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Neurodegenerative diseases, notably Alzheimer's and Parkinson's diseases, are amongst the most devastating disorders afflicting the elderly. Currently, no curative treatments or treatments that interdict disease progression exist. Over the past decade, immunization strategies have been proposed to combat disease progression. Such strategies induce humoral immune responses against misfolded protein aggregates to facilitate their clearance. Robust adaptive immunity against misfolded proteins, however, accelerates disease progression, precipitated by induced effector T cell responses that lead to encephalitis and neuronal death. Since then, mechanisms that attenuate such adaptive neurotoxic immune responses have been sought. We propose that shifting the balance between effector and regulatory T cell activity can attenuate neurotoxic inflammatory events. This review summarizes advances in immune regulation to achieve a homeostatic glial response for therapeutic gain. Promising new ways to optimize immunization schemes and measure their clinical efficacy are also discussed.
Parkinson's disease (PD) is second only to Alzheimer's disease (AD) as the most prevalent neurodegenerative disorder and the most common neurodegenerative movement disorder. Profound tremors, rigidity, postural instability, and bradykinesia (Olanow et al. 2009) seen in advanced clinical stages commonly lead to societal, interpersonal, and economic hardships (Dauer & Przedborski 2003, Savitt et al. 2006, Minati et al. 2009). No interdictive treatments exist (Korecka et al. 2007) and current therapies are palliative. Moreover, drug effects are transient and often induce untoward side effects (Fox & Lang 2008). Thus, new therapies that slow or reverse disease pathobiology are desperately needed.
One means to slow nigrostriatal degeneration is through clearance of misfolded proteins. As misfolded proteins accumulate within the brain, clearance mechanisms become overwhelmed leading to aggregate formation, increased inflammatory environments and oxidative stress with subsequent neuronal injury and death. In an attempt to clear these protein aggregates, early AD immunization studies showed effective clearance of beta-amyloid (Aβ) plaques and improved cognition in rodent models of human disease. However, although the results from multiple animal models seemed promising (Janus et al. 2000, Lemere et al. 2000, Morgan et al. 2000, Schenk et al. 1999, Weiner et al. 2000) the first AD vaccine trial, AN-1792, highlighted a major peril of using this strategy in humans. Indeed, a subset of AD patients (6%) developed meningoencephalitis (Senior 2002) that was shown to be mediated by proinflammatory effector T cells (Teffs) (Pride et al. 2008). Such engagement of the adaptive immune system exacerbated neuroinflammatory responses and subsequent neurotoxicity most likely by oxidative modification of Aβ and induction of inflammatory T cell-mediated meningoencephalitis. Indeed, while post-mortem analyses showed extensive cortical regions largely devoid of plaques (Vellas et al. 2009), vaccine-treated patients often retained long-term cognitive dysfunction (Kokjohn & Roher 2009). On balance, a subset of those vaccinated who showed strong antibody responses did retain cognitive function (Gilman et al. 2005). Overall, these findings demonstrate that clearance of misfolded protein aggregates alone may be necessary, but not sufficient to halt neurodegenerative responses.
These results have recently led to a novel immunization approach designed to regulate CNS immunity by pharmacologically directing neuroprotective effects in glia (Reynolds et al. 2007, Reynolds et al. 2009b) which target two pathogenic steps in disease progression: accumulation of misfolded protein aggregates or microbial infection and neuroinflammation (Benner et al. 2008, Masliah et al. 2005). Our laboratory has demonstrated that targeting both innate and adaptive processes by boosting regulatory T cell (Treg) activity with the immunosuppressive vasoactive intestinal peptide (VIP), can restore brain homeostasis. Treg were also shown to attenuate microgliosis and protect against MPTP-induced nigrostriatal neurodegeneration (Reynolds et al. 2010). We posit that using such a transformative therapeutic strategy for PD, which could easily be applied to other neurodegenerative diseases, substantively slows disease progression.
Herein we discuss recent advancements in immunization strategies that both affect misfolded protein clearance as well as modulate glial biology towards a neurotrophic signature. First, we summarize the contribution of innate and adaptive immunity to the pathobiology of neurodegenerative disease and how neuroinflammation can be harnessed effectively towards therapeutic outcomes. Second, we propose a multi-disciplinary research direction that leads to protein clearance and Treg-induced neuroprotection. Third, the means to optimize immunization approaches and advanced bioimaging techniques to monitor disease outcomes after immunization are outlined.
Innate and adaptive immune responses remove pathogenic agents and maintain tissue homeostasis within the CNS. In early innate immune responses, activated microglia phagocytose pathogens, clear debris, and secrete proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, IL-12, chemokines, proteases, and redox proteins (Suzuki et al. 2005) primarily to enhance or accelerate clearance mechanisms. These cytokines affect T and B function and cell entry into the CNS by disrupting blood brain barrier (BBB) integrity and neural homeostasis (Shriver et al. 2009). The mechanisms involve autocrine and paracrine production of neurotoxic factors (including proinflammatory cytokines and chemokines) that upregulate endothelial adhesion molecules, affect migrating cell shape and volume, and continuously attract activated leukocytes to sites of neuroinflammation, perpetuating disease pathobiology (Babcock et al. 2003). In parallel, activated microglia increase interferon gamma (IFN-γ) production by type 1 T helper cell (Th1) effector T cells (Teffs), which coordinate pathogen killing (Adams & Hamilton 1987). Following this proinflammatory phase, microglia normally enter anti-inflammatory and neurotrophic states, during which they orchestrate repair and neural reconstruction (Edwards et al. 2006, Mosser 2003).
