The Relaxin-3 Receptor, RXFP3, Is A Modulator Of Aging-Related Disease Ⅱ

As discussed, the aging process is largely driven by glucometabolic dysfunction. Hence, conditions such as T2DM or MetS are potent triggers of multiple forms of aging-related disease. Therefore, it is important to consider that the mechanisms through which glucose metabolism becomes dysregulated over time require more in-depth investigation. While the insulinotropic system is the primary mechanism for controlling glucose uptake and use, it has been shown in recent years that there are many other receptor systems (especially GPCRs) that also potently regulate glucose metabolism [86,128,134–137]. Here, we propose that the RXFP3 system, especially when it is actively interacting with GIT2, also forms part of this glucometabolic family [2,5,24,133]. Even though there has been a signifificant focus on investigating the role of glucose metabolism in the aging process (potentially via the profound link with mitochondrial support in aging), there is also the strong impact of the adipose tissue system in this paradigm [138–140]. Reinforcing the potential importance of RXFP3 in the aging process, it has been demonstrated that RLN3 can play an important role in adipogenesis and maturation [141]. This functionality may not be entirely unexpected as RXFP3 appears likely to be a manager of the energy metabolism in times of aging-associated metabolic disruption [10]. Hence, RXFP3 has been associated with the functionality of energy metabolic systems involving dietary-related weight gain, insulinotropic functions, and adipogenic activities that are strong players in the aging process [3,10,29,138,142–144]. In the next sections, we will highlight the contributions and activities of RLN3/RXFP3 signaling in aging-related diseases. These insights strengthen the concept that the RXFP3-GIT2 signaling system may represent a novel signaling relationship system that can be developed for novel and effective anti-aging therapeutics. 

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3.1. Alzheimer's Disease 

It is now well appreciated that many classical central nervous system neurodegenerative disorders, such as Alzheimer’s, Parkinson’s, and Huntington’s disease, share many common etiological features with perhaps the primary one being metabolic dysfunction [126,128,145,146]. With respect to AD, it has been shown that RXFP3 levels are signifificantly altered in the neocortex of depressed Alzheimer’s patients [147]. Alzheimer’s disease is primarily represented as a dysfunctional capacity for short-term memory formation and then, at a later stage, a dysfunction in long-term memory recollection. It is relevant to note that depletion of RXFP3 levels in the brain have been associated with long-term memory regulation in adult mice [27]. In addition to long-term memory recall, RXFP3 functionality has also been associated with spatial memory formation [148,149]. 

3.2. Anxiety and Post-Traumatic Stress Disorder 

Anxiety, along with associated disorders such as post-traumatic stress disorder (PTSD), have in recent years been strongly associated with premature aging conditions [150–153]. With respect to the impact of RXFP3 upon anxiety-related disorders, it has been shown that specific central stimulation generates an anxiolytic effect in model organisms [154]. While acute effects of RXFP3 stimulation can generate anxiolytic effects, it has recently been shown that chronic localized RXFP3 stimulation instead can actually promote anxiety behavior [26]. Thus, it appears that the anxiety-related activities of RXFP3 may be highly context-specific in experimental animal models [155]. Such a phenomenon therefore entails a more detailed view in human patients of this specific situation and therefore RXFP3-based interventions could potentially be therapeutic targets for certain forms of anxiogenic activity. 

It is interesting to note that the RXFP3-GIT2 signaling axis seems to be a priority signaling system for central anxiety/stress conditions, as not only is GIT2 involved in anxiety-related behavior directly, it is also a potent regulator of the glucose metabolic system that intertwines with anxiety-related conditions [156,157]. Moreover, it has been shown that both RXFP3 and GIT2 are highly expressed in the amygdala [123,158,159]. There is also evidence to suggest that through common activities related to stress responses RXFP3 and GIT2 together may contribute in a coordinated manner to interconnect anxiety behaviors [158] and stress responses such as hyperphagia or binge-eating [160–162]. This anxiety-related condition will likely then feed into the generation of metabolic dysfunction via metabolic or diabetic syndromes. While impulsive behavioral responses in response to stress are seen with food, there is also considerable evidence that this stress-induced activity also includes augmented alcohol-seeking activity [163].

3.3. Schizophrenia

Our recent work has begun to provide evidence for the aging-related control of schizophrenia and schizophrenia-related conditions [164–166]. Relaxin ligands, as well as RXFP3 itself, have been suggested by some researchers to be implicated in schizophrenia-related conditions [167,168]. Again, demonstrating the metabolic basis of aging, it has been shown that relaxin-3, RXFP3, and RXFP4 polymorphisms have been linked to metabolic disruptions in patients treated with antipsychotic medications [169]. Schizophrenia and other affective conditions are typified by periods of mania and heightened activity states, and it has been demonstrated that cognitive arousal states can also be strongly affected by RXFP3 activity in experimental animal models [144]. In regard to the potential for a specifific RXFP3-GIT2 signaling axis in the aging process, it is interesting to note that epigenetic modifications (hypermethylation) of GIT2 have recently been shown by the creation of schizophrenic differential methylation networks (SDMNs) from schizophrenia patient data [170]. The specifific effects of this modification of GIT2 in this paradigm has, however, yet to be shown [164]. 

