Overview: Evidence from clinical and preclinical studies indicates that basal inflammatory status increases as a function of normal aging, and progressive development of a mild pro-inflammatory state closely associates with the major degenerative diseases of the elderly (Holmes et al., Neurology 73:768-74, 2009; Heneka et al., Lancet Neurol 14:388-405, 2015). Hallmarks of aging include increased oxidative stress, lipid peroxidation and mitochondrial and DNA damage, particularly within the brain. Microarray studies indicate an overall rise in inflammatory and pro-oxidant genes with a decline in growth, anti-inflammatory and anti-oxidant genes in the brain of older versus adult rodents (Cribbs et al., J Neuroinflammation 9:179, 2012). In line with this, levels of brain pro-inflammatory cytokines have been found elevated with age in rodents and humans, and several regulatory molecules and anti-inflammatory cytokines reduced (Deleidi et al., Front Neurosci 9:172, 2015). As a source of these pro- and anti-inflammatory molecules, microglia are thereby implicated as the major culprit of this neuroinflammation. Correcting the overproduction of pro-inflammatory cytokines by microglia may mitigate a broad number of neurodegenerative disorders prevalent in the elderly, and, in particular Alzheimers disease (AD). However, finding an appropriate drug target to safely and effectively achieve this has thus far proved difficult, and likely accounts for many of the numerous failures of clinical trials of anti-inflammatory agents in AD and associated disorders. Tumor necrosis factor-alpha (TNF-alpha) is one of the primary pro-inflammatory cytokines synthesized and released by microglial cells. Once TNF-alpha is released, it may initiate a self-propagating cycle of unchecked inflammation (Frankola et al., CNS Neurol Disord Drug Targets 10:391-403, 2011). Pharmacological intervention to interrupt this cycle may be of significant benefit in the setting of neuroinflammation-mediated diseases. In 1993, Moreira et al., (J Exp Med 177:1675-80, 1993) described a series of studies showing that the drug thalidomide (THAL) was able to lower TNF-alpha protein levels post-transcriptionally by accelerating the degradation of its mRNA. Unfortunately, THAL is not a particularly potent TNF-alpha lowering agent and is associated with serious teratogenic adverse effects to embryos in utero, sedation and peripheral neuropathy at clinical doses (Calabrese & Fleischer, Am J Med 108:487-95, 2000; DeCourt et al., Curr Alzheimer Res. 14:403-411, 2017). Nevertheless, the observation of THALs TNF-alpha lowering activity has given rise to studies to differentiate these actions, understand THALs TNF-alpha structure/activity relationship and develop more potent analogs. In principle, the identification of analogs with enhanced anti- TNF-alpha activity and reduced teratogenic and neurotoxic effects may provide a viable treatment strategy for CNS neuroinflammatory and other forms of inflammatory disease. Our medicinal chemistry modifications to the backbone of THAL and newer analogs (namely pomalidomide (POM)) have generated an extensive library of novel agents (issued US patents owned: 7,973,057 and 8,927,725, and U.S. Patent Application No. 62/235,105). Our focus is to identify well-tolerated drug-like compounds with more potent anti- TNF-alpha activity from our generated library and develop these as experimental drugs to characterize the role of the neuroinflammatory component in and to treat AD and associated disorders. Problem and Focused Aims: AD is a complex disorder that manifests as progressive dementia with few other symptoms. With a long meandering course, the disease is associated with deposits of amyloid-beta protein (ABeta) of 40 and 42 amino acids as much as 20 years prior to the development of dementia. It also induces intracellular accumulation of the microtubule-associated protein Tau (MAPT) as neurofibrillary tangles (NFTs) that correlate more closely with the extent of dementia (Sambamurti et al., Curr Alzheimer Res 3:81-90, 2006; Baranello et al., Curr Alzheimer Res 12:32-46, 2015). NFTs arise some 10 years after ABeta, and brain atrophy follows after five further years. However, the resilience and redundancy of the nervous system protects the affected subject from dementia for around five further years after the detection of atrophy by brain image analysis. The discovery that familial AD (FAD) mutations in ABeta precursor protein (APP) and presenilins (PSEN1) and 2 (PSEN2) increase ABeta42, have placed amyloid at the Occams razor of AD. The finding that the E4 variant of apolipoprotein E (APOE), detected in almost half the AD population, also fosters ABeta deposition further boosts the amyloid hypothesis. Despite the consistency of this finding, the time-dependent ABeta-triggered mechanisms of neuronal dysfunction and degeneration remain unclear; thereby making therapeutic intervention difficult. As ABeta oligomers and aggregates are tolerated over an extended time, their toxicity may not be the direct cause of neurodegeneration but, rather, the initiator of a cascade of events that become self-propagating and then drive disease progression. This premise may account for the failure of anti-amyloid therapies in clinical trials when administered late in the disease course (Becker et al., Nature Rev Drug Discov 13:156, 2014). The presence of soluble and insoluble ABeta and MAPT can induce microglia activation (McGeer & McGeer, Acta Neuropathol 126:479-97, 2013), and direct evidence of neuroinflammation in AD brain has been shown by in vivo PET imaging (Schuitemaker et al., Neurobiol Aging 34:12836, 2013). Notably, levels of pro-inflammatory cytokines are elevated in serum and CSF from AD patients, for TNF-alpha by as much as 25-fold (Tarkowski et al., J Clin Immunol 19:223-30, 1999). In MCI subjects that progress to develop AD, a rise in CSF TNF-alpha levels correlates with disease progression (Tarkowski et al., J Neurol Neurosurg Psychiatry 74:1200-5, 2003). Paralleling this, elevated expression of TNF-alpha is reported within the entorhinal cortex of 3xTg-AD mice prior to the appearance of amyloid and tau pathology, and this increase associates with the onset of cognitive deficits in these mice and later neuronal loss (Janelsins et al. J Neuroinflamm 2:23, 2005). We hypothesize that failure of protein homeostasis leads to accumulation of proteins (e.g., ABeta, APOE and MAPT) that induce microglial activation and a proinflammatory M1 response to instigate their removal. The continuing generation of protein (ABeta, APOE and MAPT) leads to maintenance of a chronic M1 response, an impairment of transition to an anti-inflammatory M2 response (particularly in the aging brain that is already vulnerable to inflammation) with ensuing neuronal impairment observed in the animal models and in preclinical AD that eventually leads to cell death. Proinflammatory cytokines, like TNF-alpha, induce vascular changes to allow lymphocyte infiltration that may underpin reported cerebral vasculature leakiness of AD patients and related Tg mouse models. Moreover, TNF-alpha induces ABeta production in cellular and animal AD models, further increasing its accumulation and the entire cascade. Our focus is understanding the time course of development of neuropathology accumulation of inflammatory cytokines and behavioral deficits in unique mouse models that may reflect the disease pathology more than the currently available ones. We also evaluate the treatment of these deficits with a clinically approved immunomodulator, POM, which lowers TNF-alpha generation as well as with the new more potent small molecule TNF-alpha synthesis inhibitors synthesized and patented for NIH by our research collaborative group within the Intramural Research Program of NIA.