The brain environment that is operative during PD is outlined in Figure 1. Under homeostatic conditions, misfolded or damaged proteins are cleared by the ubiquitin-proteasome pathway. However, during PD these proteins are not adequately cleared and accumulate intracellularly to levels sufficient to imbalance homeostatic conditions. Neuron-associated proteins, particularly alpha-synuclein (α-syn) become oxidatively-modified, forming nitrated α-syn (N-α-syn), which misfolds and aggregates to form intracellular inclusions, or Lewy bodies (Olanow et al. 2009). Although the etiology of these protein aggregates remains controversial, modification due to oxidative stress by increased reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive carbonyl species (RCS) can readily alter their composition and conformation (Stone et al. 2009). Increased levels of reactive species in the brain commonly result from high oxygen demand and relatively low antioxidant defense mechanisms which further contribute to high levels of oxidative stress, greater vulnerability of proteins to modification by reactive species, and greater degrees of aggregation and accumulation (Stone et al. 2009). These protein aggregates are commonly released into the extracellular environment from damaged or dying neurons and as such, exert pathogenic effects during PD either as non-conjugated species or as conglomerates from Lewy bodies (Kakimura et al. 2001, Lee et al. 2005, Sung et al. 2005).
Extraneuronal N-α-syn crosses the BBB to the CSF and draining lymph nodes wherein it activates antigen presenting cells (APCs) (Benner et al. 2008) and is processed and presented to naïve T cells by APCs via major histocompatibility complex (MHC) class II molecules (Fig 1). With appropriate co-stimulatory signals, naïve T cells differentiate into effector T cells (Teff) which clonally expand into different effector cell subtypes, such as Th1 and Th17 cells to drive disease processes toward a proinflammatory environment based on expression of unique cytokine profiles and functions. Th1 cells that express IL-2, IFN-γ, and TNF-α are proinflammatory and induce neurotoxic microglial release of ROS and nitric oxide (NO) (Weiner 2008). Similarly, the more recently discovered Th17 cell lineage also exhibits a proinflammatory phenotype producing IL-17 (IL-17A), IL-17F, IL-21, and IL-22 which induce tissue non-hematopoietic reactions due to distribution of the receptors for IL-17 and IL-22 among eptithelial and endothelial cells (Aggarwal et al. 2001, Korn et al. 2009) and IL-21/IL-21R-mediated signaling by T, B, natural killer (NK), and myeloid-derived cells (Monteleone et al. 2009, Hecker et al. 2009). In contrast, Th2 effectors secrete IL-4, IL-5, and IL-13 and support anti-inflammatory immune responses which enhance microglial-mediated neuroprotective activities (Weiner 2008). Additionally, Th1, Th2, and Th17 subsets provide support for production of antibodies, which specifically target pathogenic agents, including modified proteins, for subsequent removal by microglia (Aloisi 2001, Iwakura et al. 2008, Nakae et al. 2003, Hsu et al. 2008). Peripherally induced and expanded Th1 or Th17 Teffs crossing the BBB to inflammatory foci along the nigrostriatal axis would recognize N-α-syn/MHC II complex presented by antigen-presenting microglia. Induction of those Teffs to produce and secrete proinflammatory cytokines drive microglia and innate immune responses to a classically-activated state that serves to thwart invasion of pathogenic organisms, but is also neurotoxic. This state can also be acutely induced by agents such as aggregated N-α-syn, Aβ, MPP+, or lipopolysaccharide (LPS).
Simultaneously, gradients of inflammatory cytokines or weaker stimuli from injured or dying neurons, microglia, and Teffs also induce a persistent reactive state in surrounding microglia. Prolonged stimulation eventually leads to chronically-activated microglia. Accumulation of misfolded protein aggregates compromises cellular integrity, overrides the intracellular clearing mechanisms, and exerts neurodegenerative effects. Thus, persistent stimulation of the immune system will shift microglial activation states to a mixed phenotype that is more characteristic of chronic inflammation (Figure 1). As microglia attempt to initiate repair and recovery, chronic stimulation from accumulating misfolded proteins progresses to classical activation. In turn, the ascension of chronic inflammation, for instance by Th1 effectors, may promote accumulation of misfolded protein aggregates since alternative activation profiles initiated by IL-4 compromise the phagocytic activity of macrophages (Gratchev et al. 2005, Leidi et al. 2009). Chronic exposure of microglia to activating agents of neurodegenerative diseases, such as beta-amyloid (Aβ) and α-syn, are likely to exhibit a similar phenomenon that both maintains inflammation and suppresses normal microglial responses. In this way, chronic inflammation may perpetuate the neurotoxic environment observed in disease. Together with acute inflammation, chronic inflammation helps drive oxidative stress that leads to neurodegeneration in PD (Figure 1). This chronic inflammation scenario is also relevant for other neurodegenerative disorders where misfolded proteins or other stimuli play a primary role in neuroinflammation and disease pathobiology.
