Function and Pathogenic Mechanism of endocannabinoid synthase DAGLB in Parkinson's Disease

National Institute On Aging

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Abstract

Recently, Professor Beisha Tang's lab at Xiangya Hospital, Central South University, China, made significant discoveries concerning DAGLB mutations in familial early-onset recessive Parkinson's disease (PD) patients. They identified four novel loss-of-function mutations in DAGLB in Chinese patients, prompting further investigations into its role in PD pathogenesis. Through RNA sequencing data of isolated substantia nigra pars compacta (SNc) dopaminergic neurons (DANs), we found that DAGLB is the primary 2-arachidonoyl-glycerol (2-AG) synthase in both human and mouse SNc DANs, emphasizing its physiological importance in SNc DAN-dependent motor functions. To delve deeper into the pathophysiological mechanism of DAGLB dysfunction in PD, we conducted genetic knockdown (KD) of Daglb in mouse SNc DANs. This KD led to reduced nigral 2-AG levels and impaired rotarod motor skill learning. In contrast, pharmacological inhibition of 2-AG degradation increased nigral 2-AG levels, promoting DAN activity and dopamine release, and rescuing the motor deficits. These findings unveiled a novel, SNc DAN-specific pathophysiological mechanism of DAGLB dysfunction in PD and provided support for the potential benefits of supplementing endocannabinoids (eCBs) in PD treatment. To address the limitations of germline KO mice in modeling PD-related recessive mutations, we applied CRISPR/saCas9-mediated KD of Daglb specifically in SNc DANs of adult mice. This approach allowed us to avoid potential compensatory interference during development. They subjected the DAN-Daglb KD mice to a rotarod motor skill learning test to assess DAN dysfunction. Additionally, we used fiber photometry live recording to monitor 2-AG release in behaving mice, correlating it with motor performance. To enhance the efficiency of Daglb KD, a line of Daglb-LoxP knock-in (KI) mice was recently generated. By crossbreeding these KI mice with DATIRESCre or Aldh1a1CreERT2 mice, Daglb can be selectively deleted in all midbrain DANs or specifically in ALDH1A1+ midbrain DANs. This approach may reveal potentially more severe behavioral phenotypes. Since deletion also affects VTA DANs, the conditional knockout (cKO) mice will undergo additional non-motor behavioral tests, exploring non-spatial learning, reward and motivation, and stress response. To further examine the role of DAGLB, we will crossbreed DATIRESCre or Aldh1a1CreERT2 with Dagla-LoxP KI and Daglb-LoxP KI mice to genetically delete both Dagla and Daglb in all or ALDH1A1+ DANs. This will help critically evaluate the pathophysiological role of 2-AG in DAN-dependent motor and non-motor behaviors. Considering that 2-AG is implicated in inflammation, we plan to elucidate the role of DAGLB in microglia and astrocytes by selectively deleting Daglb in these cells. Additionally, we will explore how DAGLB activity is regulated. Striosome direct pathway SPN (dSPN) axons form complex structures with ALDH1A1+ DAN dendrites in the substantia nigra pars reticulata (SNr), termed striosome-dendron bouquet structures. ALDH1A1+ SNc DANs display distinct rebound activity in response to GABA-B receptor (Gabbr1)-mediated inhibitory inputs from dSPNs, triggering large dendritic Ca2+ transients likely through T-type Ca2+ channels. We will perform ex vivo electrophysiology and fiber photometry recordings in midbrain slices to evaluate whether intracellular Ca2+ elevation in dendrites induces on-site 2-AG production and release, retrogradely suppressing GABAergic inhibition from dSPNs, and further accelerating SNc DAN rebound activity. In summary, the groundbreaking research by Professor Beisha Tang and our labs presents new insights into the pathophysiological mechanisms of PD-related DAGLB mutations in mouse models. Our innovative experimental scheme contributes to a better understanding of the role of 2-AG and eCB signaling in motor functions, paving the way for potential therapeutic strategies for PD treatment.