If you don't provide the details, that doesn't make it okay!

Paper: “The Schizophrenia Susceptibility Gene Dysbindin Regulates Dendritic Spine Dynamics" by Jie-Min Jia, Zhonghua Hu, Jacob Nordman, and Zheng Li

      In this paper, the authors investigate how a protein implicated in schizophrenia, dysbindin, regulates dendritic spine dynamics in hippocampal neurons. The authors decided to look at the connection between dysbindin and dendritic spines because there is a well documented spine pathology in schizophrenia. The authors also investigate the mechanism as to how dysbindin regulates dendritic spine dynamics by looking at Ca2+/calmodulin-dependent protein kinase II (CaMKIIa) and Abl interactor 1 (Abi-1) levels. To investigate these aims, the authors utilized time-lapse imaging of the hippocampal neurons, immunoblots, and immunoprecipitation.

Despite being published in the reputable Journal of Neuroscience, this paper fails to comply with a major responsibility in publishing scientific results: stating how they analyzed their data. Though the authors put their sample size and p values under each figure, they never explicitly state what statistical tests they utilized. There is no mention of the statistical tests in the methods section. Since Journal of Neuroscience does not have a supplementary section, the analysis information cannot be hiding there either. It is crucial that readers know what statistical tests the researchers performed in order to be assured that they performed the correct tests to back up their claims. In Figure 1, the researchers wanted to test the hypothesis that the protein dysbindin regulates dendritic spine dynamics. They observed the dynamics of dendritic protrusions in neurons from WT mice and mice lacking dysbindin (sdy mice) by transfecting with a fluorescent protein construct and imaging a dendritic branch every minute for 30 minutes. They then defined whether an event was a formation, retraction, mushroom to filopodia conversion, filpodia to mushroom conversion, mushroom to stubby spine conversion, or stubby spine to mushroom conversion. Figures 1B,D,F demonstrate this categorization and assert that the dynamics in sdy mice significantly differed from WT mice. However, it is unclear if the authors correctly did a two-way ANOVA to find this significance or if they incorrectly analyzed the data with two separate t-tests.

Figures1,B,D,F

 A similar statistical question is presented with Figure 2B, where the authors quantify protrusion number per µm of mushroom, stubby, and filopodia spines for both WT and Sdy mice. Once again, this data should have been analyzed as a two-way ANOVA but appears as though it may have incorrectly analyzed with separate T-tests. Even in figures that are obviously two-way ANOVAs, such as Figure 2G and Figure 3B,D,F,H, the authors do not go into any detail regarding their multiple comparisons statistics.

Figure 2B

            Aside from the omission of specifics regarding statistical analysis, an aspect of experimental analysis was incorrect in Figure 7. In this figure, the authors sought to test the hypothesis that CaMKIIa activity is lowered in the sdy neurons due to inhibition by activity-dependent modulation of Abi interactor 1 (Abi1). The authors used western blots to determine the amount of Abi1, CaMKIIa, and dysbindin (as a control) present in WT versus sdy mice. Additionally, they did an immunoprecipitation for Abi from the P2 fractions of both genotypes. The authors present the quantification of these western blots as “normalized fold change”. However, the authors fail to define what they are normalized to. It is convention to normalize to actin levels (which are present on the western blots for whole cell lysate and P2 fraction), but the authors do not explicitly say they normalized their Abi1 and CaMKIIa levels to actin. What appears to be the biggest flaw in these sets of experiments is how they normalized their immunoprecipitation data. Typically, immunoprecipitation data is normalized to “input”, meaning that 10% of protein lysate that was used for the IP is usually run on western blot and used to normalize. However, it appears that the data for the immunoprecipitation in sdy mice is just normalized to what was immunoprecipitated in WT mice. This does not control for if there was initially more Abi1 or CaMKIIa in the input for sdy mice. If there is more protein inputted into an immunoprecipitation, then more will immunoprecipitated. Thus, it could be possible that more of the sdy sample was used for the immunoprecipitation than WT sample, which would only make it appear that CaMKIIa was increased. A loading control is NECESSARY to make any conclusion from this immunoprecipitation experiment.

Figure 7

            Though this was an interesting paper in demonstrating how dysbindin regulates spine dynamics, the authors failed to provide crucial information regarding their statistics as well as normalization procedures. Without this information, it is difficult to have full faith in their statistics and subsequent conclusions.