Microglia are primary sources of immune mediators, such as pro- and anti-inflammatory cytokines and chemokines, neurotrophic factors, glutamate, and various free radicals which, in appropriate combinations, direct mixed chronic inflammatory phenotypes (Shie & Woltjer 2007). Supporting evidence has been provided by mouse models of PD. One in vitro study found chronic stimulation of microglia by multiple LPS stimulations, compared to single acute stimulation, led to profound differences in activation of proinflammatory transcription factors such as NF-κB, as well as a mixed microglial profile, where production of NO and TNF-α were progressively reduced, but prostaglandins and cyclooxygenase-2 (COX-2) levels remained elevated (Ajmone-Cat et al. 2003). Furthermore, this mixed profile environment primes microglia for robust acute inflammatory responses upon subsequent exposures to LPS, marked by increased IL-1β, TNF-α and IL-6 production (Perry et al. 2003, Gao et al. 2003). Conceivably, accumulation of aggregated α-syn over time activates microglia to excess, which overwhelms regulatory mechanisms by predisposing microglia to chronic inflammation and the adaptive immune system to heightened activation, both of which drive neurodegeneration. Evidence that support these scenarios is that first, accumulated misfolded protein aggregates at high levels are not effectively cleared by microglia; second, accumulated protein aggregates trigger activation of microglial immune responses, which in turn, induce increased inflammation and oxidative stress that lead to secondary neurodegeneration; and third, adaptive immune responses initiated or driven by classically-activated microglia commonly become chronic and progressive, which further exacerbate neuroinflammation, oxidative stress, and neuronal death. Indeed activation of microglia by modified proteins and protein aggregates often leads to exacerbated inflammation and increased neuronal death (Benner et al. 2008, Stone et al. 2009, Reynolds et al. 2010, Reynolds et al. 2009a) (Figure 1). Data from the MPTP model of acute inflammation suggest that modified α-syn primes microglia to elicit responses from Teff that, in turn, sustain chronic inflammation. Upon exposure to persistent stimulation by N-α-syn in vitro, microglia enter the classically-activated state and exhibit pathogenic profiles of proinflammatory genes and expression of corresponding cytokines (Reynolds et al. 2008a, Reynolds et al. 2008b). However, N-α-syn-stimulated microglia also exhibit profiles reminiscent of neurotrophic and anti-inflammatory states, including upregulation of gene expression for IL-10, known to initiate an acquired-deactivation microglial state. Genomic, proteomic, and physiological analyses demonstrate that aggregated N-α-syn shifts innate immune responses from neurotrophic to neurotoxic. N-α-syn stimulation of microglia induces a neurotoxic phenotype marked by increased transcription of genes whose expression of inflammatory and redox protein products were correspondingly upregulated. Concurrently, robust amounts of such proteins were detected in the substantia nigra (SN) and basal ganglia of PD patients (Reynolds et al. 2008a). Interestingly, microglia also secrete neurotrophic factors, suggesting that while inflammatory pathways and oxidative stressors are active in tandem as a reactive microglia phenotype, compensatory mechanisms remain intact and may explain the slow clinical progression characteristic of PD. Most recently, we demonstrated that the neurodegenerative effects of such a mixed profile are elicited by Th17 cells (Reynolds et al. 2010), which play a major role in the pathogenesis of multiple sclerosis (MS) characterized by chronic neuroinflammation (Weiner 2009). Taken together, this evidence indicates that dysregulation of innate and adaptive immunity greatly contributes to neurodegenerative disease pathogenesis; thus modulating microglial immunity provides an attractive therapeutic target.
Having a demonstrable role in the pathogenesis of neurodegenerative diseases, inflammation represents a viable target to suppress neurotoxic phenotypes and interdict neurodegeneration. As such, various anti-inflammatory therapies that target inflammatory processes, in general, and microglia, in particular, have been proposed. Commonly used non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, ibuprofen, and naproxen, have been investigated due to their inhibitory actions against both COX-1 and COX-2, enzymes that function to amplify proinflammatory responses via production of prostaglandins (Blasko & Grubeck-Loebenstein 2003). COX-1 is constitutively expressed in most tissues and cell types, including microglia (Hoozemans et al. 2001), whereas COX-2 is an inducible form present during inflammatory processes (Yasojima et al. 1999). In early AD, levels of COX are elevated and upregulated by proinflammatory mediators such as TNF-α (Yamamoto et al. 1995). During disease progression, COX is overexpressed in neurons of the hippocampus, frontal cortex, and thalamus regions (Bazan et al. 2002) and induces production and release of reactive oxygen intermediates (Gebicke-Haerter 2001). In animal models of PD and in PD patients, the normally low expression of COX in dopaminergic neurons becomes elevated and promotes oxidative stress (Teismann et al. 2003, Feng et al. 2003, Wang et al. 2005, de Meira Santos Lima et al. 2006, Tyurina et al. 2006). These observations suggest that suppression of COX activity by NSAIDs may prevent the inflammation driving the progression of neurodegenerative diseases.
