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Postulated vasoactive neuropeptide immunopathology affecting the blood-brain/blood-spinal barrier in certain neuropsychiatric fatigue-related conditions: A role for phosphodiesterase inhibitors in treatment?

Neuropsychiatric symptoms occur in a number of neurological fatigue-related conditions including multiple sclerosis (MS), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and chronic fatigue syndrome (CFS). These conditions have been attributed variably to neuroinflammatory and neurodegenerative processes. While autoimmune pathology, at least in part, has long been suspected in these conditions proof has been elusive. Autoimmune pathomechanisms affecting the blood–brain barrier (BBB) or blood–spinal barrier (BSB) may predispose the BBB/BSB to ‘leakiness’ and be a precursor to additional autoimmune events resulting in neuroinflammatory or neurodegenerative processes. The aim of the paper is to postulate immunopathology of the cerebrospinal perivascular compartment involving certain vasoactive neuropeptides, specifically pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP), in the etiology of certain neuropsychiatric fatigue-related conditions such as MS, ALS, PD, and CFS. Vasoactive neuropeptides (VNs) such as PACAP and VIP have critical roles as neurotransmitters, vasodilators including perfusion and hypoxia regulators, and immune and nociception modulators. PACAP and VIP are widely distributed in the central nervous system (CNS) and have key roles in CNS blood vessels including maintaining functional integrity of the BBB and BSB. Autoimmunity affecting these VNs would likely have a detrimental effect on BBB and BSB functioning arguably predisposing to further pathological processes. Virchow–Robin spaces (VRS) are perivascular compartments surrounding small vessels within the CNS which contribute to the BBB and BSB integrity and contain PACAP and VIP receptors. Autoimmunity of these receptors would likely affect BBB and VRS function and therefore may contribute to the etiology of these conditions by affecting CNS and immunological homeostasis, including promoting neuropsychological symptomatology. PACAP and VIP, as potent activators of adenylate cyclase (AC), have a key role in cyclic adenosine monophosphate (cAMP) production affecting regulatory T cell (Treg) and other immune functions. Phosphodiesterase enzymes (PDEs) catalyze cAMP and PDE inhibitors (PDEIs) maintain cAMP levels and have proven and well known therapeutic benefit in animal models such as experimental allergic encephalomyelitis (EAE). Therefore PDEIs may have a role in therapy for certain neuropsychiatric fatigue-related conditions.


Introduction
Neuropsychiatric symptoms occur in a number of neurological fatigue-related conditions including multiple sclerosis (MS), 1 Parkinson's disease (PD), 2

amyotrophic lateral
Staines et al sclerosis (ALS) 3 and chronic fatigue syndrome (CFS). 4 While autoimmune pathology, at least in part, has long been suspected in these conditions proof has been elusive. The present paper asserts a provocative hypothesis that autoimmune pathomechanisms affecting the blood-brain barrier (BBB) or blood-spinal barrier (BSB) may predispose the BBB/BSB to 'leakiness' and be a precursor to additional autoimmune events resulting in neuroinfl ammatory or neurodegenerative processes, compounded possibly on a genetically susceptible background or exposure to environmental factors.
The paper examines the potential role for vasoactive neuropeptides (VNs) such as pituitary adenylate cyclaseactivating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) in the possible autoimmune etiology of these disorders through effects on the blood-brain/spinal barriers (BBB/BSB) whereby neuropsychiatric symptomatology may occur. PACAP and VIP, as potent activators of adenylate cyclase (AC), have a key role in cyclic adenosine monophosphate (cAMP) production affecting BBB/BSB function along with regulatory T cell (Treg) and other immune functions.
Phosphodiesterase enzymes (PDEs) catalyse cAMP and PDE inhibitors (PDEIs) maintain cAMP levels and have proven and well known therapeutic benefi t in animal models such as experimental allergic encephalomyelitis (EAE). Therefore PDEIs may have a role in therapy for certain neuropsychiatric fatigue-related conditions.

