PERK as a Central Mediator of Neurotoxicity in Tauopathies

The overall objective of this study is to evaluate the pathogenic role of PERK in tauopathies. Our working hypothesis is that tau hampers neuronal function by chronically activating the PERK pathway. We will test our hypotheses in three aims. Aim 1 will test the impact of PERK activity on tau-mediated synaptic dysfunction. We will determine changes in various measures of brain function in tau transgenic mice. PERK activity will be manipulated in two ways: chemically, we will use a novel PERK inhibitor (GSK2606414); genetically, we will cross the tau transgenic mouse we currently have (rTg4510) with a conditional PERK knockout mouse model. The primary outcome measures are: to measure changes in the rate of protein synthesis using SUnSET (a non-radioactive pulse assay performed in vivo in the brain), changes in broad neuronal function using manganese-enhanced MRI (to measure changes in brain function longitudinally), changes in synaptic plasticity using electrophysiology (e.g. LTP, LTD, etc.), and changes in cognition using a battery of behavioral tests (e.g. RAWM, NOR, etc.). Secondarily, we will measure changes in the levels of pathological tau species and proteins that mediate the PERK pathway to confirm that the inhibitor treatment was effective. These proteins will be measured using Meso Scale Device (MSD) plates, western blots (WB), and immunohistochemistry (IHC). We expect PERK inhibition will reduce the damage induced by tau in the brain. Aim 2 will test the extent to which PERK activation induces tau pathogenesis. We will measure changes in the levels and distribution of 11 pathological tau species in tau transgenic mice treated with a PERK activator or in tau transgenic injected with lentiviral particles coding for PERK. Tau expression in rTg4510 mice is regulated by a TET/OFF system, where addition of tetracycline to the diet suppresses production of tau. Therefore, to establish whether PERK activation stimulates tau pathogenesis we will suppress tau expression in the tau transgenic mice using tetracycline. Once the chemical and genetic manipulations to activate PERK are performed, we will remove tetracycline from the diet and allow tau to be produced. Chemically, we will perform acute and chronic treatment paradigms, in which the PERK activator will be injected once, when mice are two months of age, and pathological tau species will be measured after 7d. Chronic treatment involves the continuous infusion of the PERK activator for 3 months (starting at 2mo). Genetically, mice will be injected with lenti-PERK as neonates while on tet diet. At 2 months, when robust lentiviral protein expression is evident, we will remove the tetracycline diet. For an acute paradigm, we will measure changes in protein levels after 7d post-tau expression. For a chronic paradigm, we will perform protein measurements 3mo after removal of the tet diet. The primary outcome measures (pathological tau species) will be established using standard biochemical techniques (MSD and WB) as well as IHC, the latter of which will reveal changes in distribution of tauopathic proteins as a consequence of PERK activation. Secondary measures include determination of the levels of PERK pathway proteins. We expect PERK activation will increase the levels of pathogenic tau species. To confirm activation of the downstream PERK-mediated attenuation of translation as well as adaptive and pro-apoptotic consequences of PERK activity, we will measure protein synthesis (SUnSET) and changes in the levels of PERK pathway proteins, including upregulation of protein expression with RT-PCR. Aim 3 will test the correlation between the progression of human tauopathy and the extent of activation of the PERK pathway in different tauopathies. Our hypothesis is that progression of tauopathy correlates with the transition of adaptive response of the PERK pathway to the pro-apoptotic response. We will measure changes in the levels of proteins involved in the PERK pathway and tau burden in post-mortem tauopathic human brains from the UK-ADC biospecimen repository. Proteins will be measured using IHC imaged using an unbiased system called the Aperio ScanScope, which is housed in the Sanders-Brown Center on Aging.