NSAIDs may also exert anti-inflammatory and neuroprotective properties independent of COX mechanisms on Aβ-activated microglia by acting on post-translational processing of inducible nitric oxide synthase (iNOS) to decrease production of NO (Stratman et al. 1997) and by activation of peroxisome proliferator-activated receptor-gamma (PPAR-γ) to inhibit proinflammatory cytokine secretion (Combs et al. 2000). Indeed, in rats treated with NSAIDs, Aβ infusion into the lateral ventricle resulted in an attenuated inflammatory response in activated microglia surrounding the subsequently formed plaques (Netland et al. 1998). Moreover, in experimental models of PD, COX activity plays a crucial role in microglial activation and induction of oxidative stress and inflammation that directs dopaminergic neuronal death as demonstrated by attenuation of neuroinflammation and increased neuroprotection subsequent to treatment with COX inhibitors (Vijitruth et al. 2006, Aubin et al. 1998, Ferger et al. 1999, Mohanakumar et al. 2000, Casper et al. 2000, Teismann & Ferger 2001, Carrasco et al. 2005, Maharaj et al. 2006, Di Matteo et al. 2006b, Di Matteo et al. 2006a, Wang et al. 2005). Thus, NSAIDs may mitigate proinflammatory microglial responses that lead to neurodegeneration. In contrast, anti-inflammatory therapeutics have shown conflicting results in experimental models and clinical trials of neurodegenerative diseases, wherein some NSAIDS exacerbate neurodegeneration (Lleo et al. 2007). Co-culture of PC12 with indomethacin, ibuprofen, ketoprofen, or diclofenac, but not aspirin or NS-398, a selective COX-2 inhibitor, enhances MPP+-induced cell death (Morioka et al. 2004). Seemingly this effect was attributed to NSAID-mediated diminution of multiple drug resistance transporters and accumulation of MPP+ and potentiation of MPP-induced neuronal cell death.
Data from laboratory, animal, and epidemiological studies of AD and PD that demonstrate the anti-inflammatory efficacy of NSAIDs have not always been reproduced in recent clinical trials. In a large cohort of over 142,000 who regularly used non-aspirin NSAIDs, in particular ibuprofen, exhibited a lower risk of PD than non-regular users of NSAIDs; findings that have withstood an extended period of monitoring and meta-analysis of seven studies (Chen et al. 2005, Chen et al. 2003, Gagne & Power 2010) (Gao, X., Chen, H., Schwarzschild, M., and Ascherio, A. 2010. Use of non-steroidal anti-Inflammatory drugs and risk of Parkinson's disease: a prospective study and meta-analysis. Oral presentation S03.003, 62nd American Academy of Neurology, Toronto, Canada, April 13, 2010). In vitro pre-incubation of cells obtained from PD patients with NSAIDs reduces microglial numbers (Alafuzoff et al. 2000) and prevents the release of toxic proinflammatory molecules (Klegeris & McGeer 2005). In mouse models of AD and PD, NSAIDs are associated with decreased neuronal degeneration (Etminan et al. 2003). Moreover, epidemiological studies suggest that chronic users of certain NSAIDs have a decreased risk for AD and PD (Rich et al. 1995, Zandi et al. 2002, Stewart et al. 1997, t' Veld et al. 2001, Chen et al. 2003, Chen et al. 2005). However, results from clinical trials of NSAIDs in AD suggest otherwise. Over 2,500 patients age 65 and older were given daily standard doses of ibuprofen, naproxen, or indomethacin for 2 years and followed for up to 12 years (Breitner et al. 2009). Contrary to results from animal and epidemiological studies (Etminan et al. 2003, McGeer & McGeer 2007, Szekely et al. 2004), decreased risk for dementia was not detected. Furthermore, NSAIDs may be incapable of preventing or delaying onset of AD, at least when given past a certain age (Reines et al. 2004, Pasqualetti et al. 2009, Van Gool et al. 2001).
The difficulty of using NSAIDs as immunotherapy is that they neither locally target the brain nor are specific to inflammation associated with neurodegenerative diseases, resulting in major side effects. Moreover traditional NSAIDs, by targeting both COX-1 and COX-2, generate adverse effects on the gastrointestinal tract (Singh et al. 2009). Subsequent NSAIDs were developed to selectively target COX-2 without interfering with normal COX-1 activity. However, those inhibitors lack cardioprotective effects mediated by COX-1 inhibition (Frankish 2002). In the Alzheimer's Disease Anti-Inflammatory Prevention Trial (ADAPT), which sought to determine if COX-2 selective NSAIDs could prevent or delay onset of AD, patients had increased risk of cardiovascular events that prompted its discontinuation (Meinert et al. 2009). Additional concerns have arisen about the non-specificity and BBB penetration by NSAIDs. To ensure sufficient NSAIDs cross the BBB, high dosages are typically required to elevate drug concentrations in the brain to therapeutic levels (Chen et al. 2005). However, such dosages may be toxic to peripheral tissues. Thus, given the widespread and differing roles of COX, restricting the anti-inflammatory effects of NSAIDs to only the brain may prove difficult.
Minocycline is a third generation lipophilic tetracycline analog noted for its ability to easily cross the BBB, attenuate inflammation, and potentiate neuroprotection via action on activated microglia (Kim & Suh 2009). Minocycline's mechanisms of action include inhibition of microglial activation and proliferation, secretion of pro-inflammatory cytokines and chemokines, release of cytochrome C from mitochondria, and expression/activation of caspase-1 and caspase-3 (Tikka et al. 2001, Tikka & Koistinaho 2001, Griffin et al. 2006, Chen et al. 2000, Zhu et al. 2002). In animal models of ALS, AD, and PD, minocycline is efficacious in preventing neurodegeneration in the respective models (Du et al. 2001, Kriz et al. 2002, Tomas-Camardiel et al. 2004, Seabrook et al. 2006, Quintero et al. 2006, Noble et al. 2009, Wu et al. 2002, Cuello et al. 2010), however reports have indicated that minocycline has little efficacy in Aβ plaque deposition or protection of striatal fibers (Yang et al. 2003, Diguet et al. 2004, Seabrook et al. 2006, Sriram et al. 2006). At issue, is the seemingly lack of robust therapeutic efficacy of minocycline in clinical trials as demonstrated by patients treated with minocycline leading to accelerated disease in ALS patients and trends toward worse, though not futile, Unified Parkinson's Disease Rating Scale (UPDRS) scores in PD patients compared to placebo-treated patients (NINDS NET-PD Investigators 2006, NINDS NET-PD Investigators 2008, Gordon et al. 2007). Thus, questions remain unresolved as to the efficacy of minocycline as a viable PD modality, and although future clinical trials cannot be ruled out, none are currently in progress and may require the resolution of dose, formulation, stage of disease, and endpoint determinations before trials can proceed.