Functions of PACAP and VIP
PACAP and VIP are widely distributed in the central (CNS) and peripheral (PNS) including autonomic (ANS) nervous systems and peripheral tissues including heart, lung, pancreas, adrenal gland, gonads, and gastrointestinal tract as well as immune cells and lymphatic system. 5,103 PACAP and VIP have critical roles as neurotransmitters, vasodilators including perfusion and hypoxia regulators, and immune and nociception modulators. They have key roles in blood vessels in the CNS 84 and VIP is associated with maintaining functional integrity of the BBB. 83 PACAP and VIP infl uence regulatory T cell (Treg) 29 and other immune functions. Their role as antiinfl ammatory modulators is of particular interest 89 considering the implications for loss of immune and infl ammatory regulation should they fail, for example through autoimmunity of their receptors. PACAP and VIP are potent activators of AC and thus have a key role in cAMP production.
Virchow-Robin spaces (VRS) are perivascular compartments surrounding small vessels within the CNS which contribute to BBB and BSB integrity and modulate immune responses. 90 VRS contain microglia and these cells are known to be infl uenced by PACAP and VIP in immunoregulation. 8 Autoimmunity of these VNs or their receptors would be expected to affect BBB/BSB and VRS function and therefore may contribute to the etiology of neuropsychiatric-related neurodegenerative and other conditions by affecting CNS and immunological homeostasis.
Autoimmunity as an etiology in MS, ALS, PD, and CFS remains controversial. While autoimmunity is reasonably well established in MS, 88 an autoimmune etiology in PD, 85 which may involve the BBB, 86 and ALS 87 is only just emerging. Autoimmunity affecting PACAP and VIP has been postulated as a possible contributing factor in MS and ALS, 79 PD 80 and CFS. 81

Role of PACAP and VIP in the blood-brain/blood-spinal barrier
The multiple functions of PACAP and VIP impact on maintaining BBB/BSB function. Moreover PACAP and VIP have specifi c and complex roles in the CNS including high level neurological functioning such as memory and learning. 6 PACAP and VIP have a well described neuroprotective role 7 as well as being infl ammatory mediators in microglial activation, 8 therefore their failure may be linked to neuroinfl ammatory and other disease processes in the BBB/BSB and the CNS.
PACAP and VIP exert potent effects in metabolism as they have a vital role in cAMP production and regulation through AC activation. Immunological dysregulation of vital biochemical and/or epigenetic mechanisms affecting PACAP and VIP resulting in down-regulation of cAMP are possible pathways by which disease entities become manifest. Their role along with other neurotrophic factors, 9 in maintaining cAMP levels 10 are important for maintaining the integrity of the BBB and the BSB.
Conditions such as MS, PD, and ALS are increasingly being investigated for possible disruption of the BBB or the BSB. 11-13 Zlokovic 107 asserts BBB breakdown due to the disruption of the tight junctions may initiate and or contribute to a "vicious circle" of disease process resulting in progression synaptic and neuronal dysfunction and loss in disorders including PD, ALS, and MS. The BBB and the BSB are the main barriers between the brain and spinal cord parenchyma and the intravascular compartment and are therefore important in keeping immune hydrophilic macromolecules and neuroactive hormones from interfering with brain and spinal neurological processes. Generally the CNS parenchyma does not express MHCII molecules consequently the CNS has long been regarded as an immune privileged site. However certain cells such as pericytes and microglia within the BBB and BSB are able to express MHCII molecules on activation and initiate T helper cell (Th4) immunological responses. 66 PACAP and VIP suppress the expression of MHCII molecules 102 and this likely has an important role in maintaining the immuneprotected status of the CNS through maintaining the integrity of the BBB/BSB. Disruption of BBB and BSB are well known in certain pathological states, for example, injury and infl ammation and this disruption may involve VRS.
