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 [18
F]-fluoroDOPA (FD), which is taken up in the nigrostriatal system; β-carbomethoxy-3 β-[4-iodophenyl]tropane (β-CIT) and [99m
Tc]-[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 [11
C]-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
I]-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 [99m
Tc]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 [11
C]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.