Maintenance of mitochondrial integrity in midbrain dopaminergic neurons governed by a conserved developmental transcription factor

Abstract

Progressive degeneration of dopaminergic (DA) neurons in the substantia nigra is a hallmark of Parkinson’s disease (PD). Dysregulation of developmental transcription factors is implicated in dopaminergic neurodegeneration, but the underlying molecular mechanisms remain largely unknown. Drosophila Fer2 is a prime example of a developmental transcription factor required for the birth and maintenance of midbrain DA neurons. Using an approach combining ChIP-seq, RNA-seq, and genetic epistasis experiments with PD-linked genes, here we demonstrate that Fer2 controls a transcriptional network to maintain mitochondrial structure and function, and thus confers dopaminergic neuroprotection against genetic and oxidative insults. We further show that conditional ablation of Nato3, a mouse homolog of Fer2, in differentiated DA neurons causes mitochondrial abnormalities and locomotor impairments in aged mice. Our results reveal the essential and conserved role of Fer2 homologs in the mitochondrial maintenance of midbrain DA neurons, opening new perspectives for modeling and treating PD.

Introduction

Midbrain dopaminergic (mDA) neurons play a central role in controlling key brain functions, including voluntary movement, associative learning, motivation and cognition¹. The progressive and selective degeneration of mDA neurons in the substantia nigra (SN) reduces nigrostriatal DA transmission, leading to the motor symptoms of Parkinson’s disease (PD). Although PD was first described over 200 years ago and is the most prevalent neurodegenerative movement disorder, the exact pathologic mechanisms remain unclear. Treatment options are limited to symptomatic relief with DA replacement therapy^2,3. Thus, there is an urgent need to identify targets for disease-modifying therapies that reverse or hinder neurodegeneration in PD.

PD is a multifactorial disorder caused by a combination of genetic and environmental factors⁴. Increasing evidence indicates that PD risk factors lead to common pathological processes, including excessive production of reactive oxygen species (ROS), axonal pathology, neuroinflammation, defects in the ubiquitin-proteasome system, and mitochondrial dysfunction^2,5. In particular, mitochondrial impairment is integral to most other pathological cellular processes and thought to play a central role in dopaminergic neurodegeneration. Complex I activity is reduced in the brains of PD patients, and mitochondrial toxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and rotenone potently induce SN degeneration. The findings that multiple genes linked to familial PD, including PINK1, Parkin, LRRK2, SNCA and DJ-1, directly or indirectly affect mitochondrial physiology further support this notion^6,7.

Although several genes linked to rare monogenic forms of PD have been identified, genetic susceptibility factors for sporadic PD, which accounts for ~90% of cases, remain largely unknown. In this regard, it is noteworthy that dysregulation of transcription factors required for mDA development is implicated in PD. Numerous transcription factors required for various aspects of mDA neurons development, including En1, En2, Lmx1a, Lmx1b, Foxa1, Foxa2, Nurr1, Pitx3 and Otx2, remain expressed in the adult brain and are crucial for the survival and function of differentiated mDA neurons in mice^{8,9,10,11,12,13,14,15,16}. Genome-wide association studies (GWAS) have shown that genetic variants of developmental transcription factors are highly represented in sporadic PD patients^17,18. Moreover, overexpression of Nurr1, Foxa2, En1 or Otx2 prevents the loss of DA neurons in murine PD models^19,20,21. Therefore, a better comprehension of the genetic networks controlled by developmental transcription factors in differentiated mDA neurons may advance our molecular understanding of neurodegeneration in PD and open new therapeutic options.

Drosophila offers a powerful model system to investigate molecular mechanisms of PD pathogenesis in vivo, due to the abundance of genetic tools, the conservation of most PD-related pathways and familial PD-linked genes, and its relatively simple nervous system^22,23. The adult fly midbrain contains approximately a dozen clusters of DA neurons projecting to different areas²⁴. Although the anatomical arrangements of DA neurons in Drosophila and vertebrates differ considerably, recent works have highlighted the functional homology of some of the dopaminergic circuits²⁵. The protocerebral anterior medial (PAM) cluster is the largest subgroup containing ~80% of all Drosophila brain DA neurons. PAM neurons are highly heterogeneous in their function and projection patterns, modulating various behavior including associative learning, sleep, and locomotion^26,27. Remarkably, some of the PAM neurons are required for the startle-induced climbing, a locomotor behavior found to be defective in multiple PD models^27,28,29, rendering the PAM cluster partially analogous to the mammalian SN. Dopaminergic innervation from the PPL1 and PPM3 clusters to the central complex also appear to be functionally homologous to the nigrostriatal pathway and is reported to be impaired in some PD models^25,30,31.

