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Microscopic image of nerve cells produced from stem cells of a PCH2a patient. © Theresa Kagermeier und Simone Mayer, HIH

Biological Background on PCH2

Pontocerebellar hypoplasias (PCH) are a group of neurogenetic disorders in which the cerebellum and pons are severely reduced in size. Clinically, PCH is characterized by profound and multiple disabilities causing a tremendous burden to patients and families .1234 . To date, there are no therapeutic treatment approaches for PCH. PCH2 is the most common PCH, but it is still a rare autosomal recessive disorder with a prevalence of less than 1:200,000  4. In addition to a severe developmental delay starting at birth, PCH2 patients exhibit a dyskinetic-spastic motor disorder and epileptic seizures as well as gastrointestinal problems 3. Besides pontocerebellar hypoplasia, patients develop progressive microcephaly 56.

It has been hypothesized that PCH2 is a neurodegenerative disorder due to development of microcephaly and reduction of neocortical volume after birth and remnants of the glial scaffold found in regions of the cerebellum devoid of neurons 5 . However, the early onset of the disease also points towards a neurodevelopmental dysfunction.

A subset of PCH is caused by genetic variants in members of the TSEN complex 4. Most (90%) of PCH cases are caused by a homozygous missense variant in the TSEN54 gene (p.TSEN54 A307S). These are referred to as PCH2A4,7 . PCH2A is, therefore, a genetically homogenous disorder.

TSEN54 encodes a subunit of the tRNA splicing endonuclease (TSEN) complex, which is required to remove introns from pre-tRNAs to generate mature tRNAs and is conserved from archaea to eukaryotes 8. A subset of tRNAs requires splicing before being able to serve their role in providing amino acids to the ribosome during translation. 28 intron-containing tRNAs are predicted in humans 9 . The TSEN complex consists of TSEN2, TSEN34, TSEN54, and TSEN15. TSEN2 and TSEN34 are the catalytic subunits, while TSEN54 and TSEN15 are structural proteins 8,10. TSEN54 has been proposed to act as a „molecular ruler” recognizing the tRNA structure and hence the position of the splice site 11.

Recent structural data of the TSEN complex supports this notion and suggests that TSEN54 additionally serves as an organizational scaffold for the catalytic subunits and supports tRNA binding 5,12,18. Interestingly, it has recently been found that PCH mutations do not affect the endonuclease activity of the TSEN complex 19. Instead, the thermal stability of the complex is reduced in patient fibroblasts leading to altered tRNA pools¹⁹. The structural analysis corroborates this finding by showing that mutations are distant from the active site. However, the structure around TSEN54 A307S has not been resolved because it is in an unstructured region 5,12,18, which mediates the interaction with CLP1 5 .

Efforts to establish the cellular mechanism of pathophysiology in animal models have been inconclusive so far. In zebrafish, loss of TSEN54 function leads to cell death in the brain during development7. A complete loss of TSEN54 in mice leads to early embryonic lethality13. Moreover, variants of TSEN54 in dogs lead to a neurological disorder characterized by leukodystrophy 14. Recently, a fly model of PCH2 was published, but the relevance to the clinical phenotype is unclear 15. Additionally, while the catalytic subunits of the TSEN complex are conserved from archaea, the non-catalytic subunits play essential roles in eukaryotes.

Particularly TSEN54 has undergone evolutionary changes in the primate lineage16. These findings imply a species-specific effect of TSEN54 malfunction on brain development. Taken together, the lack of appropriate models of PCH2a has to date precluded the elucidation of its molecular pathology. In order to close this gap in knowledge, a human in vitro model of PCH2A has been recently established using patient-derived pluripotent stem cells and brain region-specific organoids 17.

TSEN54 protein is expressed throughout the body at varying levels (Human Protein Atlas, 8. At the RNA level, TSEN54 is expressed throughout the developing human brain (Human Brain Transcriptome) 9, starting in the first trimester of gestation 7. In the second trimester of gestation, TSEN54 is expressed highly in the developing cerebellum, pons, and olivary nuclei  4. Its expression does not seem cell type-specific at the level of the RNA in the neocortex and cerebellum 10,11. It is, therefore, unclear why specifically cerebellum and pons are affected in PCH2A 7.

In line with the findings that abnormalities in tRNA metabolism cause a plethora of neurodevelopmental disorders 12, it has been hypothesized that specific brain areas, such as the cerebellum and brain stem, are especially vulnerable to TSEN malfunction since they have a stronger requirement for this complex during the last trimester of gestation and early postnatal development 4.


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