The Mechanism Of Metabolic Dysfunction Induced By Dopaminergic Neuron Conditional Knockout Of ALAS1 In Mice

Background: Parkinson’s disease(PD)is the second-most common neurodegenerative disorder,characterized by the loss of dopaminergic neurons in the substantia nigra compact and the formation of Lewy bodies.Lewy bodies are protein aggregates composed of many proteins,including α-synuclein.Although the pathogenesis of PD is still unclear,numerous studies have shown that mitochondrial dysfunction plays a pivotal role in both sporadic and familial forms of the disease.There is evidence that exposure to mitochondrial toxins such as MPTP triggers dopa-responsive parkinsonism.In addition,mutations in PD-related pathogenic genes such as PINK1 、 Parkin and LRRK2,can cause mitochondrial dysfunction.In this study,we screened the upstream regulatory genes of PINK1 using a CRISPR/Cas9 library and showed that deletion of the mitochondrial protein delta-aminolevulinate synthase 1(ALAS1)resulted in an increase in the full length of PINK1,suggesting that ALAS1 may be associated with PD.ALAS1 is the first and rate limiting enzyme in the heme biosynthetic pathway,catalyzing the condensation of glycine and acetyl-coenzyme A to form ALA.Otherwise,a blood transcriptomic meta-analysis has shown that some enzymes of the heme synthesis pathway are downregulated in PD,including ALAS2,an isoenzyme of ALAS1.But the relationship between ALAS1 and PD has not been reported yet.Objective: investigate the relationship between ALAS1 and mitochondrial function and the role of ALAS1 in the pathogenesis of PDMethods: 1.We constructed a PINK1-GFP cell line,in which the upstream regulatory genes of PINK1 were screened by CRISPR/Cas9library;2.We constructed an ALAS1 knockout SH-SY5 Y cell line to investigate whether mitochondrial function is impaired in the absence of ALAS1;3.The metabolic phenotype was studied by using immunohistochemistry,CLAMS comprehensive lab animal monitoring system,GTT and ITT methods in dopaminergic neuron conditional knockout of ALAS1 mouse model;4.RNA sequencing of ALAS1 knockout cell lines was performed to explore the potential mechanism of metabolic dysfunction in ALAS1 conditional knockout mice.Results: 1.Full-length PINK1 increase in the ALAS1 knockout SH-SY5 Y cell line;2.The absence of the ALAS1 causes the increase of mitochondrial ROS,the reduction of membrane potential,the decrease in the ATP level,the affection in the function of the mitochondrial electron transfer chain,and a significant decrease in the expression levels of MTCO1 and MTCO2 in mitochondrial complex IV;3.ALAS1 conditional knockout mice shows significant loss of dopaminergic neurons in the substantia nigra and phenotype of metabolic dysfunction;4.RNA sequencing results showed that ALAS1 deletion resulted in decreased expression of the gene encoding the dopamine transporter.Conclusion: This study demonstrates that ALAS1 deletion leads to mitochondrial dysfunction,while ALAS1 conditional knockout mice show significant loss of dopaminergic neurons in the substantia nigra,providing important evidence for the association between ALAS1 and PD.In addition,ALAS1 conditional knockout mice showed metabolic dysfunction,similar to the metabolic characteristics exhibited by PD patients.Meanwhile,ALAS1 deletion cause downregulation of the encoding gene of dopamine transporter,suggesting that ALAS1 may be associated with the metabolic phenotype by affecting dopamine level.This provides an idea for further studies on how ALAS1 participates in PD occurrence.