Joe F. Abisambra

TAU MODIFIES RIBOSOMAL DYNAMICS SHIFTING TRANSLATIONAL PROFILES IN AD

human MAPT against a Mapt background and manifests early (w3m) somatodendritic relocalisation of tau and late (>12m) pathological and behavioural changes reminiscent of AD. We produced AAV9 vectors for CMV promoter-driven expression of a full-length MAPT-AS1 transcript (tNAT1-FL) and aminimised artificial variant as well as an inactive deletion variant (DM). Adult htau mice (912m) were injected into right hippocampus with AAV9-CMV vectors for tNAT1-FL and DM and AAV9-CMV-eGFP as control. Brains harvested 8 weeks later were analysed by Western blot and real-time qRT-PCR. Results: With AAV9-CMV-eGFP, we showed extensive CNS spread of GFP without any site-specific tropism. Eight weeks post-injection, both ipsiand contralateral sides of htau mice injected with AAV9-CMV with tNAT1-FL showed robust reduction (up to 70%) of tau protein levels that correlate with spread and levels of the tNAT1-FL transcript. In contrast, therewere no changes in tau protein levels with expression ofDM transcript. Conclusions:MAPT-AS1 presents a novel opportunity for therapeutic tau reduction with the advantage of exploiting physiological repression of CNS tau. The excellent safety profile and robust CNS spread and persistence of AAV has made them vector of choice for CNS-targetted gene therapy. The htau mouse model gives us the platform for the pre-clinical study of the benefits of this tau reduction in slowing pathological tau progression and alleviating behavioural deficits displayed by these transgenic mice. [1] Andorfer, C., et al., J Neurochem, 2003. 86:582-90.

TRANSCRIPTOMIC PROFILING OF TAUOPATHY REVEALS GENE POPULATIONS RESPONSIVE TO TAU EXPRESSION AND A SUBPOPULATION OF THERAPEUTICALLY RELEVANT GENES

Background:Metformin is used for the treatment of insulin resistant diabetes. Diabetics are at an increased risk of developing dementia and insulin resistance in the CNS has been found in Alzheimer’s disease (AD). Recent epidemiological studies suggest in diabetics that metformin treatment prevents cognitive decline. A pilot clinical study found cognitive improvement with metformin in patients with MCI. Preclinical studies have found that metformin increases PKC, a regulator of the Ab and Tau pathways. In addition, studies have found that metformin reduces AD-like pathology in mouse models of AD. The SAMP8 mouse is a model of spontaneous onset of AD. The SAMP8 mice have age-related impairment in learning and memory that correlates with an elevation of amyloid precursor protein (APP), amyloid beta (Ab), and hyperphosphorylated tau (pTau). Methods: In the current study, we used 11 month old SAMP8 mice. Mice were given daily injections of metformin at 20mg/kg or 200mg/kg for 8 weeks. After 4 weeks mice were tested in T-maze foot shock avoidance, object recognition and Barnes maze. At the end of the study brain tissue was collected for analysis of mitochondrial BAX, PKCz, PKCi, PKCa, PKCb, PKCg, PKCε,GSK-3b, pGSK-3Bser9, pGSK-3btyr216, pTau404 and APP analysis. Results:Metformin improved both acquisition and retention in SAMP8 mice in T-maze foot shock avoidance, retention in novel object recognition and acquisition in the Barnes maze. Biochemical analysis indicated that metformin decreased mitochondrial BAX, increased both atypical and conventional forms of PKC; PKCz, and PKCa at 20 mg/kg (P<0.05 and P<0.01 respectively). Metformin significantly increased pGSK3bser9 at 200mg/kg (P<0.05), and decreased pTau404 (P<0.05) and APPc99 (P<0.001) at both 20mg/kg and 200mg/kg. There was no difference in blood glucose levels between the aged vehicle treated mice and the mice that received metformin. Conclusions: Metformin improved learning and memory in the SAMP8 mouse model of spontaneous onset AD. Biochemical analysis indicates that metformin improve memory by increasing PKC which lead to alterations in pGSK-3b which decreases pTau and APP. The current study lends support to the therapeutic potential of metformin for AD.

