Over the last couple of decades, general knowledge on the biological role of neuropeptides in the central nervous system (CNS) has increased substantially. Currently, more than a hundred different neuropeptides have been described in the CNS, most of which are involved in the modulation of different brain functions [1, 2, 3, 4, 5]. Neuropeptides are small, amino acid-based molecules that can influence neuronal activity, neuro-immune responses and whose dysregulations have been implicated in the pathogenesis of several mental illnesses such as Alzheimer’s or Parkinson’s disease, depression, anxiety, stroke, migraines, epilepsy and multiple sclerosis [6, 7, 8, 9, 10, 11, 12, 13, 14, 15].

It has become increasingly clear that certain neuropeptides such as neuropeptide Y (NPY), somatostatin, calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) exert anti-inflammatory effects in the CNS [16]. This has resulted in research focusing on these neuropeptides as potential therapeutic targets for the treatment of neuroinflammatory diseases [17, 18, 19, 20, 21].

The neuropeptides pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are widely distributed throughout the CNS and the peripheral nervous system (PNS) and are involved in neuroprotection and immunomodulation [22, 23, 24, 25, 26, 27]. The activities of PACAP and VIP are mediated by three G protein-coupled receptors (GPCRs), namely PAC1, VPAC1 and VPAC2 [28] (Fig. 1).

Fig. 1.

PACAP and VIP and their downstream effects. A schematic overview of the binding of VIP and PACAP to the PAC1, VPAC1 and VPAC2 receptor with its main down-stream effects.

PACAP binds with high affinity to both PAC1, VPAC1 and VPAC2 receptors and its activity is believed to be predominantly neuroprotective [29, 30, 31]. For example, PACAP can prevent neuronal cell death after ischemia [32] and can promote axonal regeneration after spinal cord injury [33]. In contrast, VIP binds less efficiently to PAC1 receptors, whereas its exhibits similar high affinities for VPAC1 and VPAC2 receptors as PACAP [34]. The latter two receptors’ activities are thought to be mainly associated with immune modulatory roles in the CNS as well as in peripheral organs [35, 36, 37]. Given VIP binding preference towards VPAC receptor subtypes, this peptide has emerged as a potential anti-inflammatory target to treat multiple sclerosis (MS) and perhaps, other inflammatory diseases [38, 39]. For example, in human rheumatoid arthritis (RA), VIP treatment downregulated chemokines production and interleukin-6 (IL-6) and decreased the levels of other pro-inflammatory mediators in RA patients [40]. Additionally, a single intracerebroventricular injection of VIP was able to attenuate microglial activation and prevented neurodegeneration in animal models of Parkinson’s disease [41].

Recent studies have demonstrated both a higher incidence and prevalence of comorbidities in MS patients compared with the healthy population [78, 79]. MS comorbidities often appear in people suffering from concurrent vascular and/or metabolic diseases, as well as certain neurological and psychiatric disorders [80, 81, 82, 83, 84]. Epidemiological studies in MS patients have demonstrated that afflicted people also have a higher chance of developing epilepsy [83], migraines [84], as well as affective/emotional disturbances such as depression or anxiety [79]. In view of the comorbidities often seen in MS patients and the critical role of neuropeptides in many pathological domains of these comorbidities, exploring the mechanisms and the extent at which both PACAP and VIP peptides can contribute to ameliorate the comorbidities of MS is becoming a hot topic. For this purpose, this review will summarise literature on the role of neuropeptides, focussing on the role of VIP and PACAP in the neurological comorbidities of MS, and how these neuropeptides could contribute to improve the clinical presentation of MS and disease course.

MS is a devastating disease that comes with physical as well as psychological hardship, which may ultimately lead to the development of mood disorders or facilitate its onset in vulnerable people. One of the most prevalent comorbidities seen in MS patients is depression (23.7%), followed by anxiety (21.9%) and bipolar disorder (BD; 5.83%) [79]. Interestingly, these disorders are all associated with neurochemical evidence of CNS inflammation, supporting a pathological link with MS [12, 85, 86, 87, 88]. Moreover, depression, anxiety and BD are all known to be influenced and triggered by stress, a risk factor able to also affect oligodendrocyte’s health [89, 90, 91, 92, 93]. Here, the role of PACAP and VIP in depression, anxiety and BD is highlighted.

