Shelby E. Meier

Pathological Tau Promotes Neuronal Damage by Impairing Ribosomal Function and Decreasing Protein Synthesis

One of the most common symptoms of Alzheimers disease (AD) and related tauopathies is memory loss. The exact mechanisms leading to memory loss in tauopathies are not yet known; however, decreased translation due to ribosomal dysfunction has been implicated as a part of this process. Here we use a proteomics approach that incorporates subcellular fractionation and coimmunoprecipitation of tau from human AD and non-demented control brains to identify novel interactions between tau and the endoplasmic reticulum (ER). We show that ribosomes associate more closely with tau in AD than with tau in control brains, and that this abnormal association leads to a decrease in RNA translation. The aberrant tau–ribosome association also impaired synthesis of the synaptic protein PSD-95, suggesting that this phenomenon contributes to synaptic dysfunction. These findings provide novel information about tau-protein interactions in human brains, and they describe, for the first time, a dysfunctional consequence of tau–ribosome associations that directly alters protein synthesis. SIGNIFICANCE STATEMENT Despite the identification of abnormal tau–ribosomal interactions in tauopathies >25 years ago, the consequences of this association remained elusive until now. Here, we show that pathological tau associates closely with ribosomes in AD brains, and that this interaction impairs protein synthesis. The overall result is a stark reduction of nascent proteins, including those that participate in synaptic plasticity, which is crucial for learning and memory. These data mechanistically link a common pathologic sign, such as the appearance of pathological tau inside brain cells, with cognitive impairments evident in virtually all tauopathies.

MW151 Inhibited IL-1β Levels after Traumatic Brain Injury with No Effect on Microglia Physiological Responses

A prevailing neuroinflammation hypothesis is that increased production of proinflammatory cytokines contributes to progressive neuropathology, secondary to the primary damage caused by a traumatic brain injury (TBI). In support of the hypothesis, post-injury interventions that inhibit the proinflammatory cytokine surge can attenuate the progressive pathology. However, other post-injury neuroinflammatory responses are key to endogenous recovery responses. Therefore, it is critical that pharmacological attenuation of detrimental or dysregulated neuroinflammatory processes avoid pan-suppression of inflammation. MW151 is a CNS-penetrant, small molecule experimental therapeutic that restores injury- or disease-induced overproduction of proinflammatory cytokines towards homeostasis without immunosuppression. Post-injury administration of MW151 in a closed head injury model of mild TBI suppressed acute cytokine up-regulation and downstream cognitive impairment. Here, we report results from a diffuse brain injury model in mice using midline fluid percussion. Low dose (0.5–5.0 mg/kg) administration of MW151 suppresses interleukin-1 beta (IL-1β) levels in the cortex while sparing reactive microglia and astrocyte responses. To probe molecular mechanisms, we used live cell imaging of the BV-2 microglia cell line to demonstrate that MW151 does not affect proliferation, migration, or phagocytosis of the cells. Our results provide insight into the roles of glial responses to brain injury and indicate the feasibility of using appropriate dosing for selective therapeutic modulation of injurious IL-1β increases while sparing other glial responses to injury.

Identification of Novel Tau Interactions with Endoplasmic Reticulum Proteins in Alzheimer’s Disease Brain

Alzheimers disease (AD) is a progressive neurodegenerative disorder that is pathologically characterized by the formation of extracellular amyloid plaques and intraneuronal tau tangles. We recently identified that tau associates with proteins known to participate in endoplasmic reticulum (ER)-associated degradation (ERAD); consequently, ERAD becomes dysfunctional and causes neurotoxicity. We hypothesized that tau associates with other ER proteins, and that this association could also lead to cellular dysfunction in AD. Portions of human AD and non-demented age matched control brains were fractionated to obtain microsomes, from which tau was co-immunoprecipitated. Samples from both conditions containing tau and its associated proteins were analyzed by mass spectrometry. In total, we identified 91 ER proteins that co-immunoprecipitated with tau; 15.4% were common between AD and control brains, and 42.9% only in the AD samples. The remainder, 41.8% of the proteins, was only seen in the control brain samples. We identified a variety of previously unreported interactions between tau and ER proteins. These proteins participate in over sixteen functional categories, the most abundant being involved in RNA translation. We then determined that association of tau with these ER proteins was different between the AD and control samples. We found that tau associated equally with the ribosomal protein L28 but more robustly with the ribosomal protein P0. These data suggest that the differential association between tau and ER proteins in disease could reveal the pathogenic processes by which tau induces cellular dysfunction.

Identification of changes in neuronal function as a consequence of aging and tauopathic neurodegeneration using a novel and sensitive magnetic resonance imaging approach

Tauopathies, the most common of which is Alzheimers disease (AD), constitute the most crippling neurodegenerative threat to our aging population. Tauopathic patients have significant cognitive decline accompanied by irreversible and severe brain atrophy, and it is thought that neuronal dysfunction begins years before diagnosis. Our current understanding of tauopathies has yielded promising therapeutic interventions but have all failed in clinical trials. This is partly due to the inability to identify and intervene in an effective therapeutic window early in the disease process. A major challenge that contributes to the definition of an early therapeutic window is limited technologies. To address these challenges, we modified and adapted a manganese-enhanced magnetic resonance imaging (MEMRI) approach to provide sensitive and quantitative power to detect changes in broad neuronal function in aging mice. Considering that tau tangle burden correlates well with cognitive impairment in Alzheimers patients, we performed our MEMRI approach in a time course of aging mice and an accelerated mouse model of tauopathy. We measured significant changes in broad neuronal function as a consequence of age, and in transgenic mice, before the deposition of bona fide tangles. This MEMRI approach represents the first diagnostic measure of neuronal dysfunction in mice. Successful translation of this technology in the clinic could serve as a sensitive diagnostic tool for the definition of effective therapeutic windows.

Non-invasive detection of adeno-associated viral gene transfer using a genetically encoded CEST-MRI reporter gene in the murine heart

Research into gene therapy for heart failure has gained renewed interest as a result of improved safety and availability of adeno-associated viral vectors (AAV). While magnetic resonance imaging (MRI) is standard for functional assessment of gene therapy outcomes, quantitation of gene transfer/expression relies upon tissue biopsy, fluorescence or nuclear imaging. Imaging of gene expression through the use of genetically encoded chemical exchange saturation transfer (CEST)-MRI reporter genes could be combined with clinical cardiac MRI methods to comprehensively probe therapeutic gene expression and subsequent outcomes. The CEST-MRI reporter gene Lysine Rich Protein (LRP) was cloned into an AAV9 vector and either administered systemically via tail vein injection or directly injected into the left ventricular free wall of mice. Longitudinal in vivo CEST-MRI performed at days 15 and 45 after direct injection or at 1, 60 and 90 days after systemic injection revealed robust CEST contrast in myocardium that was later confirmed to express LRP by immunostaining. Ventricular structure and function were not impacted by expression of LRP in either study arm. The ability to quantify and link therapeutic gene expression to functional outcomes can provide rich data for further development of gene therapy for heart failure.

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.