VRS contain interstitial fl uid and, while some functional contact with sub-arachnoid spaces may occur for solute exchange, there is doubt whether they contain CSF. 14 VRS have important connections with lymphatic drainage of the head and neck 15 as well as having intricate pial relations and providing a surface for activity of neuropeptides, hormones and cytokines. Pial cells may have a role in protecting the brain from exogenous catecholamines 16 and VRS may have a complex role in leukocyte recruitment across, and maintenance of the BBB. 17 They are a likely site for antigen presentation and antigen presenting cell (APC) engagement and MHCII recognition. Perivascular microglia in the VRS undertake APC recognition in the CNS. 18 Hence it is possible CNS infl ammation may be initiated within the CNS and not in the periphery alone. As PACAP and VIP and their receptors are located throughout the CNS parenchyma and vasculature they not only serve a role in immunomodulation they could also serve as antigens which might be subject to autoimmunity. Whether VN-or VN receptor-activated autoimmune Th cells arise from the periphery and undertake autoimmune processes in CNS and other sites is unknown and is an important question for further research.
PACAP and VIP are known to have neuroprotective effects through hypoxia protection on passage through the BBB 19 via a transport mechanism which enables the intact peptides to enter the parenchymal space of the brain. 20 Additionally PACAP and VIP have protective effects on neurons and glial cells. 21 These VNs therefore may have a signifi cant role in blood BBB/BSB function and likely assist in immune regulation of VRS in the brain and spinal cord. The present paper asserts that, in view of the many vital roles of PACAP and VIP in CNS neuroregulatory and immunological function including BBB function, autoimmunity to these VNs or their receptors will have signifi cant effects on homeostasis possibly resulting in neuropsychiatric symptoms.

PACAP and VIP in neuroimmunological dysregulation
PACAP and VIP exert antiinfl ammatory activities and loss of their function, for example through autoimmune compromise, could become manifest as unmodulated activation of immune responses. Hence effects of VN autoimmunity may extend beyond the BBB and BSB and a complex multisystem explanation for certain neuropsychiatric fatigue-related conditions is possible.
PACAP and VIP exert influence over inflammatory control mechanisms including infl uencing Th1 to Th2 shift and suppression of proinfl ammatory activity through the PKA/cAMP pathway. 23 For example tumor necrosis factor alpha (TNF-α) has a reciprocal modulating relationship with cAMP in vascular dysfunction involving endothelial cells 24 and this may prove to be an analogous mechanism in VRS. Implications for the BBB/BSB functioning are important as the VRS has been identifi ed as a location for immunoreactive lymphocytes in penetration of neuronal parenchyma. 25 Also VIP has been identifi ed in connection with neuronal function and VRS, which suggests that VIP may have a regulatory function associated with vasodilatation. 26 We assert that important immunoregulation occurs in perivascular spaces and VRS and that this may involve PACAP/VIP regulation. Many regulatory and antiinfl ammatory functions of PACAP and VIP are dependent on the Th2-directed cytokines 93 eg, interleukin-10 (IL-10) and IL-4 and these could be compromised in PACAP/VIP failure. Moreover 'leakiness' in the BBB and BSB may initiate, or encourage development of relapses in, neurological conditions such as MS. 27 Regulatory T cells (Tregs) function to control autoreactive T cells in the periphery 28 and possibly within the brain parenchyma. Treg function is substantially infl uenced by these VNs 29 and they may also have infl uence over Th17 direction. 30 Loss of Treg function in VRS will therefore have signifi cant implications for infl ammatory control. Moreover Th17 development occurs under IL-6 and transforming growth factor beta (TGF-β) infl uence 31 and this may be a key switching point from a protective Treg phenotype to an autoimmune Th17 phenotype. Certainly Th1 and Th17 ratios are important in brain and spinal infl ammation regulation. 32 Further, autoreactive Th cells within the CNS are likely to be critical determinants of disease development.
