2.4 Discussion
2.4.2 Effects of the loss of densin on brain transcription
Using RNA-Seq, we investigated changes in brain transcription in mice missing the postsynaptic protein densin. We compared transcript expression in the forebrain and hippocampus of densin knockout mice to that in wild-type mice. We found dysregulation of a number of genes.
The DE genes had a number of other commonalities. When the expression pat- tern of the genes was analyzed using localization data from the ABA, they were significantly more likely to be more highly expressed in the hippocampus than in the striatum, thalamus, hypothalamus or midbrain (p<0.01). If, as this suggests, the loss of densin has a larger effect on the hippocampus than on other brain regions, densin may be more important to the function of the hippocampus than to other brain regions. This implication is consistent with preliminary results of behavioral test suggesting a memory deficit in the densin knockout mice.
Three GABA receptor subunits are significantly decreased: α2 and β1 in the hippocampus and α5 in the forebrain. If this corresponds to a decrease in surface GABA receptors, densin knockout mice may have decreased inhibitory tone compared to wild-type mice.
A number of IEGs are upregulated in the hippocampus of the knockout mice: Arc, Egr1 (Zif268), Fos, Junb and Nptx2 (Narp). The overexpression in the hippocampus does not occur in the forebrain: The levels of Arc, Egr1 and Fos all decrease in the forebrain, although not to a significant extent. The increase in IEG expression and the decrease in GABAR subunit expression suggest that the hippocampi of densin knockout mice are in a persistent state of overstimulation.
Another pattern also suggests stimulation: Several genes coding for proteins in the heat shock family are also dysregulated. Heat shock proteins (HSPs) are molecular chaperones that respond to stress, reducing the harmful effects of misfolded proteins.
Many are induced in response to stressful conditions, but high expression of HSPs can be detrimental (Feder and Hofmann, 1999). Cryab, a member of the small HSP family, is among the most DE in both the forebrain and the hippocampus of the densin knockout, but it changes in opposite directions in the two regions. Like Arc, Egr1 and Fos, Cryab is upregulated in the hippocampus and downregulated in the forebrain. It is widely expressed, but expression is also heat-inducible. Another of the most DE genes is HSP 5 (Hspa5, also called BiP or GRP78). Like Cryab, it is downregulated in the knockout forebrain. Hspa5 is a glucose-regulated protein and a member of the HSP70 family. It is induced in response to heat and other stresses. Hspa5 is also called “immunoglobulin heavy chain-binding protein”; Igh-1b (immunoglobulin heavy chain 1b (serum IgG2c) is another DE gene in the knockout.
In the hippocampus of wild-type mice, Igh-1b is not expressed (0 RPKM) while in the knockout it is expressed at 4 RPKM. This may be part of an inflammatory
response, as NFKBIA (Iκb) is upregulated in both the forebrain and hippocampus of densin knockout mice. The analysis of interactions among differentially regulated genes places Iκb at the center of the resulting network. The NFκB pathway is a key regulator of transcription responses to extracellular stimuli such as cytokines and growth factors. Importantly, this pathway in neurons is also stimulated by NMDA, calcium influx, and seizures (Boersma and Meffert, 2008).
In the forebrain, Mt1 and Mt2 were both significantly upregulated in the densin knockout. Co-regulation of these genes has been previously observed: both are up- regulated after 3,4-methylenedioxymethamphetamine (MDMA) treatment (Xie et al., 2004). Moreover, intravitreous NMDA injection upregulates Mt2. Thus, the Mt2 up- regulation in the densin knockout could be the result of the hyperexcitability indicated by the increased CaMKII transcripts and tendency toward seizure (Suemori et al., 2006).
Amid this set of indicators of over-activated excitatory mechanisms are two ap- parently incompatible observations. Transcripts of three GABAAR subunits are de- creased, and densin knockout mice are likely to have seizures in response to adminis- tration of the GABA agonist pentobarbital. A decrease in GABA receptor expression is compatible with decreased inhibition and the general shift toward excitability sug- gested by the DE genes discussed above, but increased sensitivity to a GABAAR agonist does not predict decreased GABAAR subunit expression or enhanced excita- tion.
Although induction of seizure by pentobarbital is not commonly reported, several GABAA-regulating drugs are biphasic in effect. They either cause initial procon- vulsive effects and subsequent anticonvulsive effects, or the dosage response curve includes both proconvulsive and anticonvulsive ranges (Garant et al., 1995; Stuchl´ık, 2001). Seizures induced by the withdrawal of pentobarbital are extensively used as an experimental paradigm. It is not implausible that alterations in general excitatory tone could alter the effect of pentobarbital treatment in densin knockout mice.
On the other hand, the same caveats apply to interpreting the differential expres- sion in the GABAAR genes as other genes: the change in transcript level may not correspond to a change in protein level. Although there is a positive overall correla- tion, reports of correlation vary, by gene ontology category, with R2 values from 0.2 to 0.8 (Greenbaum et al., 2003; Kadota et al., 2003). Furthermore, proteins and mRNA have different degradation mechanisms and half-lives, both of which affect abundance.
Finally, the loss of even major regulatory proteins can have silent phenotypes (Marder and Goaillard, 2006; Piedras-Renter´ıa et al., 2004).
Densin may be involved in the regulation of dendritic structure. Shank transcrip- tion is upregulated in the hippocampus of the densin knockout mouse. Shank is a densin binding partner and core PSD scaffold that is involved in PSD organization.
Densin overexpression causes excessive dendritic branching, an effect rescued by co- expression of Shank (Quitsch et al., 2005). Shank also interacts withδ-catenin, which is also a binding partner of densin (Izawa et al., 2002). These results suggest a model in which densin regulates dendritic branching via δ-catenin in a Shank-dependent
manner (Quitsch et al., 2005). Studies of neuronal branching in the densin knockout mouse are ongoing.