Co-editing PINK1 and DJ-1 Genes Via Adeno-Associated Virus-Delivered CRISPR/Cas9 System in Adult Monkey Brain Elicits Classical Parkinsonian Phenotype

Xintian Hu,Zhifang Chen,Liqi Xu,Yingzhou Hu,Zilong Qiu,Xiao Li,Haisong Jiang,Hao Li,Xia Ma,Shihao Wu,Jing Wu,Tianlin Cheng,Yong Yin,Longbao Lv,Ling Li,Wenchao Wang

doi:10.1007/s12264-021-00732-6

Abstract

Whether direct manipulation of Parkinson’s disease (PD) risk genes in the adult monkey brain can elicit a Parkinsonian phenotype remains an unsolved issue. Here, we used an adeno-associated virus serotype 9 (AAV9)-delivered CRISPR/Cas9 system to directly co-edit PINK1 and DJ-1 genes in the substantia nigras (SNs) of two monkey groups: an old group and a middle-aged group. After the operation, the old group exhibited all the classic PD symptoms, including bradykinesia, tremor, and postural instability, accompanied by key pathological hallmarks of PD, such as severe nigral dopaminergic neuron loss (>64%) and evident α-synuclein pathology in the gene-edited SN. In contrast, the phenotype of their middle-aged counterparts, which also showed clear PD symptoms and pathological hallmarks, were less severe. In addition to the higher final total PD scores and more severe pathological changes, the old group were also more susceptible to gene editing by showing a faster process of PD progression. These results suggested that both genetic and aging factors played important roles in the development of PD in the monkeys. Taken together, this system can effectively develop a large number of genetically-edited PD monkeys in a short time (6–10 months), and thus provides a practical transgenic monkey model for future PD studies.

Highlights

Parkinson’s disease (PD), with a prevalence of 2%–3% in people [65 years old, is the second-most common neurodegenerative disorder worldwide [1]
Based on our and others’ previous studies, it is well known that the loss of nigral dopaminergic neurons has to be at least 50% in one or both substantia nigras (SNs) to elicit PD symptoms [25, 26]
We found that all the single-guide RNAs (sgRNAs)-PINK1-A, sgRNAPINK1-B, sgRNA-DJ-1-A, and sgRNA-DJ-1-B effectively caused missense mutations in the protein coding regions of the COS7 cells (Fig. S2)

Summary

Introduction

Parkinson’s disease (PD), with a prevalence of 2%–3% in people [65 years old, is the second-most common neurodegenerative disorder worldwide [1] It is typically characterized by a cluster of specific motor symptoms, including bradykinesia, rigidity, postural instability and tremor, and by a set of unique pathological hallmarks, including severe dopaminergic neuron loss and clear morphological changes of the surviving dopaminergic neurons in the substantia nigra pars compacta (SNpc), and intracellular inclusions containing a-synuclein aggregates, which are named Lewy bodies in neuronal somata or Lewy neurites in neuronal processes in the SN and other brain regions [1, 2]. No breakthroughs have been reported in this area yet

Methods

Results

Conclusion