Interestingly a number of seemingly unrelated disorders may be implicated in postulated PACAP/VIP autoimmune pathology. For example, Crohn's disease (CD) and MS are infl ammatory disorders with known Th1-directed cytokines and loss of Foxp3 Treg function. 33,34 In one study in MS patients Treg numbers were unchanged but their function may have been diminished. 92 As VRS have important roles in controlling macrophage and perivascular infi ltrates in MS, 35 VRS immunological function in relation to VNs becomes of considerable interest. Complement-fi xing myelinolytic antigens have been identifi ed in the VRS in early MS, 36 which indicates the possible involvement of VRS in immune activity. However autoimmunity directed at VN guanine nucleotide protein-coupled receptors (GPCRs) is currently unproven and loss-of-function autoimmunity to GPCRs generally is not well documented, 22 although parallels exist with other conditions eg, Sjogren's syndrome which has T cell and/or B cell antibody targeting of acetylcholine GPCRs. 37 The role of PACAP and VIP in linking the innate and acquired immune systems 38 suggests there would be signifi cant effects on immunological and neurological homeostasis if they are compromised and include their complex infl uences on infl ammatory regulation involving microglia. 105 As noted above, macrophages and microglia in VRS express MHC class II molecules, albeit in low levels constitutively, and interact with lymphocytes from the blood in initiating and promoting immune responses to foreign antigens in the brain. However not all infl ammatory events within the CNS lead to MS. 104 Nevertheless, sites defi cient in the BBB include the subarachnoid space and pial surface, and circumventricular organs may be more prone to macromolecule penetration of CSF and this may have implications regarding autoimmune dysfunction within the CNS. 39 Collections of macrophages may occur in VRS following trauma in a pro-infl ammatory context 40 and pathological dilatation of VRS may occur from a variety of causes including ischemia. 41 These pathological features rely on PACAP/VIP protection to ameliorate their effects.
The distribution of VRS in anatomically specifi c locations implies that compromise of VRS will affect the functions of those anatomical locations. For example VRS located in the nucleus tractus solitarius in the dorsal medulla oblongata suggests that their compromise may have a role in impairing viscerosensory and autonomic functions. 42 Capillary diversity within the subfornical organ (SFO) and area postrema (AP) may function as low-resistance pathways for the rapid dispersion of blood-borne hormones inside their organ boundaries and this may have a role in regulation of blood pressure and body fl uids. 43 Hence these functions linked to VRS microanatomy may be particularly susceptible to PACAP/VIP compromise.
Vascular compromise within the CNS, possibly as a result of PACAP/VIP compromise, may give rise to features consistent with certain forms of dementia. Fronto-temporal dementia (FTD) is a neurodegenerative disease in which a vascular component is suggested and immunoreactivity of Bax, a proapoptotic protein regulated in part by PACAP/ VIP in astrocytes, suggests a role for autoimmunity in the pathology of FTD. 44 Astrogliosis in FTD corresponds with SPECT hypoperfusion, suggesting that astrocyte disruption may be related to disturbances of cerebral perfusion in FTD. 45 Cognitive dysfunction is associated with reduced cerebral blood fl ow in different types of dementia. 46 Moreover VRS dilatation associated with microvessel abnormality may contribute to the diagnosis of vascular dementias. 47 Changes in social behavior occur in cerebrovascular comprise and may result from an FTD-like syndrome. 48 Similarly, reduction in cortical blood fl ow has been identifi ed in CFS patients; 49,50 however these fi ndings were not replicated in a study of twins with CFS. 51 FTD however is recognized in ALS. 91 Other mechanisms such as water channel function are infl uenced by PACAP/VIP. The astroglial water channel aquaporin (AQP4) is essential for the maintenance of BBB integrity. 52 Antibodies to AQP4 have a highly specifi c role in neuromyelitis optica (NMO) and characteristically bind to cerebral microvessels, pia mater and VRSs. 53 Secretin is important for other aquaporin expression via vasopressin, secretin receptor-null mice for example have reduced renal expression of AQP2 and AQP4. 54 Thus VIP impairment may infl uence water channel function. Additionally an association with VRS has been identifi ed supporting the view that cortical nerve cells release VIP in the perivascular space during periods of activity and thus contribute to local vasodilatation associated with neuronal function. There is an important relationship whereby ATP over-expression has a downregulatory impact on AQP4 expression. 55 Adenylate cyclase compromise could have an impact on ATP levels by failure to convert to cAMP, arguably maintaining elevated levels of ATP with adverse consequences for AQP4 function.

