Methamphetamine-induced Neuroinflammation and Neurotoxicity:a Role for Interleukin-1β

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Neurobiology and Anatomy, 2012.Methamphetamine (MA) is a potent, addictive psychostimulant abused by millions of people worldwide. MA exposure induces neurotoxicity, particularly in the striatum, where it damages dopaminergic terminals. In parallel with this neurotoxicity, MA also induces neuroinflammation, characterized by activation of striatal microglia and astrocytes, leading to production of pro-inflammatory cytokines and reactive oxygen species. It is unclear whether MA-induced neuroinflammation contributes to MAinduced neurotoxicity. To address this issue, we examined the time course and dose response of MA-induced neurotoxicity and neuroinflammation to search for time- and dose-specific links between the two, using a binge dosing paradigm of four injections given every two hours to mimic human use patterns; we also focused on the specific contributions of the pro-inflammatory cytokine interleukin-1β (IL-1β) and the inflammation-associated oxidative enzyme NADPH oxidase 2 (NOX2). We found that treatment with at least 4 mg/kg MA per injection decreased striatal dopamine, tyrosine hydroxylase, and dopamine transporter levels within one day, and that these decreases persisted for at least one week. Striatal microglia and astrocytes were both activated one day after binge MA treatment of all doses, including those that did not induce measurable neurotoxicity; while astrocyte activation persisted, microglial activation attenuated during the two weeks of the study. Despite this gliosis, we did not find altered mRNA expression of the pro-inflammatory cytokines tumor necrosis factor α, interleukin 6, or chemokine (C-C motif) ligand 2 in the striatum. IL-1β knock-out (KO) mice were resistant to MA-induced neurotoxicity, and local overexpression of IL-1β exacerbated MA-induced neurotoxicity. However, we were unable to find an increase in striatal IL- 1β expression in the hours and days following MA exposure, suggesting that MAinduced IL-1β upregulation and signaling may primarily occur elsewhere, or may be too subtle or rapid for us to detect. Finally, although MA exposure increased NOX2 mRNA expression in the striatum, NOX2 KO mice were not protected against MA-induced neurotoxicity or glial activation. These findings give insight into the complex relationship between MA-induced neuroinflammation and neurotoxicity

An Azobenzene Photoswitch Sheds Light on Turn Nucleation in Amyloid-β Self-Assembly

Amyloid-β (Aβ) self-assembly into cross-β amyloid fibrils is implicated in a causative role in Alzheimer’s disease pathology. Uncertainties persist regarding the mechanisms of amyloid self-assembly and the role of metastable prefibrillar aggregates. Aβ fibrils feature a sheet-turn-sheet motif in the constituent β-strands; as such, turn nucleation has been proposed as a rate-limiting step in the self-assembly pathway. Herein, we report the use of an azobenzene β-hairpin mimetic to study the role turn nucleation plays on Aβ self-assembly. [3-(3-Aminomethyl)­phenylazo]­phenylacetic acid (AMPP) was incorporated into the putative turn region of Aβ42 to elicit temporal control over Aβ42 turn nucleation; it was hypothesized that self-assembly would be favored in the <i>cis</i>-AMPP conformation if β-hairpin formation occurs during Aβ self-assembly and that the <i>trans</i>-AMPP conformer would display attenuated fibrillization propensity. It was unexpectedly observed that the <i>trans</i>-AMPP Aβ42 conformer forms fibrillar constructs that are similar in almost all characteristics, including cytotoxicity, to wild-type Aβ42. Conversely, the <i>cis</i>-AMPP Aβ42 congeners formed nonfibrillar, amorphous aggregates that exhibited no cytotoxicity. Additionally, <i>cis</i>-<i>trans</i> photoisomerization resulted in rapid formation of native-like amyloid fibrils and <i>trans–cis</i> conversion in the fibril state reduced the population of native-like fibrils. Thus, temporal photocontrol over Aβ turn conformation provides significant insight into Aβ self-assembly. Specifically, Aβ mutants that adopt stable β-turns form aggregate structures that are unable to enter folding pathways leading to cross-β fibrils and cytotoxic prefibrillar intermediates

Neural Precursor Cell Proliferation Is Disrupted Through Activation of the Aryl Hydrocarbon Receptor by 2,3,7,8-Tetrachlorodibenzo-p-Dioxin

Neurogenesis involves the proliferation of multipotent neuroepithelial stem cells followed by differentiation into lineage-restricted neural precursor cells (NPCs) during the embryonic period. Interestingly, these progenitor cells express robust levels of the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that regulates expression of genes important for growth regulation, and xenobiotic metabolism. Upon binding 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a pervasive environmental contaminant and potent AhR ligand, AhR, is activated and disrupts gene expression patterns to produce cellular toxicity. Because of its widespread distribution in the brain during critical proliferative phases of neurogenesis, it is conceivable that AhR participates in NPC expansion. Therefore, this study tested the hypothesis that AhR activation by TCDD disrupts signaling events that regulate NPC proliferation. The C17.2 NPC line served as a model system to (1) assess whether NPCs are targets for TCDD-induced neurotoxicity and (2) characterize the effects of TCDD on NPC proliferation. We demonstrated that C17.2 NPCs express an intact AhR signaling pathway that becomes transcriptionally active after TCDD exposure. 3H-thymidine and alamar blue reduction assays indicated that TCDD suppresses NPC proliferation in a concentration-dependent manner without the loss of cell viability. Cell cycle distribution analysis by flow cytometry revealed that TCDD-induced growth arrest results from an impaired G1 to S cell cycle transition. Moreover, TCDD exposure altered p27 kip1 and cyclin D1 cell cycle regulatory protein expression levels consistent with a G1 phase arrest. Initial studies in primary NPCs isolated from the ventral forebrain of embryonic mice demonstrated that TCDD reduced cell proliferation through a G1 phase arrest, corroborating our findings in the C17.2 cell line. Together, these observations suggest that the inappropriate or sustained activation of AhR by TCDD during neurogenesis can interfere with signaling pathways that regulate neuroepithelial stem cell/NPC proliferation, which could adversely impact final cell number in the brain and lead to functional impairments