The Problem of Averaging
Traditional "bulk" sequencing methods take a piece of brain tissue, grind it up, and average the gene expression across millions of cells. But the brain is not a smoothie; it's a fruit salad. A SHANK3 mutation might devasate a specific subtype of striatal projection neuron while leaving a neighboring astrocyte completely untouched. To understand the true pathology of ASD, we need cellular resolution.
Single-Cell RNA Sequencing (scRNA-seq)
Recent studies utilizing scRNA-seq on human brain organoids and Shank3 mutant mice have revealed a striking heterogeneity. The dysregulation is not uniform. In the striatum, the "D2-type" Medium Spiny Neurons (MSNs) appear to be disproportionately affected compared to "D1-type" MSNs. These D2-MSNs are crucial for the "indirect pathway" of movement control, which suppresses unwanted actions. Their selective vulnerability explains the repetitive behaviors and motor stereotypies seen in SHANK3-deficient models.
Proteomics: Beyond the Transcriptome
Gene expression (mRNA) is only half the story; proteins are the functional workhorses. Using cell-type-specific proximity biotin-labeling (a technique where a specific enzyme tags nearby proteins with biotin), researchers have mapped the "proteome" of the postsynaptic density in vivo. This revealed that SHANK3 loss doesn't just remove SHANK3; it destabilizes an entire complex of interacting proteins, including Homer1, PSD-95, and specific glutamate receptor subunits. This "interactome collapse" is the true molecular lesion.
Organoids as a Window into Development
Human induced pluripotent stem cells (iPSCs) derived from PMS patients can be grown into 3D brain organoids ("mini-brains"). Single-cell analysis of these organoids has shown that SHANK3 deficiency disrupts neurogenesis itself, altering the trajectory of how neurons mature and migrate. This suggests that the roots of ASD lie deep in early fetal development, further emphasizing the need for early intervention.
Excerpt from: Harnessing Single-Cell Omics, CRISPR, MSCs, miRNAs, and Valproic Acid Targeting SHANK3 Mutations and Associated Pathways by Peter De Ceuster
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