3.4. Obesity and Metabolic Dysfunction

Multiple experimental animal and longitudinal studies have demonstrated that diet-induced obesity promotes pro-aging phenotypes [84,97,171,172]. A considerable component of the obesity-based drive of aging likely results in alterations in insulin sensitivity as well as the drive towards alternative sources of energy, such as lipid- or protein-mediated metabolism that can incur a greater level of oxidative stress [173–175]. 

RXFP3 expression and activity have been shown to be closely associated with both eating behavior alterations [161,176] as well as the physiological responses to augmented food intake [160,177–179]. In many of these experiments, it has been noted that the role of RXFP3 in these scenarios is more pronounced in females compared to males [177]. Concordant with this, it has been shown that female RXFP3 knock-out mice present with more heightened anxiety behavior than male RXFP3 knock-out mice in assessments of anxiety, such as the elevated plus maze. Hence, male RXFP3 knock-out mice spent more time in the open arms of the maze indicating their lower levels of anxiety than their female RXFP3 knock-out counterparts [144]. Experimental animals fed a high fat/glucose diet (a common mechanism to accelerate metabolic aging) displayed signifificant alterations in the CNS expression of RLN3 and RXFP3 [178]. These diet-induced obese (DIO) male rats displayed signifificantly higher levels of RLN3 expression compared to the control diet ad libitum-fed animals. This increased expression of RLN3 in DIO rats likely engenders the hyperphagic condition found in this experimental cohort. This study found that during a metabolic challenge of refeeding after food deprivation, the DIO rats only exhibited an increased expression of RXFP3 receptors in brain regions involved in food intake regulation [178]. With respect to the links between the RLN3/RXFP3 system and human obesity, it has been shown that RLN3 genetic polymorphisms are signifificantly associated with traits including obesity, hypercholesterolemia, and diabetes [169]. The intersection of RLN3/RXFP3 signaling between stress-responsive binge eating and this greater role of RXFP3 in predisposition to obesity demonstrates the importance of this system in the control of neurometabolic dysfunction in the aging context. Given these associations, considerable activity has since focused on the development of RLN3-based interventions for obesity paradigms [162,179,180].

3.5. Ischemic Stroke

Aging is considered to be one of the strongest independent risk factors for ischemic stroke-based injuries [181,182]. Hence, almost three-quarters of all strokes occur in people aged ≥65 years. Ischemic damage is cellular destruction associated with altered nutrient or oxygen support–resulting in energy deprivation and ROS-based damage. A recent study reported that relaxin peptides can protect tissues from ischemic damage. Using a rat stroke model, it was demonstrated that RXFP3 activation (using RLN2 and RLN3) reduced the extent of cellular/tissue damage induced by the application of vascular ligation [53]. In this study, it was reported that the ability to reduce the size of infarcts induced by transient middle cerebral artery occlusions was primarily mediated by selective activation of RXFP3. In addition to this, RXFP3 stimulation also demonstrated the capacity to reduce the damaging effects of oxygen and glucose deprivation in cellulose-cultured primary astrocytes. 

3.6. Reproductive Aging

The control of reproductive behavior is tightly linked to the functional cellular/tissue mechanisms associated with energy metabolism and food availability [59,98,122,183–185]. As reproductive behavior and physiology are tightly controlled at certain points in the lifespan, it is not surprising that the broader relaxin system likely intersects with this aging–reproduction nexus. The role of relaxin in the reproductive process is one of the best-studied aspects of its molecular biology [186–188]. In a recent study that investigated the effects of premature defects in the female reproductive system (i.e., ovariectomy) it was found that in areas of the brain that demonstrated dysfunctional network connectivity, there was a signifificant alteration in the levels of both RXFP3 expression and its potential preferred partner, GIT2 [122]. Thus, it is likely that this receptor system [10] can also form a functional bridge between the aging process and the reproductive system. 

3.7. Alcohol Abuse

Alcohol use disorders are a leading cause of preventable deaths worldwide. Sobering patients often experience alcohol use relapses in times of physical and psychosocial stress. Both RLN3 and RXFP3 have been shown to modulate stress-induced relapse to alcohol seeking in rats. The amygdala is one of the most crucial areas of the CNS that controls this pathobiology. The central nucleus of the amygdala (CeA) in the rat receives an RLN3 innervation and possesses considerable levels of RXFP3 expression. In addition to this, the CeA receives considerable input from corticotropin-releasing factor (CRF) neurons demonstrating a functional intersection between stress and this activity of the RLN3/RXFP3 system. In this specifific scenario, it is thought that CeL (lateral CeA) CRF neurons provide both local inhibitory GABA and excitatory CRF signals to the CeA neurons [189]. 