A DYNAMIC PERK-TAU COMPLEX REGULATES TAU PHOSPHORYLATION, ER STRESS, AND TREATMENT OUTCOMES IN RTG4510 MICE

Background: Despite growing awareness of putative links between head injury and CTE, the substrates and mechanisms underpinning this association, and relationships to concussion and TBI, remain largely unknown and matters of significant controversy. Most notably, there is insufficient knowledge regarding changes in brain structure and function during the acute-subacute period after head injury that may represent the earliest antecedent pathologies of CTE. Methods: Here we combined human clinicopathological correlation analysis, animal and experimental modeling, and biomechanical and computational simulations to investigate these questions.Results:We examined postmortem brains from teenage athletes in the acute-subacute period after symptomatic closed-head impact injury and found astrocytosis, axonopathy, microvascular injury, perivascular neuroinflammation, and phosphorylated tau protein pathology. To investigate causalmechanisms, we developed a mouse model of closed-head impact injury that uses momentum transfer to induce traumatic head acceleration. Experimental impact injury was associated with axonopathy, bloodbrain barrier disruption, TGFb1/pSMAD2-associated astrocytosis, TREM2+ microgliosis, monocyte infiltration, and phosphorylated tauopathy in ipsilateral cerebral cortex. Phosphorylated tauopathy was detected in ipsilateral axons by 24 hours, bilateral axons and soma by 2 weeks, and distant cortex bilaterally at 5.5 months postinjury. Impact pathologies colocalized with serum albumin extravasation in the brain that was diagnostically detectable in living mice by dynamic contrast-enhanced magnetic resonance imaging. Transient neurobehavioral deficits at the time of injury did not correlate with blood-brain barrier disruption, microgliosis, neuroinflammation, phosphorylated tauopathy, or electrophysiological dysfunction postinjury. Computational modeling showed that impact injury generated point loading on the head and high-amplitude peak shear stress in the brain. Moreover, intracerebral shear stress peaked before onset of gross head motion. Conclusions:We conclude that fast-acting, highamplitude cortical shear stress triggers acute neurobehavioral deficits associated with concussion, whereas longer duration, lower amplitude shear stress associatedwith headmotion induces structural brain damage and neuropathological sequelae. These results suggest that closed-head impact injuries, independent of concussive signs, can induce traumatic brain injury as well as early pathologies and functional sequelae associated with chronic traumatic encephalopathy. These findings also shed light on the origins of concussion anddifferentiate this condition from traumatic brain injury and sequelae.

NOVEL APPLICATIONS OF MRI TECHNIQUES IN THE DETECTION OF NEURONAL DYSFUNCTION BEFORE TANGLE PATHOLOGY IN TAU TRANSGENIC MICE

the two signals. Semi-quantitative analysis of the signal intensity in the forebrain region revealed a significant accumulation of fluorine19 MR signal in the rTg4510 mice, compared with the wild-type mice. Histological analysis showed fluorescent signals of ShigaX35 binding to the neurofibrillary tangles in the brain sections of rTg4510 mice. Conclusions: Our data indicate that fluorine-19 MRI using Shiga-X35 would be a powerful tool to evaluate the accumulation of neurofibrillary tangles in the brain.

