Increased expression of Kalirin-9 in the auditory cortex of schizophrenia subjects: its role in dendritic pathology

Abstract

Reductions in dendritic arbor length and complexity are among the most consistently replicated changes in neuronal structure in post mortem studies of cerebral cortical samples from subjects with schizophrenia, however, the underlying molecular mechanisms have not been identified. This study is the first to identify an alteration in a regulatory protein which is known to promote both dendritic length and arborization in developing neurons, Kalirin-9. We found Kalirin-9 expression to be paradoxically increased in schizophrenia. We followed up this observation by overexpressing Kalirin-9 in mature primary neuronal cultures, causing reduced dendritic length and complexity. Kalirin-9 overexpression represents a potential mechanism for dendritic changes seen in schizophrenia.

Figure 1

A) An example blot from the comparison of subjects with schizophrenia (S) and Control (C) subjects. S1/C1 refers to Pair 2 in Supplemental Table 2. S2/C2 refers to Pair 4. B) Comparison of Kalirin-9 expression (normalized to tubulin expression) for the 26 pairs of subjects. Each point represents a pair of subjects. The diagonal reference line represents a control : schizophrenia ratio of one. Points above the line indicate increased expression in schizophrenia subjects. The increase in Kalirin-9 expression in schizophrenia subjects was significant (p=0.0038) C) Adjusted mean pairwise difference in Kalirin isoform expression (normalized to tubulin expression). Error bars represent adjusted standard errors. ** p=0.0038

Figure 2. The effect of age on Kalirin isoform expression (normalized to tubulin expression) in Schizophrenia and Control subjects

Increasing age was associated with significantly lower expression of all isoforms: Kalirin-5 (t₃₄ = −4.77, p < 0.001), Kalirin-7 (t₃₂ = −3.16, p = 0.003), Kalirin-9 (t_35.1 = −5.04, p < 0.001), and Kalirin-12 (t_35.2 = −6.75, p < 0.001). There were no significant interactions between diagnosis and age for any of the isoforms (all p > 0.1).

Figure 3

A) DIV26 cultured cortical neurons were immunostained for endogenous kalirin-9 and endogenous PSD-95. Left images show the dendritic tree of a representative neuron. The images to the right show a high magnification image of spines protruding from the dendritic shaft. The kalirin-9 and PSD-95 channels are shown separately, and together, to reveal colocalization. Kalirin-9 colocalized with PSD-95 in spines. B) DIV26 cultured cortical neurons overexpressing either GFP alone or in combination with 2-week kalirin-9 overexpression. Representative images of dendrite tracings and spine images are shown. C) Quantification of the effects of 2-week kalirin-9 overexpression on spine density and dendrite morphology. Kalirin-9 overexpression resulted in a significant increase in spine density (p<0.001) with no effect on spine area. Kalirin-9 overexpression reduced total dendritic length and reduced apical dendritic length (p<0.05). Basal dendrite length was not affected. Sholl analysis revealed a reduction in the complexity of the total dendritic tree with reduced complexity evident in both basal and apical dendrites. *p<0.05; **p<0.01; ***p<0.001

Figure 4. Example western blot of Kalirin isoforms in post-synaptic density (PSD) enriched fraction from human cortical gray matter from a 53-year-old female subject with a PMI of 13 hours

15 μg of protein from human samples and 10 μg of protein from mouse (Kal KO and WT) were loaded. Spinophilin and n-methyl-D-aspartate receptor 1 (NR1) are postsynaptic markers while synaptophysin indicates a presynaptic marker. H=total protein extracted from a cortical gray matter homogenate from the same human subject, P=presynaptic fraction, D=PSD, KO= total protein extracted from brain homogenate from a Kalirin knock-out mouse, WT= total protein extracted from brain homogenate from a wild-type mouse. A previously described(Penzes et al., 2000) non-specific band with an apparent MW of ~150 can be detected in the KO.