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

National Institute On Aging

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Abstract

Recently, Professor Beisha Tangs lab at Xiangya Hospital, Central South University, China, identified four novel loss-of-function mutations in DAGLB in Chinese patients with familial early-onset recessive PD. From RNA sequencing data of isolated SNc DANs, we found that DAGLB is the dominant 2-AG synthase in both human and mouse SNc DANs, suggesting the physiological importance of DAGLB-mediated 2-AG biosynthesis in SNc DAN-dependent motor functions. Indeed, we found that genetic knockdown (KD) of Daglb in the mouse SNc DANs led to reduced nigral 2-AG levels and impaired rotarod motor skill learning, whereas pharmacological inhibition of 2-AG degradation increased nigral 2-AG levels, promoted DAN activity and dopamine release, and rescued the motor deficits. Therefore, we reveal a previously undescribed, SNc DAN-specific pathophysiological mechanism of DAGLB dysfunction in PD pathogenesis and provide the rationale and additional preclinical evidence for the beneficial effects of eCB supplementation in PD treatment. To overcome the futility in modeling the PD-related recessive mutations with germline KO mice, we applied CRISPR/saCas9-mediate KD of Daglb selectively in the SNc DANs of adult mice. This approach avoids any potential compensatory interference during development. We also subjected the DAN-Daglb KD mice to the SNc DAN-dependent rotarod motor skill learning test to examine any DAN dysfunction. Finally, we employed fiber photometry live recording technique to monitor the 2-AG release with genetically encoded eCB sensor in behaving mice in correlation with the motor performance. Together, we offer a new innovative experimental scheme to study the pathophysiological mechanism of PD-related genetic mutations in mouse models. Since the overall efficiency of CRISPR/saCas9-mediated Daglb KD is about 70-80% in the current study, we recently generated a line of Daglb-LoxP knock-in (KI) mice. By crossbreeding the KI mice with DATIRESCre or Aldh1a1CreERT2 mice, we can selectively delete Daglb in all or ALDH1A1+ midbrain DANs, which would be useful to reveal potentially more severe behavioral phenotypes. Since the deletion also occurs in VTA DANs, we will also subject the conditional KO (cKO) mice to additional non-motor behavioral tests, such as non-spatial learning, reward and motivation, and stress response. Considering DAGLA, although a minor 2-AG synthase in SNc DANs, may also contribute to the residual 2-AG production in Daglb-deficient DANs, we will also 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, which may provide the means to critically evaluate the pathophysiological role of 2-AG in DAN-dependent motor and non-motor behaviors. In addition, as 2-AG is also implicated in inflammation53, we will further elucidate the role of DAGLB in microglia and astrocytes through selective deletion of Daglb in microglia or astrocytes. Although the rotarod motor training paradigm promotes the on-demand 2-AG production by DAGLB, how the DAGLB activity is regulated remains to be determined. Striosome dSPN axons are intermingled with the dendrites of ALDH1A1+ DANs perpendicularly protruding in the substantia nigra pars reticulata (SNr) and form so-called striosome-dendron bouquet structures. The ALDH1A1+ SNc DANs display distinct rebound activity in response to the GABA-B receptor (Gabbr1)-mediated inhibitory inputs from dSPNs, which then trigger large dendritic Ca2+ transients likely through T-type Ca2+ channels. We will perform ex vivo electrophysiology and fiber photometry recording in midbrain slice to further evaluate whether the intracellular Ca2+ elevation in dendrites is required to induce on-site 2-AG production and release, which in turn retrogradely suppress the GABAergic inhibition from dSPNs and further accelerate the rebound activity of SNc DANs.