POST-INJURY PERK INHIBITION IN MOUSE MODEL OF TAUOPATHY

Background:Pathological aggregations of tau and amyloid beta proteins are the hallmarks of Alzheimer’s disease. Previous work from our group has shown Abl-selective tyrosine kinase inhibitors stimulate beclin-mediated autophagy and promote clearance of neurodegenerative proteins. Specifically, the drugs nilotinib and bosutinib decrease levels of pathological proteins and reverse motor and cognitive decline in mouse models of neurodegenerative disease. Further, we have shown pazopanib, an FDA-approved inhibitor of the tyrosine kinases VEGFR, PDGFRa, PDGFRb, and c-KIT, penetrates the blood-brain barrier and decreases p-tau levels in TauP301L mice. These experiments aim to confirm and expand upon the initial findings as well as determine the effects of pazopanib on amyloid beta in 3x-APP mice. Methods:Male and female TauP301L, 3x-APP, and non-transgenic littermates approximately 12-17 months old were treated with 5mg/kg pazopanib (roughly half the clinically-used dose) or vehicle (DMSO) intraperitoneally (IP) for 3-4 weeks. Phosphorylated tau levels were measured by Western blot and enzyme-linked immunosorbent assay in brain homogenates, and immunohistochemistry in 20mm brain sections, fixed in 4% paraformaldehyde. Serum was collected to assess kidney and liver function. Ab levels and inflammation were measured using MILLIPLEX ELISA. Autophagy markers were measured via Western blot. Results: Pazopanib treatment does not alter weight, liver (ALT), or kidney injury markers in TauP301L and 3x-APP mouse cohorts. Pazopanib significantly reduces levels of p-tau (T181, T231) in TauP301L mice. Further, brain levels of beclin-1 and p-mTOR/mTOR were unchanged. In 3x-APP mice, treatment does not alter Ab40 or Ab42levels. However, pazopanib significantly reverses levels of IP-10, MIP-1a, MIP-1b, and RANTES toward control levels. Conclusions: Pazopanib (5mg/kg) appears to be a safe, well-tolerated drug that significantly reduces p-tau levels in TauP301L mice in a manner likely independent of beclin or mTOR-mediated autophagy and reverses inflammation in 3x-APP mice. Future studies aim to determine which specific tyrosine kinase target(s) is complicit in p-tau clearance.

MANGANESE-ENHANCED MAGNETIC RESONANCE IMAGING (MEMRI)-BASED IDENTIFICATION OF NEURONAL DYSFUNCTION BEFORE THE APPEARANCE OF TAU PATHOLOGY IN RTG4510 MICE

Background:Alzheimer’s disease (AD) is considered to be one of the most devastating disorder known for the symptoms as weaken in memory, motor ability, and cognitive ability. AD is characterized by the deposition of amyloid tangles, more specifically, amyloidbeta (Ab), a polypeptide approximately 4 kDa. It is produced from the cleavage of amyloid precursor by secretases into two forms: Ab1-40 and Ab1-42, which Ab1-40 was found to be more abundant in AD.Monomeric formsofAb aggregateswith eachother to formprotofibrils and oligomers which eventually becomes fibrils that causes amyloid tangles in the brain. A humanized antibody Solanezumab was developed by Eli Lilly targeting the neurotoxic Ab peptides for anti-amyloid treatment. Although the outcome of the treatment is still under investigation, the interaction of Solanezumab and Ab became crucial in both the study ofAD. Surface PlasmonResonance analysis was conducted to determine the affinity of such interaction between the antibody and different forms of the Ab protein. Methods: Surface Plasmon Resonance (SPR) binding experiments were performed using a Biacore X100 SPR (GE Healthcare). Guided by the Biotin CAPture Kit (GE Healthcare), The sensor chips were activated using the Biotin CAPture Reagent and immobilized with Biotinylated Ab (Anaspec, CA). Analytes (Solanezumab-scFv) were injected at designated concentrations. To determine the full kinetic profile, protein-small molecules binding spectrograms were evaluated using the Biacore X100 Evaluation Software. Results: Analyses were conducted for both Ab1-40 and Ab1-42. The binding affinity (KD) between Solanezumab and Ab1-40 oligomer were detected to be 0.346 and 0.832 nM at 25 C and 37 C, respectively. On the other hand, the KD between Solanezumab and Ab1-42 oligomers were 0.103 nM and 0.061 nM at 25 C and 37 C, respectively. However, no detectable interactions were observed between the antibody and mature fibril forms of Ab. Conclusions:SPR analyses were performed for the interaction of Solanezumab andAb. The affinity of Ab1-42 oligomer was 3 to 5 times stronger than of Ab1-40 indicating the high efficacy of eliminating the toxic oligomers of Ab1-42. No detectable interactions of Ab fibrils were observed suggesting an unavailability of binding site for the antibody.