Several neuropeptides such as NPY, somatostatin, galanin and orexin, at different extents, have been implicated in the pathology of affective disorders and have been suggested as potential therapeutic targets [21, 94, 95, 96, 97, 98, 99, 100, 101]. The VIP/PACAP system has also been implicated with depression and anxiety, although there are some conflicting findings. In some studies using PACAP-deficient mice, PACAP gene ablation increased depressive-like and anxiety-like behavior under stress conditions [102, 103, 104], whereas in a study by Lehmann and colleagues it was found that PACAP${}^{-/-}$ mice had reduced anxiety and did not develop depressive-like behaviors [105]. However, this study used social defeat to trigger chronic stress whereas the other studies either used naïve mice or mice exposed to a milder form of stress. Thus, PACAP${}^{-/-}$ mice might exhibit a whole spectrum of behavioral disorders, depending on the initial source of stress, type and duration. In a clinical study, VIP serum levels negatively correlated with depression and anxiety state and positively correlated with brain volume of the left amygdala [106]. Similarly, decreased serum levels of VIP were detected in a rat depression model [107]. Moreover, single nucleotide polymorphisms (SNPs) in the VIPR2 gene and VIP gene were found to be associated with unipolar major depression and BD, respectively [108]. In addition, a VIP injection into the CA1 region of the hippocampus of rats showing anxiety-like behaviors attenuated the symptoms [109], although, for unexplained reasons, VIP antagonist was not able to abolish VIP-mediated behavioral improvements.

Corticosterone, one of the hormones produced by the adrenal gland as part of the hypothalamic-pituitary-adrenal axis (HPA-axis), is known to play a critical role in the development of depression, anxiety and bipolar disorder [92, 110, 111]. Under stress conditions, PACAP-deficient mice exhibited an attenuated corticosterone response, which occurred irrespectively of the development of depressive-like behaviors [105, 112]. Moreover, PACAP injections into the amygdala and bed nucleus of the stria terminalis (BNST) have been found to reliably increase corticosterone levels and anxiety-like behaviors in rodents [113, 114, 115]. Interestingly, under chronic stress conditions there was a region-specific increase of PACAP and PAC1, but not VIP and VPAC1-2 levels in the BNST [116]. VIP has also been implicated in the control of glucocorticoid hormones release, although in a work focused on studying its effects in relationship to circadian rhythmicity [117]. Thus, there appears to be a link between PACAP, stress and the HPA axis, which could be relevant for the development of certain affective disorders.

In summary, the VIP/PACAP system appears to be a key player in the modulation of mood and other affective disturbances. Each peptide exerts intrinsic regulatory functions in brain homeostasis and it is not surprising that dysfunctional regulation of PACAP or VIP in specific brain regions or cell populations may be critical for the development of conditions such as depression, anxiety and bipolar disorder. Future research is warranted to explore if targeting this neuropeptide system can be used as an effective therapeutic strategy to treat mood disorders.

Psychotic disorders encompass a broad range of mental illnesses such as schizophrenia, affective psychosis, delirium and drug-induced psychosis. Approximately 2–4% of MS patients have reported to experience psychotic episodes at some point during the course of the disease, which is a considerably higher rate than in the general population [118]. In line with MS pathology, neuroinflammation and increased oxidative stress events in the CNS are pathogenic events that are associated with the occurrence of certain psychotic disorders [119, 120].

Several neuropeptides have shown to activate signalling pathways that are implicated in the genesis of psychosis. Clear associations have been found between schizophrenia and neuropeptide Y, neurotensin, somatostatin and oxytocin and the number of psychotic episodes (reviewed in [121, 122]). Moreover, a genetic link between neuregulin-1 (NRG1), cholecystokinin A and schizophrenia has been described [123, 124, 125, 126, 127]. Similarly, genetic polymorphisms of genes encoding PACAP peptide or its receptors have also been correlated with schizophrenia [128, 129, 130, 131, 132], although for the former gene target (PACAP gene, aka Adcyap1), a replication study failed to reproduce the same findings [133].

There are certain indications that neuroinflammation can lead to the development of schizophrenia and psychotic episodes. In schizophrenia and first-time psychosis patients, studies have reported increased levels of pro-inflammatory cytokines and decreased levels of the anti-inflammatory cytokine IL-2 in the CNS [134, 135]. Additionally, increased microglia activation has been observed in recent-onset schizophrenic patients [136, 137]. However, since no comprehensive animal model exists for psychiatric diseases, reliable strategies to study neuroinflammation at a molecular level in psychiatric illnesses remains a daunting task. Furthermore, since neuroinflammation in schizophrenia is a relatively novel concept, the exact mechanisms through which it could contribute to disease aetiology remain uncertain. However, it would be interesting to explore if the anti-inflammatory activities of the VIP/PACAP system could potentially attenuate the CNS inflammation seen in people with psychotic disorders.

