Rett Syndrome’s Genetic Secrets Unveiled by Growing Brains in a Dish

While numerous investigations have traditionally examined Rett syndrome, a developmental disorder, as a singular entity stemming from generalized loss of function in the MECP2 gene, recent research conducted by neuroscientists at The Picower Institute for Learning and Memory at MIT reveals that two distinct alterations within this gene precipitate markedly different abnormalities in laboratory-generated cultures. Furthermore, rectifying the specific deviations caused by each mutation necessitated unique therapeutic interventions.

“The specific nature of each mutation carries significant weight,” stated Mriganka Sur, the senior author of the aforementioned study published in Nature Communications and a Newton Professor at The Picower Institute and the Department of Brain and Cognitive Sciences. “This represents a pathway toward individualized therapies, even for a disorder caused by a single gene.”

For this investigation, advanced three-dimensional human brain tissue cultures, referred to as “organoids” or “minibrains,” were utilized. These were derived from skin or blood cells provided by individuals diagnosed with Rett syndrome, with each sample harboring a specific genetic mutation. Tatsuya Osaki, the lead author and a research scientist at The Picower Institute, explained that the organoids’ capacity to model the precise ramifications of individual mutations permitted him to acquire mutation-specific insights that had not been elucidated in previous studies where researchers generally inactivated MECP2 without differentiation. These organoids also provided an unprecedented avenue for comprehending how each mutation impacted diverse cell types and their interrelationships.

Divergent Manifestations

Although over 800 mutations in MECP2 can precipitate Rett syndrome, a mere eight account for more than 60 percent of prevalent cases. Sur and Osaki elected to investigate one of these prevalent mutations, R306C, characterized by a single DNA base pair alteration (916C>T), which is implicated in approximately 7-8 percent of Rett syndrome diagnoses. The second mutation examined, V247X, is significantly rarer and more severe, as it truncates the production of the gene’s protein product due to a single DNA base deletion (705Gdel), resulting not only in an aberrant protein but also an incomplete one.

Following three months of cultivation, the organoids exhibited certain shared yet also distinct consequences compared to control organoids containing the non-mutated MECP2 gene. For a substantial portion of their experimental protocols, the research team employed “three-photon” microscopy. This technology provides cellular-level resolution throughout the organoids’ approximate 1mm thickness, enabling visualization of their structural integrity via “third-harmonic generation” imaging, as well as the dynamic activity patterns of their neurons through calcium fluorescence.

For illustration, the researchers observed that the V247X organoids displayed several structural disparities when contrasted with their control counterparts; they were larger and exhibited variations in the thickness of different layers. Conversely, the R306C organoids presented structural characteristics far more akin to the controls. Organoids bearing either mutation demonstrated less developed axon projections from their neurons in comparison to their control subjects.

Upon scrutinizing the properties of neural activity and connectivity within the organoids, the scientists identified some analogous deficiencies across both mutation types. Both groups exhibited diminished neuronal firing rates and a reduction in synchronicity between neurons when compared to controls.

However, when the investigators examined other characteristics, the organoids began to exhibit divergence. Notably, an indicator of network structure efficiency, termed “small-world propensity” (SWP), was found to be reduced in R306C organoids and elevated in V247X organoids relative to controls. This observation signifies that both mutations perturbed the typical development of network architectures essential for information processing, albeit in opposing directions.

To validate the relevance of these findings for individuals with Rett syndrome, the team collaborated with Charles Nelson at Boston Children’s Hospital. His group assessed electroencephalogram (EEG) readings from several children afflicted with different Rett mutations. Although the patient cohort was small, the researchers detected indicators suggesting that the SWP characteristic in the EEG recordings was altered in the study participants, mirroring the changes observed in the organoids.

Finally, by selectively labeling excitatory neurons to emit light in one color and inhibitory neurons in another, the scientists were able to ascertain that the interconnections between these distinct neuronal types differed significantly from controls within the V247X organoids.

Therapeutic Exploration

All experimental results consistently demonstrated that each mutation induced multiple alterations in organoid structure, activity, and connectivity, and that these deviations were frequently specific to the particular mutation.

To elucidate the origins of these differences and devise potential corrective strategies, Sur and Osaki’s team proceeded to analyze differential gene expression patterns in the cells of each organoid type compared to controls. Discrepancies in gene expression frequently lead to modifications in critical cellular molecular pathways, which can consequently disrupt cellular activity and function. Analysis employing single-cell RNA sequencing indeed revealed hundreds of expression differences in each organoid type, with some genes exhibiting increased expression relative to controls while others showed reduced expression.

For instance, the analyses indicated that in R306C organoids, a gene known as HDAC2 was overexpressed. This protein is recognized for its role in suppressing the expression of other genes. Concurrently, in the V247X organoids, the scientists detected diminished expression of genes associated with certain receptors for the inhibitory neurotransmitter GABA. These organoids also exhibited functional impairments in astrocyte cells, which are crucial for supporting numerous aspects of neural function.

Organoids harboring either mutation also displayed anomalies in the molecular pathways responsible for the development of circuit connections between neurons, known as synapses.

Based on the specific defects identified, the researchers administered treatments to the organoids using a pharmaceutical agent capable of inhibiting HDAC2 activity and another designed to enhance GABA’s effectiveness. The HDAC2 inhibitor successfully restored neuronal activity and SWP to normal levels in the R306C organoids, while the GABA “agonist” baclofen normalized SWP to control levels in the V247X organoids.

Tatsuya highlighted that the therapeutic drugs used have already undergone investigation for other disease indications, signifying that they are well-characterized agents potentially amenable to repurposing.

Having now established an organoid platform for dissecting the consequences of individual mutations, identifying their underlying causes, and evaluating potential treatments, the researchers intend to apply this methodology to the study of four additional mutations, Sur reported, comparing each against a standardized control organoid.

In addition to Sur, Osaki, and Nelson, the study’s co-authors include Chloe Delepine, Yuma Osako, Devorah Kranz, April Levin, and Michela Fagiolini.

Financial contributions for this research were provided by the National Institutes of Health, a MURI grant, The Freedom Together Foundation, and the Simons Foundation.