1,4-Dihydroxy-2-naphthoic acid (1,4-DHNA) prevents MPTP-induced motor function deficits

Introduction

Parkinson’s disease (PD) is the second most prevalent progressive neurodegenerative disorder that significantly impacts the quality of life of approximately 1 to 2% of individuals over the age of 65 (Twelves et al., 2003, Marras et al., 2018, Simon et al., 2020). Common motor impairments in PD include tremors, bradykinesia, rigidity and postural instability (Mazzoni et al., 2012). Dopamine acts as a crucial neurotransmitter involved in motor control, and death of dopaminergic neurons in the substantia nigra pars compacta (SNpc) is a hallmark of PD. However, the etiology of PD is still elusive. Genetic factors have been identified in familial forms of PD (Day et al., 2021); however given that the majority of PD cases are sporadic, environmental factors were indicated to also have significant contribution (Ball et al., 2019). Disease progression has been linked to dysfunction in mitochondrial processes, resulting in increased oxidative stress leading to cellular damage and inflammation (Beal, 2003, Subramaniam et al., 2013). Additionally, oxidative stress exacerbates the aggregation of α-synuclein, and accumulation of Lewy bodies, which in turn disrupts cellular functions (Maguire-Zeiss et al., 2005, Olivares et al., 2009).

Interestingly, dopamine and carbidopa, a drug used for treating PD, are aryl hydrocarbon receptor (AhR) ligands (Safe, 2017, Park et al., 2020). Moreover, abnormal kynurenine pathway metabolism was associated with aging and the pathology of PD (Venkatesan et al., 2020, Fathi et al., 2022, Qin et al., 2022), and its major metabolite, kynurenine, is also an endogenous ligand for the AhR (Opitz et al., 2011, Bessede et al., 2014). Additionally, Parkin (RBR E3 Ubiquitin Protein Ligase, PRKN), which was linked to mitochondrial dysfunction and PD (Lim et al., 2012), is transcriptionally regulated by AhR (González-Barbosa et al., 2019). AhR is a ligand-activated transcription factor that binds structurally-diverse ligands and is expressed across many organs and tissues (Safe et al., 2018, Safe et al., 2020, Grifka-Walk et al., 2021, Stockinger et al., 2021). Additionally, AhR plays a key role in the microbiome-gut-brain axis (Lee et al., 2017, Barroso et al., 2021). This is noteworthy, because altered microbiota composition has been observed in PD patients, and is believed to be associated with gut dysbiosis and inflammation (Romano et al., 2021) and the development of PD (Cryan et al., 2020, Zhu et al., 2022). Moreover, Lewy bodies and abnormal aggregation of α-synuclein were observed in the enteric nervous system of PD patients (Gold et al., 2013), supporting the hypothesis that, at least in some forms of PD, the pathological process is interconnected from the gut to brain (Holmqvist et al., 2014, Kim et al., 2019).

1,4-Dihydroxy-2-naphthoic acid (1,4-DHNA) is a bacteria-derived metabolite produced by Propionibacterium freudenreichii and Lactobacillus casei, which stimulates bifodobacterial growth (Bentley et al., 1982, Mori et al., 1997, Isawa et al., 2002). 1,4-DHNA is an AhR agonist (Cheng et al., 2017, Vermillion Maier et al., 2021), and given the suggested roles of gut microbiota and AhR in PD, this study employed the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) rodent model to examine the effectiveness of 1,4-DHNA in preventing motor deficits linked to Parkinson.

MPTP is a prodrug that damages the nigrostriatal dopaminergic pathway and produces motoric deficits which resembles PD, and it is the most common mouse model used for investigating PD (Huang et al., 2017). MPTP crosses the blood brain barrier where it is modified to the neurotoxin MPP+ by reactive astrocytes (Ransom et al., 1987). MPP+ was demonstrated to displace dopamine from vesicular stores, inhibit the mitochondrial respiratory complex I, and increase the formation of reactive oxygen species, resembling pathological processes in PD (Beal, 2003). In this study, we examined the ability of 1,4-DHNA to prevent MPTP-induced Parkinson-like state and focused on the ability of 1,4-DHNA to prevent MPTP-induced deficits in stride length/walking/balance as well as to ameliorate MPTP-induced deficits in fine motor coordination. Moreover, to determine the role of AhR in mediating the prevention of MPTP-induced symptoms, we compared the effects of 1,4-DHNA to the effects observed for 3,7-dihydroxy-2-naphthoic acid (3,7-DHNA), a structurally similar derivative that is predominantly inactive at the AhR (Cheng et al., 2017).