While NSAIDs and minocycline may provide anti-inflammatory therapeutics, an effective option may be an immunization strategy that clears misfolded protein aggregates, while targeting both innate and adaptive immune-mediated neuroinflammation. One approach that could accomplish this goal is the modulation of Tregs (Figure 2). In this scenario, regulation of adaptive immunity is carried out by mutually opposing activities of Teffs and Tregs. However, in the chronic inflammation model that is responsible for much of the neurodegeneration in PD, Teff activity is unopposed. Naturally occurring CD4+CD25+Foxp3+ (forkhead homeobox P-3 immunoreactive) Tregs antagonize Teff and myeloid activities and attenuate immune-mediated inflammation either by cell-cell contact, secretion of immunosuppressive cytokines such as IL-10 and TGF-β, or by direct killing of effector cells (Huppa & Davis 2003). A primary role of Tregs is the installation and maintenance of immune tolerance to self (Onishi et al. 2008, Sakaguchi 2004, Sakaguchi et al. 1995). Deficiencies in Treg number and function lead to exacerbated lesions or accelerated disease progression in animal models of multiple sclerosis, inflammatory bowel disease, and rheumatoid arthritis (Atassi & Casali 2008). Treg development and regulatory activity is under control of the FOXP3 gene and a mutation in humans results in the lack of functional Tregs which leads to a propensity for early development of the autoimmune disorder immunodysregulation, polyendocrinopathy, and enteropathy, X-linked (IPEX) (Gambineri et al. 2008). The phenotypes that prevail with the lack of Treg underscore the necessity of Treg for the prevention of autoimmunity and control of chronic inflammation.
Currently, the effects of Tregs on innate and adaptive immunity in neurodegenerative diseases are a relatively new avenue. However, this strategy is supported by both in vivo and in vitro models wherein Treg-induced attenuation of microglial inflammatory responses is protective against nigrostriatal dopaminergic neurodegeneration (Mosley et al. 2006, Stone et al. 2009). In the MPTP mouse model, immunization with glatiramer acetate (GA, copolymer-1, COP-1, Copaxone), a randomly-synthesized polymer of four amino acids, serves as an immunomodulatory agent prescribed for relapsing-remitting multiple sclerosis and induces GA-specific CD4+ cells that attenuate activated microglia and protect against neurodegeneration (Benner et al. 2004, Laurie et al. 2007). More recently, we demonstrated the capacity of relatively few CD4+CD25+Foxp3+ natural Tregs to abate MPTP-induced neuroinflammation and ameliorate virtually all dopaminergic neurodegeneration in both the substantia nigra and striatum (Reynolds et al. 2007). In vitro experiments further show that co-culture of Tregs and microglia before stimulation with N-α-syn suppresses microglial responses, as measured by diminished NF-κB activation, proinflammatory cytokine production, and oxidative stress (Reynolds et al. 2007, Reynolds et al. 2009b, Reynolds et al. 2009a). Moreover, co-culture of Tregs with microglia prior to N-α-syn stimulation suppresses microglial-induced ROS, inhibits activation and nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) to suppress Teff-induced inflammation and neurotoxicity (Reynolds et al. 2009a). On the other hand, co-culture of Tregs after activation of microglia with N-α-syn induces apoptosis of microglia that is mediated largely by Fas/FasL interactions. Proteomic analyses revealed that the Treg-mediated suppressive activities are accomplished by modulating microglial production of neurotoxic redox-active enzymes and bioenergetic proteins linked to metabolism, migration, phagocytosis, and protein transport and degradation by microgla (Reynolds et al. 2009a, Reynolds et al. 2009b). These data suggest that with disease progression of PD, Treg function becomes compromised leading to dysregulation of adaptive immunity, especially since restoration of Treg activity protects against neuroinflammation and subsequent neurodegeneration. Indeed, Tregs isolated from mice immunized with N-α-syn are deficient in their capacity to suppress polyclonally- or antigen-activated Teff proliferation, and adoptive transfer of N-α-syn specific Teffs to MPTP-intoxicated mice exacerbates neuroinflammation and amplifies nigrostriatal dopaminergic neuronal loss (Benner et al. 2008, Reynolds et al. 2010). Such findings, coupled with known roles of Tregs in maintaining self-tolerance and suppression of myeloid APC function and Teff responses (Ozdemir et al. 2009), have inspired immunization strategies aimed at using pharmacological adjuvents to increase de novo Treg number and/or activity.
