Reversibility of motor dysfunction in the rat model of NGLY1 deficiency

(2021) 14:91 Asahina et al. Mol Brain https://doi.org/10.1186/s13041-021-00806-6 Open Access RESEARCH Reversibility of motor dysfunction in the rat model of NGLY1 deficiency Makoto Asahina1,3, Reiko Fujinawa2,3, Hiroto Hirayama2,3, Ryuichi Tozawa1,3, Yasushi Kajii1 and Tadashi Suzuki2,3* Abstract N-glycanase 1 (NGLY1) deficiency is a rare inherited disorder characterized by developmental delay, hypolacrima or alacrima, seizure, intellectual disability, motor deficits, and other neurological symptoms. The underlying mechanisms of the NGLY1 phenotype are poorly understood, and no effective therapy is currently available. Similar to human patients, the rat model of NGLY1 deficiency, Ngly1−/−, shows developmental delay, movement disorder, somatosensory impairment, scoliosis, and learning disability. Here we show that single intracerebroventricular administration of AAV9 expressing human NGLY1 cDNA (AAV9-hNGLY1) to Ngly1−/− rats during the weaning period restored NGLY1 expression in the brain and spinal cord, concomitant with increased enzymatic activity of NGLY1 in the brain. hNGLY1 protein expressed by AAV9 was found predominantly in mature neurons, but not in glial cells, of Ngly1−/− rats. Strikingly, intracerebroventricular administration of AAV9-hNGLY1 normalized the motor phenotypes of Ngly1−/− rats assessed by the rota-rod test and gait analysis. The reversibility of motor deficits in Ngly1−/− rats by central nervous system (CNS)-restricted gene delivery suggests that the CNS is the primary therapeutic target organs for NGLY1 deficiency, and that the Ngly1−/− rat model may be useful for evaluating therapeutic treatments in pre-clinical studies. Keywords: Ngly1, Motor dysfunction, Ngly1 deficient rats Introduction N-glycanase 1 (NGLY1), also known as peptide:N-glycanase, is an evolutionarily conserved enzyme among eukaryotes that plays a crucial role in quality control for newly synthesized N-glycoproteins [1, 2]. N-glycanase 1 (NGLY1) deficiency is a rare inherited disorder that is caused by mutations in the NGLY1 gene. In 2012, the first patient harboring mutations in NGLY1 was identified [3]. Since then, more than 60 similar patients have been confirmed worldwide and several clinical reports have been published [4–10]. NGLY1 deficient patients show a broad spectrum of clinical features including developmental delay, hypolacrima or alacrima, seizure, intellectual disability, and motor deficits [4–14]. The mutant form of Ngly1 and its orthologs have been analyzed to elucidate *Correspondence: 2 Glycometabolic Biochemistry Laboratory, RIKEN Cluster for Pioneering Research, RIKEN, 2‑1 Hirosawa, Wako, Saitama 351‑0198, Japan Full list of author information is available at the end of the article the molecular function of NGLY1 in various organisms [1, 15–31]. However, the pathological mechanisms leading to the varied symptoms of NGLY1 deficiency remain obscure, and no effective therapy is currently available. Our previous study demonstrated that, similar to human patients, Ngly1−/− rats show developmental delay, movement disorder, somatosensory impairment, scoliosis, and learning disability [32]. Histological analysis identified prominent pathological abnormalities including necrotic lesions, mineralization, intra- and extra-cellular eosinophilic bodies, astrogliosis, microgliosis, and significant loss of mature neurons in the thalamus of Ngly1−/− rats [32]. In endoplasmic reticulum-associated degradation processes, NGLY1 cleaves N-glycans from misfolded glycoproteins in the cytosol while they are degraded by the proteasome; the loss of Ngly1 led to accumulation of cytoplasmic ubiquitinated proteins, a marker of misfolded proteins, in the neurons of the central nervous system (CNS) in Ngly1−/− rats [32]. In © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Asahina et al. Mol Brain (2021) 14:91 addition, Ngly1−/− rats showed axonal degeneration in peripheral nerves [32], similar to human patients. The face validity of Ngly1−/− rats as a model animal for NGLY1 deficiency has been verified by phenotypic analysis [32]. However, to utilize Ngly1−/− rats as an animal model for exploring therapeutic options, it is necessary to evaluate the reversibility of symptoms. In addition, it is important to clarify the target organs and cells for the development of therapeutic agents and modalities for NGLY1 deficiency. While Ngly1 is ubiquitously expressed throughout the body with the highest expression in the testis [33], the neurological symptoms in most patients suggest abnormalities in the CNS [4–11, 14]. However, it remains unclear whether loss of NGLY1 in the CNS primarily causes the symptoms of NGLY1 deficiency. The adeno-associated virus (AAV) is a powerful tool for gene delivery, with features that include the ability to infect different tissues with various injection routes, long-term foreign gene expression, and a lack of pathogenicity in animal models [34]. AAV vectors are also the leading platform for gene delivery for the treatment of a variety of human diseases, with approximately 200 interventional clinical trials involving AAV in human patients registered at ClinicalTrials.gov [35]. In the present study, we investigated whether a single intracerebroventricular (i.c.v.) administration of AAV9 expressing human NGLY1 cDNA would normalize the most apparent and translatable symptoms of Ngly1−/− rats. By expressing the hNGLY1 gene specifically in the CNS of Ngly1−/− rats by i.c.v. injection, we verified that the motor symptoms are reversible. Our findings clearly demonstrate that the Ngly1−/− rat model is useful for evaluating therapeutic options in pre-clinical studies, and suggest the CNS as a target organ for therapies for NGLY1 deficiency. Results NGLY1 transgene expression pattern after intracerebroventricular injection of viral vectors In this study, we aimed to evaluate the reversibility of disease phenotypes in Ngly1−/− rats. To this end, (...truncated)