Methods

Results

Discussion

This study demonstrated that 1,4-DHNA can prevent MPTP-induced motor function deficits. Interestingly, it appears that 1,4-DHNA’s ability to mitigate various motor deficits is likely mediated via different underlying molecular mechanisms. PD patients exhibit bradykinesia, or slowness of movement, and hypokinesia, or abnormally decreased movement, that are not due to weakness (Schwab et al., 1959, Fahn, 2003). It was suggested that both symptoms might have the same origin, and that the reason for the longer movement duration is due to inadequate scaling of the motor command and reduced level of motor motivation (Mazzoni et al., 2012). Specifically, PD patients move slower because of alterations in motor control of speed selection, as they naturally select for lower speeds than healthy controls (Mazzoni et al., 2007). Additionally, PD causes the total movement duration to increase because the movement is initially scaled too short, requiring additional commands and corrective measures. Hence, PD patients exhibit significantly reduced stride length (Zanardi et al., 2021). In this study, MPTP reduced stride length, and this motor control deficit was prevented by 1,4-DHNA. In contrast, 3,7-DHNA was not as effective in preventing MPTP-induced reduction in stride length. Additionally, 1,4-DHNA, but not 3,7-DHNA, reduced the time to cross the balance beam. Thus, because 1,4-DHNA is an AhR agonist while 3,7-DHNA is inactive at the AhR (Cheng et al., 2017, Vermillion Maier et al., 2021), the results suggest that the AhR might play a role, in part, in the control of motor motivation, movement planning, and natural speed selection, as related to bradykinesia and shortened step length observed in PD.

PD patients suffer from rigidity which is experienced as a stiffness and resistance to passive movement. Although it is hard to attribute particular behavioral deficits to a certain deficit in motor control, rigidity was suggested to explain, at least in part, the impairment in fine motor function skills, dexterity, and motor coordination observed in PD patients (Norman et al., 2013, Dahdal et al., 2016). Removal of an adhesive strip requires certain levels of dexterity. Similarly, turning around on a very narrow pole and descending down in a controlled fashion requires certain motor coordination. Both 1,4-DHNA and 3,7-DHNA prevented the deficits in the motor pole test and in the adhesive strip removal test. This indicates that AhR is likely not mediating the beneficial effects of DHNA isomers in preventing MPTP-induced impartment in fine motor skills, and perhaps rigidity.

DHNA is a natural product of gut microbiota Propionibacterium freudenreichii and Lactobacillus casei (Bentley et al., 1982, Mori et al., 1997, Isawa et al., 2002). Interestingly, alterations in gut microbiome composition have been reported in PD patients and are induced by MPTP (Lai et al., 2018, Torres et al., 2018). Specifically, an enrichment of the genera Lactobacillus was observed in PD patients (Romano et al., 2021) and in a response to MPTP (Torres et al., 2018). This could represent an adaptation to increase levels of DHNA, and might further supports the hypothesis that pathological and mitigating process in the gut could play an essential role in the development of PD.

This study demonstrated that 1,4-DHNA counteracts the development of MPTP-induced motor deficits. Although MPTP is a widely used rodent model to precipitate symptoms resembling PD, it does not represent the etiology of the disorder and has limitations in its predictability. (Dauer et al., 2003, Huang et al., 2017, Konnova et al., 2018). For example, death of dopaminergic neurons in the substantia nigra is observed both in the MPTP model and in PD (Kopin et al., 1988), yet the etiology and timeline differ significantly. Specifically, PD takes years to manifest, while the effects of MPTP on dopaminergic neurons and symptoms appear within a short time (Donnan et al., 1987, Mori et al., 1988). The aim of this study was to establish that DHNA can prevent the motoric deficits caused by MPTP. Given the general timeline of the effects of MPTP, DHNA administration began 3 weeks prior to MPTP. Clearly, this study was not designed to establish a timeline, given the short timeline of symptom progression that does not resemble PD progression. Thus, future studies should examine DHNA using other PD models which better resemble the lengthy process of symptom precipitation to further establish the therapeutic potential of DHNA in PD and the ability of DHNA to reverse or prevent the progression of symptoms. Future studies should also closely examine 1,4-DHNA’s mechanism of action, including confirming the AhR-dependent therapeutic effects by examining its action in AhR knockout animals. Additionally, future studies should evaluate the action of other AhR ligands, including gut-derived tryptophan metabolites, in order to fully understand their therapeutic potential and to reveal the role of AhR and other signaling pathways in motor control and PD.