To test one such pharmacological strategy, we utilized VIP, a known inducer/enhancer of Treg responses (Delgado et al. 2005, Gonzalez-Rey et al. 2006) in a model of N-α-syn-mediated Treg diminution and exacerbated neurodegeneration (Reynolds et al. 2010). Co-adoptive transfer of N-α-syn Teffs with spleen cells or CD4+CD25+Foxp3+ T cells from VIP-treated mice enhanced protection of dopaminergic neurons along the nigrostriatal axis compared to spleen cells or CD4+CD25+Foxp3+ T cells from naive animals. Such neuroprotection was achieved by VIP-induced Treg abrogation of proinflammatory Th17 response, a mechanism by which Tregs exert immunosuppressive activity (Leceta et al. 2007). Based on these results, a proposed vaccine, possibly using N-α-syn as an antigenic anchor to clear aggregated proteins would be formulated with an adjuvant, such as VIP to stimulate induction of Treg expansion and/or activity (Figure 2). Expanded Tregs which are summoned to neuroinflammatory sites, would traverse the BBB and migrate to areas of neuroinflammation, release IL-10, TGF-β, and FasL to drive activated microglia toward restoration of a homeostatic state or to induce their apoptosis. This would inhibit the production and secretion of pro-inflammatory cytokines, ROS, RNS, peroxynitrite (OONO-), and glutamate with concomitant attenuation of neuroinflammation. Thus, antibody-mediated clearance of existing aggregated α-syn, synergized with attenuated neuroinflammation, which alleviates oxidative stress and diminishes production of aggregated α-syn would ameliorate further neuronal injury and apoptosis, and interdict PD progression. Adaption of this strategy would require resolution of several issues. For instance, as Tregs suppress dendritic cell function, antigen presentation, and Teff proliferation, VIP induction of Treg before and during antigen priming, may inhibit Teff-assisted antibody formation as the extent of interplay between Treg and Th2 effector cells have yet to be completely defined (Chapoval et al. 2010). Alternatively, increased Treg number and function have been shown to shift immune responses to favor Th2 effectors (Xinqiang et al. 2010). Thus, timing of adjuvant administration relative to antigen priming and boost will need to be resolved for an efficacious vaccine strategy. On the other hand, increasing Treg number or function alone with VIP or other pharmacological agents such as non-mitogenic, Treg-inducing anti-CD3 antibody may be sufficient to inhibit neuroinflammatory responses and aggregated protein accumulation (Chatenoud & Bluestone 2007, Herold et al. 2009, Notley et al. 2010, Chatenoud 2010).
In addition to direct neuroprotection, modulation of glial biology during immunization also may support reparative and regenerative processes, thus affording additional benefits. Two days after adoptive transfer of GA-specific T cells to MPTP-intoxicated mice increases local expression of glial cell line-derived neurotrophic factor (GDNF) (Benner et al. 2004), whereas transfer of activated Tregs, but not Teffs (Reynolds et al. 2007), increases both GDNF by 2 days post transfer and brain-derived neurotrophic factor (BDNF) by 7 days after transfer. Sources of these neurotrophic factors were astrocyte-derived, rather than microglia- or T cell-derived. This suggests that Tregs may be involved in regulating reparative processes in both microglia and astrocytes which may contribute to greater than 90% protection of dopaminergic neurons. Indeed, postmortem PD brains exhibit decreased neurotrophic factor levels suggesting that glia-derived neurotrophic factors as well as T cell-derived cytokines play an important role in the pathogenesis of PD (Imamura et al. 2005). In vitro, GDNF is secreted by activated astrocytes and acts as the most potent factor supporting dopaminergic neurons (Burke et al. 1998). In vivo, GDNF induces dopaminergic nerve fiber sprouting upon injury to the rat striatum (Batchelor et al. 1999), which becomes markedly reduced once GDNF expression is inhibited by antisense oligonucleotides (Batchelor et al. 2000). Studies show that GDNF reduces dopaminergic neuronal death and significantly enhances their function in both MPTP-intoxicated mice and monkeys (Gash et al. 1996, Kordower et al. 2000, Eberhardt et al. 2000). Thus, GDNFmodulation of microglia biology to support dopaminergic neurons in the nigrostriatal axis may yet yield promising clinical outcomes despite mixed results in clinical trials (Nutt et al. 2003, Patel et al. 2005, Lang et al. 2006, Slevin et al. 2007). Future studies should explore the mechanisms by which Tregs upregulate GDNF in the context of protection against progression of PD and other neurodegenerative disorders.
In order to quickly and effectively advance the science of neurodegenerative disease vaccines, multi-disciplinary research advances are needed, beginning with the development of animal models of higher recapitulative value. The importance of chronic inflammation in the pathogenesis of PD warrants the development of chronic MPTP models to determine whether immunization can alleviate chronic neuroinflammation and afford neuroprotection. One possible chronic mouse model may involve multiple small dosages of MPTP intoxication over time, which would allow N-α-syn to aggregate and sustain the mixed activation states. Detection of similar classical and alternative pathogenic profiles of microglia would provide supportive evidence for the proposed late-stage pathogenesis in PD during which protein aggregates accumulate and dysregulated adaptive immunity predominates to yield a chronic inflammatory state leading to neurodegeneration. However, the ability of mice to recover from multiple MPTP intoxications and the appropriate dosages levels would need to be resolved. Furthermore, in acute exposure to low dosages of MPTP, mice have been observed to recover from intoxication. Low dosages of MPTP may lead to less production of N-α-syn, but levels sufficient to trigger inciting events and the long term effects that eventually lead to the dysregulation of adaptive immunity and development of chronic neuroinflammation have yet to be studied.