As discussed previously, alcohol-seeking behavior can be a major component of PTSD/anxiety phenotypes [190]. Recent research has also indicated that the response to alcohol intake is also affected by the age of the afflicted individual [191]. As we have contended that these stress-related conditions are potentially driven by metabolic disruption, it is unsurprising that recent research has started to propose that alcoholism behavior is also linked to pathological aging [192–194]. Recent evidence has indicated that alcoholism can even lead to Alzheimer-like conditions that have a strong neuroinflammatory component [195]. Excessive and inappropriate alcohol abuse results in the generation of multiple co-morbidities, including neurodegenerative atrophy, dysfunctional immune responses, and accelerated or premature aging [194,196]. One of the better-studied functions of RXFP3 has been its regulatory capacity in alcohol-seeking behavior [26,163]. In contrast to the RXFP3-based actions on feeding behavior [177], it has been shown that only male RLN3 knock-out animals showed an increase in alcohol preference [197]. Thus, it is possible that RXFP3-based therapeutics could be a potential future target for the treatment of alcoholism. 

4. Conclusions 

Aging is one of the largest risk factors for nearly every type of major mortality-causing disease in the world today. Therefore, tractable mechanisms for controlling this highly complex process are urgently required as a molecular target for intervention. The therapeutic interdiction of the aging process is currently one of the most studied therapeutic areas. Interventions in the aging process often fall into either lifespan extension strategies or damage/disease reduction strategies. While lifespan extension is an interesting goal [198] interventions that seek to reduce the accumulation rates of damage [199,200] may be more likely to impact medicine more immediately. With respect to lifespan extension, one of the most studied current modes of intervention is the cellular rejuvenation process. In this context, it is often proposed that through selective genetic modulation of longevity-regulating factors a reversal of aging-related damage can be achieved [198,201]. Several prominent reports have indeed suggested that reversal of aging damage can occur, e.g., in vivo ectopic expression of three (Oct4, Sox2, Klf4) of the four Yamanaka reprogramming factors [202] was able to promote axon regeneration after previous eye injury [203] and also stem multiple aspects of the aging-related disease such as renal failure, cardiomyopathies, and diabetic conditions [201]. While generating dramatic results these interventions are still at the experimental animal phase and will be unlikely to transition to the human stage soon. 

Lifestyle (e.g., exercise) and dietary interventions (e.g., caloric restriction) have been shown to be effective in slowing down the molecular aging process [95,98,99,204,205] in controlled experimental conditions, however, the adherence of human patients to these is often poor and difficult to maintain for long periods of time [206]. Exercise and caloric restriction are proposed to exert beneficial effects through natural augmentations of cytoprotective systems through the introduction of mild stress. An alternative method to induce this stress is via controlled exposure to other stressors such as heat, cold, or mild irradiation [207–209]. While interventions such as caloric restriction and exercise are still problematical for adherence, these even more drastic mild stressors are much less likely to be accepted by a clinical audience.

In addition to genetic or lifestyle interventions the use of polypharmacological natural compounds, e.g., quercetin or resveratrol [171,210], has gained signifificant interest in recent years but has often stalled following the transition from experimental conditions to more clinical settings [211,212]. It is likely that the complex polypharmacological actions of these natural agents could be the issue with respect to this transition, as many of these effects may be specifific to smaller groups of patient populations and may also be highly influenced by diet and compound metabolism variation. The concept of tackling complex disorders, e.g., pathological aging, in a polypharmacological manner, may indeed be a good strategy as many systems may need remediation. The natural compounds may indeed exert systemic beneficial effects, but as that could be through a variety of molecular targets a coherent response profile in patients may be hard to obtain. A pragmatic approach may be to identify natural receptor signaling systems that present an ability to interdict pathological aging at a systemic level, e.g., the RXFP3/RLN3 system, especially when combined with the GIT2 signaling paradigm. Our research, as well as that of others, has identified RXFP3 as a potentially crucial factor in controlling both the classical hallmarks of molecular aging and the etiological process of multiple forms of aging-associated disease (Figure 2). Moreover, our research outlined here also indicates that there are multiple points of intersection between the RXFP3 signaling paradigm and molecular signaling mechanisms of aging-associated disease. We have previously shown that RXFP3 possesses a strong functional relationship with the aging keystone, GIT2. Thus, this synergistic relationship presents as a completely novel therapeutic mode for attenuating aging pathology in a multidimensional manner. In this intervention avenue, there is a strong systemic anti-aging component combined with the capacity to generate more selective and specifific molecular intervention, compared to naturally occurring compounds. Hence, this approach is a form of engineered polypharmacology. To further advance this research it would be interesting to generate signal-selective compounds that target the RXFP3 in a manner that specifically stimulates the RXFP3 to generate GIT2-dependent signaling outputs. This agent could then be introduced to ex vivo or in vivo experimental paradigms to demonstrate the capacity of such agents to ameliorate aging-associated damage and also aging-induced disease phenotypes.

Figure 2. The human RXFP3 receptor is involved in multiple disorders associated with dysfunctional aging. Alterations in the activity and expression of the human RXFP3 receptor have been shown by multiple research teams to play a pivotal role in the disease processes depicted. The participation of RXFP3 in these disorders indicates a role for perturbed natural aging signaling mechanisms in these conditions. Hence, it is likely that further investigation of the diverse signaling capacity of RXFP3 may help generate novel therapeutics for these conditions that work via altering the rate of aging in these disorders. 

Conflicts of Interest: The authors declare no conflict of interest. 

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