Zoology/Biology session

Schizophrenia (SCH) is a severe neuropsychiatric disorder with poorly understood etiology. Based on our previous findings, the density of calretinin (CR) interneurons in the dorsolateral prefrontal cortex is lower in SCH. However, single nucleus mRNA sequencing suggests that the expression level of the gene encoding CR (CALB2) is not significantly lower (Batiuk et al., 2022).

One possible explanation may be that in schizophrenia, CR mRNA is transcribed, but the translation process is disrupted in a subset of cells. To test this, we designed an in situ mRNA hybridization experiment (RNAScope technique) in which we could simultaneously label the CR mRNA and protein. Our hypothesis was that in the SCH group, we would find more cells in which CR mRNA is present, but the protein is either not expressed or expressed at very low levels.

One part of my work included reanalysing the data from those samples for which both protein and single-cell sequencing mRNA level data were already available (N=5 SCH, 5 CTR), to compare the density of CR-immunoreactive cells and the level of CALB2 gene expression. Furthermore, we performed the RNAscope experiment on tissue samples from the same cases. The sections were digitized using a fluorescent scanner and manually annotated, during which the cells were classified into five main categories: 1) high protein – high mRNA 2) high protein – low mRNA 3) low protein – high mRNA 4) low protein – low mRNA and 5) no protein – high mRNA.

From the single-cell mRNA sequencing data, I subsetted the CALB2-expressing interneuron clusters following a multi-step cluster analysis, and calculated the average CALB2 gene expression value for each case. Based on this, we concluded that the density of immunoreactive CR cells and the average nucleus CALB2 mRNA levels are often not inferable from one another. In case of the RNAScope experiment, the largest differences were observed between the first category (high protein – high mRNA) and the fifth category (no protein – high mRNA). The proportion of cells in the first category was generally lower in the SCH group, while the proportion of cells containing only mRNA was higher. A notable portion of the latter group of cells was concentrated in the first cortical layer. We did not detect any significant differences in the other categories.

The experiment generally supported our hypothesis; however, the difference did not arise from the density of low protein and high RNA content cells, but rather from the presence of cells in layer 1 of the SCH group that expressed only mRNA. Further investigations are needed to interpret this finding. As CR is an important calcium-binding protein that plays a crucial role in neuronal signalling, elucidating its potential role in SCH may help us to understand its elusive etiology.