An alternative chronic mouse model that avoids multiple low dosages of MPTP may involve sustaining the activity of a larger dose of MPTP. Recently, the organic acid transporter (OAT) inhibitor probenecid, which blocks rapid clearance of MPTP, has been used to provide chronic regimens of MPTP that ablated enough dopaminergic neurons to establish hallmark motor and pathobiological markers of PD (Meredith et al. 2008). This model was later used to demonstrate behavioral and cytokine expression differences in acute versus chronic inflammation (Luchtman et al. 2009). The advantage of this model over multiple low dosages of MPTP is that continuous presence of MPTP can aid in N-α-syn accumulation, which better ensures trigger of chronic inflammation. Moreover, retaining MPTP activity over the long term more closely recapitulates the slow clinical progression of AD, which has not been achieved by either transgenic or MPTP mouse models (von Bohlen Und Halbach 2005). Such MPTP mouse models of chronic inflammation are ideal for testing whether immunization can target both acute and chronic inflammation in PD.
An enticing therapeutic strategy combines nanomedicine delivery with immunogenicity to optimize vaccine delivery. We have recently determined that only a few ex vivo CD3-activated Tregs are necessary to attenuate MPTP-induced inflammatory microglia and protect dopaminergic neurons along the nigrostriatal axis (Reynolds et al. 2007). Thus, delivery of strategically-targeted pharmacological agents which upregulate Treg function or number would provide personalized and profound neuroprotection for PD patients. In vivo Treg expansion and anti-inflammation can be accomplished by administration of agents such as trichostatin A (TsA), a histone deacetylase inhibitor (HDACi) (Koenen et al. 2008, Tao et al. 2007) or activin A (Huber et al. 2009), and non-mitogenic anti-CD3 (Kohm et al. 2005). Substances such as GM-CSF and 1α,25-dihydroxyvitamin D3, generate Tregs and stimulate the production of TGF-β and IL-10 (Taher et al. 2008, Vasu et al. 2003, Gregori et al. 2002, Gangi et al. 2005). We have shown that immunomodulatory agents, such as VIP, increase Treg function and suppress neuroinflammation and dopaminergic neurodegeneration (Reynolds et al. 2010). By combining nanomedicine delivery with immunization technology, aggregated proteins such as misfolded α-synuclein can be cleared more efficiently and inflammation reduced earlier to halt disease progression.
However, nanoformulation and targeting strategies are not without complications. For instance, targeting agents to increase Treg function, such as TGF-β or inclusion of TGF-β in the nanoformulations, may attenuate and diminish inflammation, but may also increase proinflammatory Th17 induction as TGF-β is a known stabilizer of Th17 effectors. The consequence may yield a harmful imbalance between Tregs and Th17 effectors, which results in increased inflammatory responses, increased oxidative stress, and greater dopaminergic neurodegeneration. Of course, this could be countered using Th17-inhibitory/anti-inflammatory cytokines such as IL-10, or via RNAi knockdown of IL-6, IL-1β, or IL-23 at inflammation sites to interdict this aspect of the adaptive immune system. Although attractive, the use of nanoformulated therapeutics will require resolution not only of an optimized delivery formulation, but also route, site of formulation action, necessity to cross the BBB, release of the vaccine components, and the effects of the formulation on vaccine antigenicity and adjuvancy.
To assess efficacy of the proposed immunization strategies, we must consider disease pathology and the resulting symptoms. Current methods for monitoring neurodegenerative disease progression and treatment efficacy include neuroimaging and numerous rating scales that assess clinical impairment and disability. Neuroimaging by positron emission tomography (PET) or single photon emission computed tomography (SPECT) is most often used to estimate the density of remaining dopaminergic neurons and termini, DOPA uptake, and peripheral benzodiazepine receptors (PBR). However, compared to symptomatic rating scales the more objective neuroimaging is vastly underused, thus diminishing the greater additive potential that neuroimaging can provide to clinical rating scales and will ultimately need to be employed for translational assessments in disease progression and the efficacy of immunotherapies.
PET or SPECT neuroimaging can be useful in determining the extent of damage to the CNS in many neurodegenerative diseases. Several radiolabeled ligands are available for use including [18F]-fluoroDOPA (FD), which is taken up in the nigrostriatal system; β-carbomethoxy-3 β-[4-iodophenyl]tropane (β-CIT) and [99mTc]-[2[[2-[[[3-(4-chlorophenyl)-8-methyl-8-azabicyclo[3,2,1]-oct-2-yl]-methyl](2-mercaptoethyl)amino]ethyl]amino]ethane-thiolato(3-)-N2,N2′,S2,S2]oxo-[1R-(exo-exo)]) (TRODAT-1), both tropanes that bind dopamine transporters; and [11C]-dihydrotetrabenazine (DTBZ), which binds vesicular monoamine transporters (Marek et al. 2001). All can provide a window of neuronal cell survival and function in the CNS. FD uptake correlates with nigral neuronal cell counts and striatal dopamine levels in MPTP-intoxicated monkeys (Pate et al. 1993), humans, and patients with sporadic PD (Marek et al. 2001, Snow et al. 2000). However, FD uptake does not appear to be a direct measure of nigral neuronal cell density, but rather a measure of aromatic amino acid decarboxylase activity within dopaminergic neurons (Ravina et al. 2005). [123I]-β-CIT SPECT images show decreased striatal uptake in PD compared to healthy controls, which suggests its utility to detect striatal survival and loss of dopaminergic neurons in PD (Seibyl et al. 1998). Additionally, decreased uptake of [99mTc]TRODAT-1 in the putamen of PD patients compared to controls indicates that this neuroimaging method can detect loss of dopamine transporters (Mozley et al. 2000). Diminished DTBZ binding levels in nigrostriatal tissue of PD patients compared to controls demonstrates its potential for improving early diagnosis of PD (Martin et al. 2008). Moreover, DTZB binding in the contralateral midputamen region has correlated with bradykinesia and rigidity scores, but not with tremor, suggesting that tremor may only be partially related to pre-synaptic dopaminergic function with etiological mechanisms differing from that of symptoms such as bradykinesia; a hypothesis congruent with the observation that tremor is often unresponsive to dopamine therapies.
