From Proteins to Dendritic Spines: Neurolucida 360 Plays a Crucial Role in Advancing Neuroscience

In the fast-evolving field of neuroscience, groundbreaking research on the intricate workings of the vertebrate brain yields new information every day. A recent study published in the Journal of Neuroscience describes the establishment of an approach for better contextualization of proteins identified through proteomic analyses to identify candidate proteins for functional validation testing. The authors examined human synaptic processes from well-characterized human post-mortem samples and showed that integration of proteomics with dendritic spine metrics could guide unbiased identification of a target protein, Twinfilin2 (TWF2), that was shown to be functionally involved in regulating dendritic spines.

The authors obtained post-mortem human brain samples from the Brodmann area 28 (BA28) entorhinal cortex (EC) of subjects exhibiting a range of Alzheimer’s disease (AD) pathology and categorized into 3 groups based on cognition and AD pathology: normal cognition, noAD pathology; normal cognition with moderate to severe AD pathology, and definite AD cases. Synaptosome fractions were characterized biochemically, and proteomic profiles were determined using liquid chromatography coupled to mass spectrometry. Weighted gene co-expression network analysis was used to generate a protein co-expression network and identify protein modules (co-expressed proteins) that were present in the different cognition/AD pathology categories.

In parallel, tissue samples from the same brain area were fixed and processed for dendrite imaging using Golgi-Cox staining. Dendritic segments of pyramidal neurons from layers 2 and 3 of BA28 from each category of cognition/AD pathology were imaged with a 60X/1.40 NA oil-immersion objective using a brightfield microscope. The resulting 3D image stacks were opened in Neurolucida 360 and dendrite and dendritic spine morphologies were reconstructed using semi-automatic and automatic functions. Spines were automatically classified as stubby, mushroom, or filopodia. Volumetric measurements of the spine, as well as the density of each spine type per dendrite length were extracted with Neurolucida Explorer.

Figure: Overview of workflow. Synaptosomes were isolated from postmortem human BA28 entorhinal cortex (EC) and subjected to liquid chromatography tandem mass spectrometry-based proteomics. Weighted Gene Co-Expression Network Analysis (WGCNA) was used to generate a network of protein co-expression modules. BA28 EC samples were also Golgi stained and z-stacks of dendritic segments were imaged and digitally reconstructed to obtain measurements of dendritic spine density and morphology. Module eigenprotein values were correlated with dendritic spine metrics. The hub protein of a module significantly correlated with a dendritic spine metric would be selected for functional validation by CRISPR activation in rat primary hippocampal neurons.

The authors then correlated dendritic spine measurements with module eigenprotein expression from the proteomic analysis to integrate the two data categories. Among the results, one particular protein module stood out; it was consistently present in both AD and non-AD tissue, and was positively correlated with thin dendritic spine length, especially thin spines. Twinfilin2, the hub protein within this module, has a well-established role in modulating the cytoskeleton, specifically the protein actin. When the authors looked at neurons from rats grown in culture with different amounts of TWF2, they found those with more TWF2 grew longer thin-spines. This was the only type of spine affected by TWF2, demonstrating what the authors call a remarkable specificity regarding the ability of their cross-platform analysis to identify the functions of proteins.

Looking ahead, the researchers have identified many proteins organized in modules with hub-proteins, some that are expressed equally in AD and non-AD cases, and some that are not. They are in a good position to determine which of these hub proteins merit further study using functional analyses.

The comprehensive workflow employed by the researchers opens up new possibilities for unraveling the mysteries of neuronal function and holds immense potential for advancing our knowledge of diverse neurological conditions. As we delve deeper into the complex world of neuroscience, the connection between technology and scientific inquiry continues to illuminate the path towards groundbreaking discoveries.

Reference:

Walker, C. K., Greathouse, K. M., Tuscher, J. J., Dammer, E. B., Weber, A. J., Liu, E., Curtis, K. A., Boros, B. D., Freeman, C. D., Seo, J. V., Ramdas, R., Hurst, C., Duong, D. M., Gearing, M., Murchison, C. F., Day, J. J., Seyfried, N. T., & Herskowitz, J. H. (2023). Cross-platform synaptic network analysis of human entorhinal cortex identifies TWF2 as a modulator of dendritic spine length. The Journal of Neuroscience. https://doi.org/10.1523/jneurosci.2102-22.2023