There is a leading hypothesis featuring schizophrenia as a neurodevelopmental disorder [138, 139]. In post mortem brain tissue from schizophrenic patients, the pool of neural stem cells (NSC) is reduced, suggesting decreased NSC proliferative activity [140]. Moreover, schizophrenia is characterised by abnormal connectivity among brain regions and axonal abnormalities [141, 142, 143]. A noteworthy link between brain development, schizophrenia and white matter can be seen for NRG1. NRG1 signals through disrupted-in-schizophrenia 1 (DISC1) and interestingly, PACAP has been found to affect DISC1 signalling as well [144]. In addition, both NRG1 and DISC1 are involved in neuronal migration, axon ensheathment and oligodendrocyte maturation [145, 146], suggesting a role for PACAP in modulating NRG1-mediated activities. Moreover, PACAP, PAC1 and DISC1 are known to be essential components of the cellular machinery that regulates neurite outgrowth [144, 147, 148]. PACAP stimulated NSC proliferation in mice and prevented the reduction of NSCs in a ketamine-induced schizophrenia-like mouse model via PAC1 receptor activation [149, 150].

Taken together, these data suggest that PACAP (and perhaps other PACAP/VIP receptor agonists) could aid in the treatment of schizophrenia. Based on the reported pro-mitotic activities of PACAP in NSCs, it is reasonable to hypothesise that PACAP treatment could aid in replenishing the depleted pool of NSCs in the brain of schizophrenic patients, hence promoting neurogenesis. Additionally, it could reduce the chronic CNS inflammation that seems to also contribute to the development of psychotic disorders. Whilst additional investigations into this topic are still needed, there is already some indication that the beneficial actions of certain neuroleptic drugs can occur by mechanisms that involve restoring the dysfunctional VIP/PACAP signalling in the brain [151].

Epilepsy is a chronic neurological disorder that is characterised by recurring and unprovoked seizures [152]. Seizures occur when the balance between excitatory and inhibitory signals in the brain is disrupted [152]. Epilepsy is one of the most common neurological disorders that is disproportionately prevalent in MS patients compared to the general population [153]. It has been reported that the prevalence of epilepsy is six times higher in MS patients than in the healthy population [154]. This is not surprising, as both lesions, inflammation and neurotransmitter imbalances within the CNS of people with MS may trigger such disabling ailment, with evidence also suggesting that the frequency of seizures tends to increase as the disease progresses [155].

As mentioned, an imbalance in neuronal activity is a critical neurochemical feature of seizure episodes [152]. It is well-documented that neuropeptides can contribute to reset this imbalance, including PACAP and VIP [156, 157]. Despite the emerging evidence, to date only one neuropeptide-based hormone, adrenocorticotropic hormone (ACTH), is currently being tested in clinical trials for the treatment of seizures in a rare disease that affects infants (West syndrome), whereas investigations on the efficacy of other neuropeptides in epilepsy has been limited to preclinical studies [152, 158].

PACAP and VIP are released during high neuronal firing activity [159, 160]. VIP exerts an overall excitatory effect on synaptic transmission, which is mediated by VPAC1 and VPAC2 receptors [161, 162]. In hippocampal surgical samples of patients suffering from human temporal lobe epilepsy, both VPAC1 and VPAC2 receptors were shown to be up-regulated [15] and up-regulation of VPAC receptor subtypes has been associated with increased neuronal survival [161]. These data support the idea that hippocampal VPAC receptors are increased as a homeostatic mechanism to prevent excessive neuronal damage/death caused by epileptic episodes.

Research into the role of PACAP in epilepsy has been centred on its ability to modulate microglia and glutamate transmission [163, 164, 165]. The expression of PACAP increases after kainic acid-induced seizures in rats, with many suggesting that this seizure-induced increase in PACAP may help to reduce excitotoxicity and promote overall neuroprotection to protect the CNS from damage [163]. Specifically, PACAP acts on microglia to promote the release of anti-inflammatory factors that polarise microglia towards an anti-inflammatory phenotype, whilst concurrently increasing the expression of glutamate transporters that promote glutamate re-uptake, resulting in two parallel protective mechanisms [166, 167].

Magnetic resonance imaging and pathological studies have proposed that cortical inflammation, demyelination and grey matter damage in MS patients may be responsible for the onset and development of epileptic seizures [155]. However, epilepsy is an active process, so it is difficult to determine if any aspect of MS pathology may specifically promote or trigger seizure episodes, especially given that MS patients often present with unique pathogenic profiles and distributions of lesion within the CNS. This should be considered as a further incentive to invest more in researching the efficacy of PACAP and VIP as therapeutic targets for epilepsy and MS, as both conditions are often comorbid and targeting these peptides or their receptors may prove to be effective in ameliorating epilepsy associated to MS.