As discussed above, microglial activation in the brain leads to increased oxidative stress and neuronal cell death in neurodegenerative diseases. While several other cell types also play roles in neurodegeneration, the overwhelming presence of microglia at the site of neuronal injury makes this cell type a strong candidate to monitor neurodegenerative disease progression and treatments that target the inflammatory component. Strategies that image microglia would therefore provide information about the extent of inflammation. One such strategy involves imaging the peripheral benzodiazepine receptor (PBR) over-expressed by activated microglia. PBRs are widely distributed in peripheral tissues, as well as in the CNS (Anholt et al. 1985, De Souza et al. 1985), but are upregulated in several neurodegenerative diseases including Huntington's disease (Schoemaker et al. 1982) and AD (McGeer et al. 1988, Owen et al. 1983). Early studies of PBRs in neuroinflammation suggested their utilization as markers for inflammation for which many radiolabeled ligands for PBRs were developed to detect areas of ischemia and tumors in humans by PET (Black et al. 1990, Junck et al. 1989, Ramsay et al. 1992). While these studies found that PBRs localize intracellularly to mitochondrial membranes in peripheral cells, the cell types expressing PBRs in the CNS following brain injury remained enigmatic until PBRs were found by PET to co-localize with activated microglia in a study that utilized a specific PBR ligand, PK11195 (Stephenson et al. 1995). A later study using PK11196 PET imaging showed elevated levels of PBRs in PD brains throughout 2 years of monitoring, suggesting an association with microglial activation and PD progression (Gerhard et al. 2006). Recently, other radiolabeled ligands for PBRs have been developed and tested (Van Camp et al.). In fact, using the [11C]DAA1106 ligand, elevated PBR levels can be detected at early stages of AD (Yasuno et al. 2008). Taken together, these data suggest that radiolabeled ligands can be used to quantify PBRs by PET as a measure of microglial activation in neurodegenerative diseases. Furthermore, this method can improve early detection of neurodegeneration, as well as provide a means to non-invasively monitor therapeutic efficacy.
To support PET and SPECT data and further improve assessment of clinical efficacy of treatments, standard clinical evaluations should be performed in tandem, but blinded to neuroimaging results. Currently, there are several rating scales for both AD and PD that measure levels of impairment and disability to support diagnosis and assessment of disease stages and therapeutic efficacy. Application of the most relevant clinical rating scale for PD could be utilized to assess the efficacy of immunomodulatory therapies. The least flawed and thus, most common rating scale used to diagnose PD is the Unified Parkinson's Disease Rating Scale (UPDRS) (Ramaker et al. 2002). A recent comparison of rating scales found that the UPDRS has relatively high internal consistency and inter-rater reliability, and satisfactory construct validity when used with the Hoehn and Yahr (Ramaker et al. 2002). However, like all rating scales, UPDRS is subjective, but is also redundant as it emphasizes bradykinesia to a greater degree than other rating scales (Brooks et al. 2003). Thus, these disadvantages warrant complementing rating scales with neuroimaging to permit a more comprehensive diagnosis and evaluation of disease progression and treatment. When assessing PD therapeutic efficacy, considering which symptoms are responsive to dopaminergic treatment and those that are not, represents a key issue. The classical clinical features of PD, bradykinesia, gait disturbances and rigidity, are all caused by decreases in dopamine levels in the striatum, and can be attenuated with the administration of L-DOPA. However, several PD symptoms are unresponsive to dopamine replacement therapy. The non-dopaminergic symptoms include freezing, falling, dementia, autonomic failure, dystonia, and possibly resting tremor (Olanow et al. 2009). The aim of immunomodulatory therapies is not only to treat the disease in a palliative manner, but to actually interdict disease progression. Thus, non-dopaminergic symptoms need to be monitored over the long-term.
In combination, the use of imaging and clinical rating scales for impairment and disability will provide sufficient data to assess the efficacy of immunomodulatory therapeutics. These methods of assessment could provide comprehensive insight into disease progression, treatment effects, and complex relationships between neurodegenerative disease pathology and the manifestation of clinical symptoms, as well as early detection of inciting disease events.
Supported by the Carol Swarts, M.D., Emerging Neuroscience Research Laboratory, the Frances and Louie Blumkin Foundation, the Community Neuroscience Pride of Nebraska Research Initiative, the Alan Baer Charitable Trust (to H.E.G.), the University of Nebraska Medical Center Patterson Fellowship (to A.D.R.), the Michael J. Fox Foundation for Parkinson's Research (to R.L.M.), and National Institutes of Health Grants R21 NS049246 (to R.L.M.), 5P01NS31492, 2R37NS36126, 2R01NS034239, P20RR15635, U54NS43011, P01MH64570, and P01NS43985 (to H.E.G.).
All authors declare no conflicts of interest.