The notion that developmental transcription factors play critical roles in the maintenance of adult DA neurons is shared between flies and mammals, as supported by our recent studies of Drosophila Fer2 gene²⁸. Fer2 encodes a basic helix-loop-helix (bHLH) transcription factor required for the development of DA neurons in the PAM cluster; thus, fewer PAM neurons are formed in Fer2² hypomorphic mutants. Additionally, Fer2² mutant flies display a progressive loss of PAM neurons in adulthood, locomotor impairment that can be improved by L-DOPA administration, and increased ROS levels in the brain. PAM neurons deficient for Fer2 accumulate abnormal mitochondria and show autophagy dysfunction. Fer2 expression persists into adulthood in PAM neurons and increases in response to oxidative stress. Importantly, the maintenance of mitochondrial integrity and the survival of PAM neurons in aged flies require the presence of Fer2 within PAM neurons in adulthood^28,29. Taken together, these lines of evidence establish the dual role of Fer2 in development and lifelong maintenance of DA neurons in the PAM cluster, and suggest that molecular studies of Fer2 function will shed light on the mechanisms of dopaminergic neurodegeneration relevant to PD.

In the present study, we ask how Fer2 maintains DA neurons throughout adulthood and whether its function is conserved in mammals. We address these questions by analyzing functional interaction between Fer2 and familial PD-linked genes, by identifying Fer2 target genes and by generating mice with a conditional deletion of Nato3, a murine homolog of Fer2, in differentiated DA neurons. This combined approach demonstrates that Fer2 is a master regulator of a transcriptional network controlling mitochondrial integrity and function, and thereby confers dopaminergic neuroprotection in multiple genetic and toxin models of PD. Nato3 is known to be required for the genesis of mDA neurons^32,33,34 but its post-developmental role has never been studied. We demonstrate here that selective ablation of Nato3 in differentiated DA neurons leads to age-dependent motor impairment and a loss of mitochondrial integrity in mDA neurons. Taken together, our results indicate that Fer2 and Nato3 share an essential role in maintaining mitochondrial health and dopaminergic function, offering new opportunities to study the selective vulnerability of mDA neurons and to develop therapeutic interventions for PD.

Results

Fer2 overexpression protects PAM dopaminergic neurons from genetic and oxidative insults

To explore the genetic mechanisms underlying the role of Fer2 in the maintenance of DA neurons, we investigated whether Fer2 loss or gain of function affects the survival of DA neurons in genetic models of PD. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of familial PD and also predispose carriers to sporadic PD³⁵. Because pathogenic LRRK2 variants are considered to be gain-of-function mutants, most fly models of LRRK2-linked PD employ overexpression of wild-type or mutant forms of LRRK2 or its fly homolog. We thus overexpressed wild-type or mutant Drosophila Lrrk via the GAL4/UAS system using PAM neuron-specific drivers, Fer2-GAL4 or R58E02-GAL4^26,28. The number of surviving DA neurons was assessed by immunostaining with anti-tyrosine hydroxylase (TH) antibodies.

Forced expression of either wild-type or Lrrk I1915T mutant, which is homologous to the human pathogenic LRRK2 I2020T variant^35,36, reduced the number of TH-positive neurons in the PAM cluster by ~10% in 1-day-old flies (Supplementary Fig. 1a). Thereafter TH-positive cell counts further decreased in an age-dependent manner (Supplementary Fig. 1a and Fig. 1a, b). To distinguish the reduction in TH expression and the genuine loss of DA neurons, we also visualized PAM neurons by expressing the nuclear RedStinger red fluorescent protein together with Lrrk. The number of RedStinger-positive cells was reduced in the flies expressing wild-type or mutant Lrrk in PAM neurons (Fig. 1c, d). Therefore, Lrrk gain of function causes not only developmental loss but also age-dependent loss of PAM neurons. Remarkably, overexpression of Fer2 in PAM neurons prevented the reduction of TH-positive cell counts induced by the expression of wild-type or mutant Lrrk (Fig. 1a, b). The analysis of cell counts using RedStinger found that PAM neuron loss caused by the mutant Lrrk, but not the WT Lrrk, was rescued with Fer2 overexpression at least partially (Fig. 1c, d). Forced expression of Lrrk in Fer2² heterozygous background resulted in PAM neuron loss to a similar extent as in wild-type background (Supplementary Fig. 1b). Lrrk overexpression in Fer2² homozygous mutants additively enhanced PAM neuron loss compared with either of the single genetic perturbations (Supplementary Fig. 1b). These results suggest that Fer2 and Lrrk act in independent pathways but Fer2 overexpression can attenuate Lrrk-induced neuronal damage….