Cross-Platform Synaptic Network Analysis of Human Entorhinal Cortex Identifies TWF2 as a Modulator of Dendritic Spine Length.

Proteomic studies using postmortem human brain tissue samples have yielded robust assessments of the aging and neurodegenerative disease(s) proteomes. While these analyses provide lists of molecular alterations in human conditions, like Alzheimer's disease (AD), identifying individual proteins that affect biological processes remains a challenge. To complicate matters, protein targets may be highly understudied and have limited information on their function. To address these hurdles, we sought to establish a blueprint to aid selection and functional validation of targets from proteomic datasets. A cross-platform pipeline was engineered to focus on synaptic processes in the entorhinal cortex (EC) of human patients, including controls, preclinical AD, and AD cases. Label-free quantification mass spectrometry (MS) data (n = 2260 proteins) was generated on synaptosome fractionated tissue from Brodmann area 28 (BA28; n = 58 samples). In parallel, dendritic spine density and morphology was measured in the same individuals. Weighted gene co-expression network analysis was used to construct a network of protein co-expression modules that were correlated with dendritic spine metrics. Module-trait correlations were used to guide unbiased selection of Twinfilin-2 (TWF2), which was the top hub protein of a module that positively correlated with thin spine length. Using CRISPR-dCas9 activation strategies, we demonstrated that boosting endogenous TWF2 protein levels in primary hippocampal neurons increased thin spine length, thus providing experimental validation for the human network analysis. Collectively, this study describes alterations in dendritic spine density and morphology as well as synaptic proteins and phosphorylated tau from the entorhinal cortex of preclinical and advanced stage AD patients.SIGNIFICANCE STATEMENT Proteomic studies can yield vast lists of molecules that are altered under various experimental or disease conditions. Here, we provide a blueprint to facilitate mechanistic validation of protein targets from human brain proteomic datasets. We conducted a proteomic analysis of human entorhinal cortex (EC) samples spanning cognitively normal and Alzheimer's disease (AD) cases with a comparison of dendritic spine morphology in the same samples. Network integration of proteomics with dendritic spine measurements allowed for unbiased discovery of Twinfilin-2 (TWF2) as a regulator of dendritic spine length. A proof-of-concept experiment in cultured neurons demonstrated that altering Twinfilin-2 protein level induced corresponding changes in dendritic spine length, thus providing experimental validation for the computational framework.

Dendritic spine head diameter is reduced in the prefrontal cortex of progranulin haploinsufficient mice.

Loss-of-function mutations in the progranulin (GRN) gene are an autosomal dominant cause of Frontotemporal Dementia (FTD). These mutations typically result in haploinsufficiency of the progranulin protein. Grn+/- mice provide a model for progranulin haploinsufficiency and develop FTD-like behavioral abnormalities by 9-10 months of age. In previous work, we demonstrated that Grn+/- mice develop a low dominance phenotype in the tube test that is associated with reduced dendritic arborization of layer II/III pyramidal neurons in the prelimbic region of the medial prefrontal cortex (mPFC), a region key for social dominance behavior in the tube test assay. In this study, we investigated whether progranulin haploinsufficiency induced changes in dendritic spine density and morphology. Individual layer II/III pyramidal neurons in the prelimbic mPFC of 9-10 month old wild-type or Grn+/- mice were targeted for iontophoretic microinjection of fluorescent dye, followed by high-resolution confocal microscopy and 3D reconstruction for morphometry analysis. Dendritic spine density in Grn+/- mice was comparable to wild-type littermates, but the apical dendrites in Grn+/- mice had a shift in the proportion of spine types, with fewer stubby spines and more thin spines. Additionally, apical dendrites of Grn+/- mice had longer spines and smaller thin spine head diameter in comparison to wild-type littermates. These changes in spine morphology may contribute to altered circuit-level activity and social dominance deficits in Grn+/- mice.

Comparison of Golgi-Cox and Intracellular Loading of Lucifer Yellow for Dendritic Spine Density and Morphology Analysis in the Mouse Brain.

Dendritic spines are small protrusions on dendrites that serve as the postsynaptic site of the majority of excitatory synapses. These structures are important for normal synaptic transmission, and alterations in their density and morphology have been documented in various disease states. Over 130 years ago, Ramón y Cajal used Golgi-stained tissue sections to study dendritic morphology. Despite the array of technological advances, including iontophoretic microinjection of Lucifer yellow (LY) fluorescent dye, Golgi staining continues to be one of the most popular approaches to visualize dendritic spines. Here, we compared dendritic spine density and morphology among pyramidal neurons in layers 2/3 of the mouse medial prefrontal cortex (mPFC) and pyramidal neurons in hippocampal CA1 using three-dimensional digital reconstructions of (1) brightfield microscopy z-stacks of Golgi-impregnated dendrites and (2) confocal microscopy z-stacks of LY-filled dendrites. Analysis of spine density revealed that the LY microinjection approach enabled detection of approximately three times as many spines as the Golgi staining approach in both brain regions. Spine volume measurements were larger using Golgi staining compared to LY microinjection in both mPFC and CA1. Spine length was mostly comparable between techniques in both regions. In the mPFC, head diameter was similar for Golgi staining and LY microinjection. However, in CA1, head diameter was approximately 50% smaller on LY-filled dendrites compared to Golgi staining. These results indicate that Golgi staining and LY microinjection yield different spine density and morphology measurements, with Golgi staining failing to detect dendritic spines and overestimating spine size.

Conditional deletion of ROCK2 induces anxiety-like behaviors and alters dendritic spine density and morphology on CA1 pyramidal neurons.

Rho-associated kinase isoform 2 (ROCK2) is an attractive drug target for several neurologic disorders. A critical barrier to ROCK2-based research and therapeutics is the lack of a mouse model that enables investigation of ROCK2 with spatial and temporal control of gene expression. To overcome this, we generated ROCK2fl/fl mice. Mice expressing Cre recombinase in forebrain excitatory neurons (CaMKII-Cre) were crossed with ROCK2fl/fl mice (Cre/ROCK2fl/fl), and the contribution of ROCK2 in behavior as well as dendritic spine morphology in the hippocampus, medial prefrontal cortex (mPFC), and basolateral amygdala (BLA) was examined. Cre/ROCK2fl/fl mice spent reduced time in the open arms of the elevated plus maze and increased time in the dark of the light-dark box test compared to littermate controls. These results indicated that Cre/ROCK2fl/fl mice exhibited anxiety-like behaviors. To examine dendritic spine morphology, individual pyramidal neurons in CA1 hippocampus, mPFC, and the BLA were targeted for iontophoretic microinjection of fluorescent dye, followed by high-resolution confocal microscopy and neuronal 3D reconstructions for morphometry analysis. In dorsal CA1, Cre/ROCK2fl/fl mice displayed significantly increased thin spine density on basal dendrites and reduced mean spine head volume across all spine types on apical dendrites. In ventral CA1, Cre/ROCK2fl/fl mice exhibited significantly increased spine length on apical dendrites. Spine density and morphology were comparable in the mPFC and BLA between both genotypes. These findings suggest that neuronal ROCK2 mediates spine density and morphology in a compartmentalized manner among CA1 pyramidal cells, and that in the absence of ROCK2 these mechanisms may contribute to anxiety-like behaviors.