The human silencing hub (HUSH) complex may be involved in complex disorders affecting the brain and neurons, but to date its mechanism of action hasn’t been understood. A study by researchers at the Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA) has now uncovered the in vivo targets and physiological functions of a component of the HUSH gene silencing complex and one of its associated proteins. The work, led by Astrid Hagelkruys, PhD, senior research associate in the Penninger group at IMBA, and conducted in laboratory mouse models and human brain organoids, links the HUSH complex to normal brain development, neuronal individuality, and connectivity, as well as mouse behavior.

Reporting on their findings in Science Advances in a paper titled. “The HUSH complex controls brain architecture and protocadherin fidelity,” the team concluded: “Our data uncover a novel role for the HUSH complex in the regulation of clustered protocadherins within the nervous system, thereby controlling brain development and neuronal fidelity in mice and humans.”

The HUSH complex was recently identified to be of key importance for silencing repetitive genetic elements including transposons in mammals. It contains MPP8, a protein that binds the histone modification mark H3K9me3. Additionally, HUSH is known to recruit other proteins including the zinc finger protein MORC2. “This recently discovered repressor complex, which contains M-phase phosphoprotein 8 (MPP8) and recruits the histone methyltransferase SETDB1 and Microrchidia CW-type zinc finger protein 2 (MORC2), has been implicated in silencing of genes, repetitive elements, and transgenes in mammals,” the authors wrote.

In humans, mutations affecting MORC2 are associated with axonal neuropathy, a type of nerve damage, as well as with neurodevelopmental disorders. However, little is known about the physiological functions of MPP8 and MORC2 or how they might affect brain health. “Functional and mechanistic studies of the HUSH complex have hitherto been centered around SETDB1 while the in vivo functions of MPP8 and MORC2 remain elusive,” the team continued.

Hagelkruys and colleagues set out to investigate the targets and functions of these two proteins, in vivo, in laboratory mouse models, and in human brain organoids. The investigators’ comprehensive in vivo approach included behavioral, motor, developmental, genetic, and transcriptomic experiments. Their results found that MPP8 and MORC2A (the mouse ortholog of human MORC2) were highly expressed in the brain, where they are exclusively found in neurons. We demonstrated that MPP8 and MORC2A play a role in normal brain development, the specification of neuronal identity and connectivity of neurons, as well as mouse behavior,” said project lead and co-corresponding author Hagelkruys.

Furthermore, deleting MPP8 or MORC2A in the nervous system of the mouse models increased brain size and altered brain architecture without major changes in transposable element expression. The results, they noted, “show that neuronal loss of murine MPP8 or MORC2A results in a morphological and neuronal expansion of defined brain areas.”

These deletions affected the mice’s motoric functions and behavior. As the authors concluded, the increased midbrain sizes and accompanying brain architectural changes in the mutant mice “… are associated with defective motor functions and spatial learning, yet improved fear-context memory.” Hagelkruys further stated, Hence, surprisingly in a living animal, we showed that MPP8 and MORC2A act beyond transposable element regulation.

So while the HUSH complex had been found to be involved in transposon regulation, Hagelkruys continued, “we showed that MPP8 and MORC2A suppressed the protocadherin gene clusters in an H3K9me3-dependent manner. At the protein level, these protocadherin gene clusters form neuronal surface proteins that mediate contact with other neurons. Although protocadherins are not transposable elements, some are expressed in the central nervous system as ‘repetitive-like’ gene clusters.”

In the mouse models, MPP8 and MORC2A specifically silenced the protocadherin cluster on mouse chromosome 18. “Mechanistically, Mphosph8 and Morc2a are exclusively expressed in neurons, where they repress the protocadherin cluster on mouse chromosome 18 in an H3K9me3-dependent manner, thereby affecting synapse formation,” the scientists commented. Deleting MPP8 and MORC2A led to more synapses forming in the neurons, which might coincide with impairment of neuronal individuality—in other words, the ability of neurons to distinguish “self” from “non-self.”

“It requires further elucidation how this is associated with the impaired motor functions, spatial learning deficits, and the improved fear-context memory observed in Mphosph8- or Morc2a-deficient mice,” Hagelkruys and colleagues noted.

By expressing different combinations of clustered protocadherins, neurons acquire a form of “barcode” that allows them to control the formation of synaptic connections with other neurons. “The combinatorial expression of clustered protocadherins in individual neurons generates barcodes for neuronal identity as well as synapse formation and thereby provides the molecular basis for neuronal diversity, neuronal network complexity, and function of the vertebrate brain,” the scientists explained. Hence, by targeting clustered protocadherins, MPP8 and MORC2A may ensure that neurons acquire the right barcode and form synapses only with the correct counterparts.

In addition, the team examined the effects of MPP8 and MORC2 deficiency in 3D human brain organoids. Using this stem cell-derived brain model, the scientists observed concordant results: the absence of MPP8 or MORC2 led to increased numbers of clustered protocadherins expressed in organoid neurons at the single-cell level. This indicated that the absence of the two proteins disrupted neuronal identity in the human brain organoids.

Through their reported work, the researchers uncovered a pivotal role of the HUSH complex in the epigenetic regulation of protocadherin expression in the nervous system. These findings link the mechanistic effect of suppressing repetitive-like genetic elements with mouse brain physiology and behavior. “In this study, we identify murine M-phase phosphoprotein 8 (MPP8) and Microrchidia CW-type zinc finger protein 2 (MORC2A) as crucial regulators of brain development and function,” they wrote.

The team’s brain organoid results also confirmed that similar effects may be found in humans. In their paper, the team concluded, “Our data identify MPP8 and MORC2, previously linked to silencing of repetitive elements via the HUSH complex, as key epigenetic regulators of protocadherin expression in the nervous system and thereby brain development and neuronal individuality in mice and humans.”

Josef Penninger, PhD, group leader at IMBA, added: “The interest of these findings on the essential function of the HUSH complex in the brain lies in the implication of protocadherins in neuronal fidelity and brain evolution. However, how this is regulated remained largely unknown. The dysregulation of clustered protocadherins has been associated with various neurological and neurodevelopmental diseases, but also multiple mental disorders in humans. Hence, our findings might help us better understand the epigenetic regulation mechanisms governing these diseases and provide a new way to study brain evolution.”

The authors concluded, “Since dysregulation of clustered protocadherins is associated with a variety of neurological and neurodevelopmental diseases as well as mental disorders including autism spectrum disorder, bipolar disorder, Alzheimer’s disease, cognitive impairments, and schizophrenia, our data on the key importance of the HUSH complex in protocadherin gene expression might provide new understanding on the epigenetic regulation of such diseases.”

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