Stroke is the leading cause of adult disability, with one in four people globally experiencing a stroke event in their lifetime [168]. Stroke occurs when there is a long-lasting interruption or severe reduction of cerebral blood flow, which triggers a cascade of pathological events including excitotoxicity, oxidative stress, blood brain barrier (BBB) leakage and neuronal cell death [169]. Inflammatory processes, including the autoimmune activities of MS are thought to contribute to endothelial dysfunction and atherosclerosis, which may promote the development of micro- and macro-vascular alterations that culminate in ischaemic or hemorrhagic stroke. As such, it should not be surprising that compared with the general population, people with MS are at increased risk of experiencing a stroke, and if they do, they tend to suffer more severe symptoms [170].

It has been shown that administration of PACAP in animal models of stroke is neuroprotective and causes a reduction of both neurological deficits and the degree of pathological change of the CNS tissue of the ischaemic brain area [26]. There is an increase in inflammation post-stroke [13, 171], and PACAP and VIP are well-known anti-inflammatory agents in the CNS [10]. Both peptides are expressed in different types of immune cells, including microglia and astrocytes [37, 172]. Masmoudi-Kouki et al. [173] suggested that the neurotrophic and neuroprotective effects of PACAP and VIP can be partly accounted for by their activities on astrocytes. This idea is supported by the increase in astrocytic PACAP and PAC1 expression immediately following cerebral ischemia [174]. Additionally, exposure of cultured astrocytes to PACAP was found to be capable of up-regulating glutamate uptake via PAC1-mediated signalling, suggesting that the PACAP-PAC1 axis can reduce post-stroke excitotoxicity caused by excessive glutamate release [167].

In a study, treatment with VIP has shown to reduce brain damage and to promote neurogenesis following ischemic injury in the rat brain [175], although it should be highlighted that most of the neuroprotective effects reported for experimental stroke are related to PACAP and not VIP. Indeed, studies in PACAP knockout mice suggest that the peptide prevents post-ischemic neuronal cell death [176, 177]. In animal models of stroke, intranasal administration of PACAP reduced infarct volume and improved functional recovery [178]. Additionally, PACAP-dependent polarisation of microglia towards an anti-inflammatory phenotype resulted in improved functional recovery in mice post-ischemic mice [179].

These studies provide evidence that both PACAP and VIP have potential therapeutic validity for the treatment of stroke, and may find application to aid in the recovery of neuronal injury post-stroke due to their neuroprotective and anti-inflammatory functions.

Neuropathic pain is defined as pain caused by a damage or disease to the somatosensory nervous system [180]. WHO defines MS as one of the main CNS diseases responsible for the development of central neuropathic pain, followed by traumatic causes and other conditions such as spinal cord injury, traumatic brain injury and stroke, with neuropathic pain reported by about 86% of MS patients [181]. Neuropeptides are key regulators of peripheral nociception and contribute to the mechanisms regulating central sensitisation to pain. In many cases, chronic inflammation, ion channel imbalance and a lack of inhibition in the dorsal horn of the spinal cord partake in maintaining pain sensation [182]. PACAP and VIP are expressed across key anatomical regions/structures that are important in somatosensory processing and the transmission of pain signals [183]. In the PNS, these includes the dorsal root ganglia and peripheral nerves, whereas in the CNS, these peptides are detected in regions that process pain sensation and the associated emotional load, including the thalamus, periaqueductal grey (PAG), parabrachial nucleus and amygdala [22, 184, 185]. Despite the anatomical relevance of PACAP and VIP in neuropathic pain pathways, their distinct roles in the pathophysiology of neuropathic pain remains to be clarified. Studies using PACAP knockout mice have indicated that the peptide plays an excitatory role in pain transmission, as knockout animals do not develop symptoms of neuropathy after a spinal nerve transection, although they retain normal nociceptive responses [186]. These findings have been supported by subsequent studies showing that PACAP up-regulation in the dorsal horn is necessary for spinal sensitation and the development of neuropathic pain [187].

There is evidence to indicate that intrathecal injections of PACAP in mice cause both hyperalgesia and allodynia, two common symptoms of neuropathy [188]. In another study, intrathecal PACAP caused prolonged allodynia that was associated with sustained astrocytic activation [189]. Administration of a PAC1 receptor antagonist inhibited PACAP- and nerve injury-induced allodynia, suggesting a crucial role of PACAP-PAC1 interaction in the induction of neuropathic pain [190].

