View all comments by Alasdair Barr

Primary Papers: A dominant mutation in Snap25 causes impaired vesicle trafficking, sensorimotor gating, and ataxia in the blind-drunk mouse.

Comment by:  Elizabeth Scarr
Submitted 12 March 2007
Posted 12 March 2007

The paper by Jeans et al. describes a viable mouse which carries a mutation (I67T) in the b isoform of SNAP-25, the blind-drunk mouse. Based primarily on the behavioral phenotype and on the results of two postmortem studies, the authors suggest that the line might be a suitable model for research into schizophrenia.

The mice show less frequent spontaneous release of glutamate than the wild-type, but the amplitude of the miniature excitatory postsynaptic currents were similar between genotypes. More severe deficits were seen with the release of glutamate in response to stimulation; the blind-drunk mice show a rapid decrease in synaptic transmission when high-frequency repetitive stimulation is applied, suggesting that these mice may have deficits in their ability to traffic synaptic vesicles.

Unlike the Coloboma mouse (a chromosomal deletion of 1-2cM near the distal end of Ch2 [Wilson, 2000], encompassing the gene for SNAP-25 amongst others), the blind-drunk mouse does not exhibit spontaneous hyperactivity. This finding is of particular interest because when the Coloboma mouse is “rescued” by cross breeding designed to correct the lack of SNAP-25, their level of activity returns to normal. However, Jeans et al. fail to address this disparity.

Compared to wild-type, the blind-drunk mouse performs less well on the rotarod; has impaired prepulse inhibition; spends more time interacting with a novel mouse on the initial presentation but less on subsequent confrontations; transitions fewer times between light/dark areas; is less rapid moving into the dark area from the light; and tends to be more inactive, less interactive, and less exploratory in an enriched environment.

The authors suggest that the impaired prepulse inhibition and the apathetic behavior observed in the enriched environment are comparable to aspects of schizophrenia and cite two postmortem studies that showed a decrease in the levels of SNAP-25 in patients with schizophrenia in support of this. Patients with schizophrenia do exhibit impaired sensorimotor gating; however, so do patients with bipolar disorder, obsessive compulsive disorder, and Huntington disease (see Braff et al., 2001 and Geyer et al., 2002). Prepulse inhibition has been used to identify drugs that might have antipsychotic actions; however, not all drugs that are effective at correcting a deficit in prepulse inhibition are useful antipsychotic drugs.

The “apathetic” behavior exhibited by the mice in an enriched environment is suggested to replicate aspects of the negative symptomatology of schizophrenia. I would suggest that inactivity in mice does not necessarily reflect apathy. It is possible that the ataxia exhibited by these animals is a contributing factor to their inactivity and lack of interaction.

The authors correctly cite two papers which showed a decrease in the postmortem levels of SNAP-25 from patients with schizophrenia; however, they have not taken into consideration the study that showed increases, decreases, and no change in levels of SNAP-25 in different brain regions from the same subjects (Thompson et al., 1998), suggesting that the decrease seen in SNAP-25 is not an all-or-none effect but rather seems to be a complex scenario.

Similarly, the situation in bipolar disorder is equivocal, with one study showing a decrease in hippocampal SNAP-25 in tissue from subjects with either bipolar disorder or schizophrenia (Fatemi et al., 2001) and another showing elevated levels of SNAP-25 in the dorsolateralprefrontal cortex of subjects with bipolar disorder, but no change in schizophrenia (Scarr et al., 2006).

The most interesting aspects of the paper by Jeans et al. is the information obtained from the blind-drunk mice regarding how the mutation changes the affinity of SNAP-25 for its binding partners and results in a lower frequency of spontaneous miniature excitatory postsynaptic currents. Further exploration of these changes is likely to generate a great deal of information that might be useful in understanding how changes in synaptic vesicle mobilization might affect synaptic function—for example, do the levels of the binding partners of SNAP-25 change? If less transmitter is released, are the number of pre- and postsynaptic receptors altered? Is there any effect on the function of the surrounding astrocytes? What are the downstream effects of these changes? Is the functionality of distal brain regions affected as a consequence of changes in function of projecting neurons?

The potential link between type 2 diabetes mellitus and low expression of SNAP-25 is also of interest given the problem of weight gain seen with the atypical antipsychotic medications. It may be that there is an endophenotype of schizophrenia that is more susceptible to developing type 2 diabetes mellitus and that these patients have impaired SNAP-25 expression, despite polymorphisms in SNAP-25 not being associated with weight gain (Muller et al., 2005).

In summary, the blind-drunk mouse may well play a role in psychiatric research, but this role is more likely to be towards understanding the consequences of changes in normal synaptic function rather than as a model of either schizophrenia or bipolar disorder.

View all comments by Elizabeth Scarr