P 1086. Role of PTRH2 in Cerebellar Development

Background: We recently identified and described homozygous nonsense or missense mutations in the peptidyl-tRNA-hydrolase 2 gene (PTRH2) in five patients with infantile-onset multisystem neurologic, endocrine, and pancreatic disease (IMNEPD; MIM#616263). Previous data showed that Ptrh2 is highly expressed in the cerebellum, especially in Purkinje cells, potentially denoting an important role of Ptrh2 in the cerebellum. In line with this finding, patients with a homozygous PTRH2 gene mutation suffer from progressive cerebellar atrophy and ataxia among further symptoms.

Aim: To better understand the role of Ptrh2 in the cerebellum, we analyzed the effect of a loss of Ptrh2 in Purkinje cells alone or in the whole cerebellum, respectively.

Methods: We generated a Purkinje cell-specific Ptrh2 knockout mouse line (Ptrh2LoxPxPcp2Cre) and a constitutive Ptrh2 knockout mouse line (Ptrh2LoxPxhCMVCre) and analyzed the effect on cerebellar and Purkinje cell development in vivo and in vitro (e.g., qPCR, IHC, western blot, and cell culture).

Results: In constitutive PTRH2 knockout mice, we detected a postnatal reduction of cerebellar volume and cerebellar foliation with stunted foliae along with delayed Purkinje cell maturation, reduced soma size, and dendritic tree at postnatal day 5. Reduced Purkinje cell size and dendritic tree were confirmed in primary mixed cerebellar neuron cultures in vitro (at day 12 in vitro). Through RNA sequencing and qPCR of cerebella from constitutive knockout mice, we delineated a significant up-regulation of intracellular inhibitors of the sonic hedgehog (Shh) pathway, and a significant down-regulation of main intracellular effectors of the Shh pathway. While expression levels for the intracellular effectors of the Shh pathway were altered, Shh expression and protein levels were unchanged. These findings indicate that PTRH2 depletion may disrupt cerebellar development by reducing intracellular SHH pathway activity, albeit without affecting Shh expression. Since constitutive PTRH2 knockout mice suffer from a runting syndrome and are lethal at postnatal days 7 to 10, we analyzed Purkinje cell–specific PTRH2 knockout mice at adult stages and detected reduced Purkinje cell size, density, and molecular layer and reduced Purkinje cell dendritic arborization resulting in a functional cerebellar deficit with ataxia and abnormal gait.

Conclusion: We delineate a crucial role of PTRH2 in Purkinje cell development and Purkinje cell survival that may result from reduced intracellular Shh pathway activity.