Treatment in PACAP/VIP postulated autoimmune neuropsychiatric fatigue-related disorders
Receptors for PACAP and VIP exhibit a number of subtypes which are located in specifi c anatomical locations. Autoimmunity may affect these different sub-types in different anatomical regions. Thus it may be possible that VN receptor subtypes specifi c for the substantia nigra (SN) may be involved in, for example, PD. PACAP activates receptors of PAC1R, VPAC1R, and VPAC2R families, and PACAP has been identifi ed widely in the brain including the SN as well as in other organs and blood cells. 56 Moreover, PACAP has been shown to be effective in treatment in rat models of PD. 57 Both PACAP and VIP have neuroprotective effects in PD models by inhibiting the production of inflammatory mediators. 58 PACAP specifically protects against the neurotoxicity induced by rotenone 59 as well as protecting against oxidative stress-induced apoptosis. 60 In a murine model for PD, Delgado and Ganea 61 note that the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) activates microglia and promotes dopaminergic neuronal loss. These pathologies are signifi cantly decreased by VIP thus suggesting that VIP may be useful in treatment of neuropathological conditions such as PD. Conversely these fi ndings might suggest that a defect in specifi c PACAP/VIP function could act adversely on SN cells and, perhaps in concert with other environmental factors, contribute to a clinical presentation consistent with PD. PACAP/VIP are critical for neuronal survival. In a series of ALS patients, CSF levels of VIP were found to be signifi cantly lower compared with controls. 62 VIP has demonstrated potent effects on neurite outgrowth in spinal cord cultures suggesting its use in treatment of ALS. 63 Interestingly Sun and colleagues 64 noted impaired VIP receptor (VPAC2) production in activated T cells in MS patients, suggesting transcription irregularity at promoter regions of the VPAC2 gene. These fi ndings may be extremely important as more widespread impairment of VIP, if proven in patients with MS, may explain the manifestations associated with this condition. Also, in another paradigm, oligodendrocyte apoptosis is suggested as the earliest change leading to MS. Tissue injury may be amplifi ed over months or years as a result of oligodendrocyte cell death associated with myelin phagocytosis initiating local macrophage scavenger activity. 65 This paradigm is consistent with PACAP/VIP failure.
There are signifi cant treatment implications from this VN autoimmune hypothesis. As noted above, PACAP and VIP exert potent effects in metabolism as they have a vital role in cAMP production and regulation through AC activation. PDEs metabolise cAMP as a means of feedback regulation of cAMP levels. PACAP/VIP compromise could result in impaired AC activation and hence impaired cAMP production. Thus phosphodiesterase inhibitors (PDEIs), novel therapeutic substances used to promote cAMP levels 106 may have a role in treatment of VN autoimmune conditions. PDEIs have been identifi ed as a mechanism to modulate neuronal activity in psychiatric and neurodegenerative disorders. 94 Drugs such as rolipram, a phosphodiesterase type 4 inhibitor, activate cAMP-response element binding proteins (CREB) signalling as well as enhancing cAMP levels through impeding cAMP catabolism. 67 Imipramine also appears to have a key role in cAMP metabolism and therefore may have a role in combination drug therapy 68,69 as this pathway may share features with activity of PACAP and VIP as cAMP enhancers.
