Researchers at Kumamoto University have identified mutations in the proteoglycan Tsukushi as a cause of hydrocephalus.

The study, which was published in the March 31, 2021, issue of Science Translational Medicine, adds to the evidence that a significant proportion of hydrocephalus are due to genetic alterations.

Classically, hydrocephalus, which is characterized by enlargements of the brain's lateral ventricles, is considered an imbalance in the production and resorption of cerebrospinal fluid (CSF) – a plumbing problem of sorts. In these cases, intracranial pressure is a major symptom of hydrocephalus.

Some cases of hydrocephalus, especially those acquired later in life, can be explained in this way, and can be successfully treated through the surgical installation of shunts to relieve intracranial pressure buildup caused by too much CSF.

But in other cases, the lateral ventricles, which are two main ventricles of the CSF system, can enlarge without an increase in CSF and intracranial pressure.

Although increased intracranial pressure, when it occurs, is the most urgent symptom of hydrocephalus, the disorder also has a behavioral component. Hydrocephalus sufferers can have behavioral symptoms that include impaired memory, increased anxiety and impaired social behavior. Hydrocephalus also has an increased chance of co-occurring with other neurodevelopmental disorders including autism spectrum disorders (ASD) and attention deficit hyperactivity disorder (ADHD).

Such cases of hydrocephalus are linked to abnormalities in the subventricular zone (SVZ), a part of the brain that borders the lateral ventricle and is a stem cell niche, where new neurons are produced after birth and into adulthood. However, the molecular mechanisms linking trouble in the SVZ to expanded lateral ventricles remain largely unknown.

Senior author Kunimasa Ohta and his team suspected that Tsukushi might be part of that molecular link because it controls signaling upstream of the Wnt pathways, which play key roles in neurodevelopment. Tsukushi is a proteoglycan, that is, a protein that has had multiple carbohydrates added to it in post-translational processing.

The team first generated Tsukushi knockout mice, and showed that such animals developed communicating hydrocephalus, meaning that CSF can flow between ventricles, but is blocked from exiting.

Follow-up experiments revealed that Tsukushi's absence was a problem specifically in ependymal cells, a type of glial cell that lines the cerebral ventricles. Animals lacking Tsukushi specifically in this cell type showed the same symptoms as overall knockouts. The team showed that in such animals, Tsukushi affected Wnt signaling, leading neural progenitors in the SVZ to behave differently than their wild-type cousins in the first few days of life. Right after birth, the stem cells of knockouts were proliferating more heavily, producing an excessive number of daughter cells. But by 10 days after birth, those daughter cells were dying off.

Injection of wild-type Tsukushi into the ventricles could partially reverse hydrocephalus in knockout mice.

Through sequencing, the team next demonstrated that 4 of 13 sequences had mutations in their TSK genes that led to amino acid changes, with some patients having multiple mutations. One of the mutations could not be reproduced in mice because the amino acid is not conserved, but the team showed that the other three mutations, alone or in combination, resulted in defective Tsukushi activity when they were engineered into mice.

Tsukushi produced by the ependymal cells affected not only progenitor cells in the subventricular zone, but also the ependymal cells themselves, where it affected ciliogenesis. Cilia on ependymal cells are important for maintaining the flow of CSF, and the neurodevelopmental disorder Bardet-Biedl syndrome, which is often accompanied by hydrocephalus, leads to ciliary dysfunction (as well as changes in neural progenitors) in mice.

The authors concluded that Tsukushi mutations "in the absence of [Tsukushi] function, the neurogenic process is altered because of overproduction and apoptosis of neural stem/progenitor cells, and [ependymal] cell ciliogenesis is affected, leading to abnormal LV [left ventricular] expansion and hydrocephalus-related neurological phenotypes, such as increased anxiety-like behavior, impaired social behavior, prepulse inhibition, motor imbalance, and loss of coordination... the data and tools generated in this study may help to elucidate the pathogenic mechanisms of hydrocephalus and to develop new diagnostic and therapeutic approaches for the prevention and treatment of this disease."