MIT Finds a Single microRNA Connecting Rett Syndrome Mutations to Leaky Brain Blood Vessels

Rett syndrome affects mostly girls, typically appearing between ages 2 and 3 as a devastating regression of previously acquired skills - language, motor function, social behavior. Its genetic origin is known: mutations in the MECP2 gene. But how those mutations produce the specific neurological damage seen in Rett has remained incompletely understood. A study published in Molecular Psychiatry from MIT's Picower Institute for Learning and Memory traces one pathway from gene mutation to brain pathology through a molecular chain linking microRNAs to blood vessel integrity.

The researchers did not work with animal models or indirect biomarkers. They built human blood vessels in the lab.

Engineering Vessels From Patient Cells

Lead author Tatsuya Osaki, a Research Scientist in the Picower Institute, used induced pluripotent stem cells (iPS cells) donated by patients with Rett syndrome to construct three-dimensional microvascular networks. The donated cells were reprogrammed into stem cells and then directed to become endothelial cells - the structural backbone of blood vessels. Embedded in a gel with fibroblast cells and connected to microfluidics to simulate circulation, these cells self-assembled into tube-like networks that functioned as small blood vessels.

Two separate sets of cultures were created, each carrying a different common Rett-causing mutation - R306C in one set and R168X in the other. For each, Osaki used CRISPR to create a control culture genetically identical except for the absence of the mutation. That design allowed direct comparison of mutant and normal vessels in human tissue, in the same culture system, with the mutation as the only variable.

What Both Mutations Did to Blood Vessels

Both mutations produced the same problem, despite affecting the MECP2 gene in different ways. The vessels harboring either mutation showed reduced expression and mislocalization of a protein called ZO-1, which is essential for maintaining tight junctions between endothelial cells - the molecular equivalent of the grout holding tiles together in a floor. Weakened ZO-1 function means the junctions between vessel cells become porous. Leak tests confirmed that the Rett-mutation vessels allowed more material through than the controls.

The team then created a third culture type adding astrocyte cells to more closely simulate the blood-brain barrier (BBB), the highly selective membrane separating circulating blood from brain tissue. That more complex culture showed similar deficiencies. When researchers exposed neurons to media from Rett vasculature cultures, those neurons showed reduced electrical activity - suggesting that secretions from the compromised endothelial cells disrupted neural function downstream.

"There is something common across these mutations," said senior author Mriganka Sur, Newton Professor of Neuroscience in the Picower Institute.

Tracing the Mechanism to a Single microRNA

MECP2's normal function is to repress gene expression. Mutations that disable MECP2 would be expected to produce overexpression of many downstream genes. But ZO-1 was not overexpressed - it was reduced. That paradox required an intermediate: some molecule that MECP2 ordinarily represses, which in turn suppresses ZO-1 when unrestrained.

The team suspected microRNAs, short RNA molecules that regulate gene expression post-transcriptionally. Profiling miRNAs in Rett and control cultures, they found that miRNA-126-3p was overexpressed in both Rett mutation conditions. RNA sequencing identified the broader molecular pathways for vascular integrity that were dysregulated in the same cultures.

The chain: MECP2 mutation - loss of MECP2 repressor function - overexpression of miRNA-126-3p - suppression of ZO-1 and related vascular integrity genes - leaky blood vessels.

Blocking the microRNA Helped

The team treated Rett-mutation cultures with an antisense molecule - a sequence designed to bind and reduce miRNA-126-3p levels. The intervention increased ZO-1 expression and partially restored endothelial barrier function, reducing leakiness. The molecular pathways tracked through sequencing also shifted toward more normal states.

Osaki and Sur note that a drug called miRisten, which inhibits miR-126, is currently in clinical testing for leukemia. They plan to administer it to mice modeling Rett syndrome to test whether the vascular rescue seen in cell culture translates to an animal model. That would be a necessary step before any human application could be considered - the current work is entirely in vitro, using engineered tissue rather than intact organisms.

The study was supported by the National Institutes of Health, a MURI grant, The Freedom Together Foundation, and the Simons Center for the Social Brain. Co-authors include Zhengpeng Wan, Koji Haratani, Ylliah Jin, Marco Campisi, David Barbie, and MIT Mechanical Engineering Professor Roger D. Kamm.