Rolipram was developed as an antidepression drug but has been found to have antiinfl ammatory and immunoregulatory activities. 70,71 Mediation of rolipram's antiinfl ammatory effects is suggested through modulation of antigen presenting functions of dendritic cells and lowering of MHC II expression. An IL-10-dependent mechanism is suggested in vitro 72 but an IL-10-independent mechanism is suggested in vivo. 77 Mediation of autoimmune protection in islet cells is associated with suppression of inducible nitric oxide synthase (iNOS) mRNA 73 and suppression of macrophage activation along with NO suppression is noted in mouse peritoneal macrophages. 74 Perhaps contrarily, NO generation might be a contributing factor to the therapeutic benefi t in EAE achieved by Rolipram in rats. 78 However there is evidence that rolipram stabilises the endothelial junctions of the BBB/BSB to reduce permeability to infl ammatory cells and reduce EAE severity. 96 The effi cacy of PDEIs in MS is being considered and clinical trials are indicated. 97 Models of PD [98][99][100] and ALS 101 also suggest a role for PDEIs in treatment. Theoretically some of the pathology of these diseases may be reversible with PDEIs. Experimental models in EAE, for example, show promising results. 95 Rolipram has a protective effect in experimental autoimmune neuritis associated with downregulation of interferon-gamma (IFN-γ) and infl ammatory chemokines as well as upregulation of IL-4 in the PNS. 75 Unfortunately side-effects such as nausea, vomiting, and headache are reported suggesting the need for less side effect-inducing analogues in therapy 76 and continuous administration may be necessary to sustain its therapeutic effect. 77 However PDEIs have proven to be of benefi t in vitro and clinical trials in humans, particularly using more recent generation drugs, may be considered provided side effects are not a barrier. 82

Conclusion
Certain neurological fatigue-related conditions such as MS, ALS, PD, and CFS often present with fatigue and other neuropsychiatric symptoms including memory and concentration loss, emotional lability, and confusion. Whether these conditions refl ect related neuroinfl ammatory and neurodegenerative molecular pathology is speculative. However multisystem involvement including cerebrospinal effects of these conditions could be explained in part through PACAP and VIP compromise. In particular, cerebrovascular and spinovascular effects at the ultramicroscopic level involving BBB/BSB may contribute to these disorders. Compromise of PACAP and VIP or their receptors may have a role and should be the subject of further research. Evidence for compromise of these VN receptors at different levels including VN receptor mRNA, protein transcription, cellular migration and traffi cking, cell membrane localization and possible antibody or T cell targeting may provide an explanation for some of the etiology of these disorders. Alternatively, antagonist short-form variants of VNs themselves may exist through faulty protein manufacture and produce a similar clinical picture.
If PACAP and VIP do indeed fail, through autoimmunity of their receptors, MHCII molecules usually expressed at low levels in BBB, eg, in microglia/pericytes, and normally kept in check by PACAP and VIP, may be at risk of becoming overexpressed. This may be the initiating event for BBB/ BSB breakdown and subsequent neuroinfl ammation and/or neurodegeneration in MS, PD, and ALS. Why CFS does not proceed to result in further demonstrable neurodegenerative pathology is not clear under this model although confi rmatory studies have not been done to date. The possibility that PACAP/VIP failure also leads to Treg impairment, possibly through impaired Foxp3 expression, is also yet to be investigated. Treg impairment has been described in MS patients suggesting possible PACAP/VIP failure.
PDEs metabolize cAMP and serve to regulate cAMP levels. PACAP/VIP compromise will result in impaired AC activation and hence impaired cAMP production. Noting the cAMP-protecting effects of PDEIs there is evidence to suggest PDEIs may have a role in treatment for certain neuropsychiatric fatigue-related conditions.

Disclosure
The authors report no confl icts of interest in this work.