The overlap between pain and inflammatory signalling has been extensively studied and reviewed [189]. Additionally, an association between axonal regeneration and pain has been hypothesised, with evidence suggesting that accelerated regeneration may ameliorate pain of nerve origin [182]. PACAP and VIP have well-described roles in promoting axonal regeneration after peripheral nerve injury [34, 191]. For example, RNA sequencing data revealed the ADCYAP1, the gene encoding for the PACAP peptide, was the most differentially expressed gene associated with post-surgical nerve regeneration in patients with carpal tunnel syndrome [192].

Most studies have suggested PACAP has a dominant role in pain pathophysiology compared to VIP. However, Dickinson and collaborators identified distinct regulatory roles of spinal PAC1 and VPAC receptor subtypes in animals with experimentally-induced neuropathy [193], also implicating the VIP-VPAC axis as a further contributor to pain-related regulatory responses. Centrally, PACAP activity in the amygdala has been linked with the emotional expression of pain [185], whereas VIP expression in the PAG has been correlated with the development of co-morbid behaviors in nerve-injured rats [22]. Nonetheless, taken together these data pinpoint the critical role of the PACAP/VIP system in perpetuating both spinal and supraspinal pathways that are involved in the transmission of pain originating from peripheral nerve damage as well as its emotional components. With this in mind, it is conceivable that strategies aimed at blocking PACAP/VIP signaling may be beneficial in mitigating neuropathic pain, a disabling comorbid event associated with MS.

Migraine is a complex and debilitating headache disorder that affects one in seven people worldwide [194]. Despite clinically manifesting as recurrent attacks of headache, migraines are associated with a range of symptoms (i.e., nausea, vomiting, and extreme sensitivity to light and sound) and are linked to other conditions like depression, anxiety, sleep disorders, chronic fatigue and cognitive dysfunction [195]. The role of neuropeptides in migraine in an active area of research. The most studied neuropeptide in relation to migraine is CGRP [196]. CGRP administration has been shown to induce migraine-like headaches that are indistinguishable from spontaneous migraine attacks [197, 198]. This has led to the discovery and use of CGRP antagonists that are effective in treating migraine symptoms [198].

PACAP has been shown to act in a similar way to CGRP, with infusion of PACAP inducing headaches in healthy volunteers [199]. Similarly, plasma levels of CGRP and PACAP are elevated following a migraine attack [200]. Interest in PACAP has a therapeutic target for migraine, stems from the discovery that PACAP-induced migraine did not results in an increase in CGRP, suggesting an alternate route of migraine initiation [201]. Human provocation studies have provided the most robust evidence describing the involvement of PACAP in migraine [201]. Intravenous administration of PACAP produced immediate headache in healthy participants, and delayed migraine in migraine sufferers [203]. Additionally, PACAP induced pronounced dilations of extra-cranial arteries [202]. This dilation was further confirmed using magnetic resonance angiography that caused dilation of the middle meningeal artery in participants who reported migraine after PACAP infusion [203].

Despite sharing similar anatomical distribution and vasodilation properties to PACAP, the role of VIP in migraine remains controversial. Previously, it was suggested that VIP was unable to induce migraine [188]. Additionally, Bertels and colleagues, investigated the impact of PACAP and VIP on functional brain connectivity using functional magnetic resonance imaging, and revealed that PACAP, but not VIP, altered connectivity that coincided with the development of migraine in migraine patients [201]. However, recent studies have reported contrasting findings. Pellesi and colleagues, demonstrated that a 2-hour infusion of VIP could induce migraine attacks in 71% of patients with migraines without aura, similarly to PACAP [204]. They revealed that this induction was caused by dilation of cranial arteries that was mediated by VPAC1 and VPAC2 receptors, contradicting previous notions that migraine was induced via PAC1 receptor activation [204]. This could explain why novel therapeutics targeting PAC1 have had low efficacy in preventing migraine attacks in clinical trials [201, 205, 206]. Therefore, more research is needed to elucidate the specific role of each receptor in migraine induction and maintenance. For example, recent studies have proposed a link between mast cell degranulation and migraine pathology in PACAP-induced attacks, however the receptor mediating this effect has not been identified [207]. Chronic inflammation has been shown to promote the maintenance of pain states. As such, both migraine and neuropathic pain demonstrate the delicate balance of targeting PACAP and VIP in neurological disease due to their pleiotropic functions and global expression.