Danielle N. Lyons

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.

Orofacial neuropathic pain mouse model induced by Trigeminal Inflammatory Compression (TIC) of the infraorbital nerve

BackgroundTrigeminal neuropathic pain attacks can be excruciating for patients, even after being lightly touched. Although there are rodent trigeminal nerve research models to study orofacial pain, few models have been applied to studies in mice. A mouse trigeminal inflammatory compression (TIC) model is introduced here which successfully and reliably promotes vibrissal whisker pad hypersensitivity.ResultsThe chronic orofacial neuropathic pain model is induced after surgical placement of chromic gut suture in the infraorbital nerve fissure in the maxillary bone. Slight compression and chemical effects of the chromic gut suture on the portion of the infraorbital nerve contacted cause mild nerve trauma. Nerve edema is observed in the contacting infraorbital nerve bundle as well as macrophage infiltration in the trigeminal ganglia. Centrally in the spinal trigeminal nucleus, increased immunoreactivity for an activated microglial marker is evident (OX42, postoperative day 70). Mechanical thresholds of the affected whisker pad are significantly decreased on day 3 after chromic gut suture placement, persisting at least 10 weeks. The mechanical allodynia is reversed by suppression of microglial activation. Cold allodynia was detected at 4 weeks.ConclusionsA simple, effective, and reproducible chronic mouse model mimicking clinical orofacial neuropathic pain (Type 2) is induced by placing chromic gut suture between the infraorbital nerve and the maxillary bone. The method produces mild inflammatory compression with significant continuous mechanical allodynia persisting at least 10 weeks and cold allodynia measureable at 4 weeks.

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.

TRIGEMINAL INFLAMMATORY COMPRESSION (TIC) INJURY INDUCES CHRONIC FACIAL PAIN AND SUSCEPTIBILITY TO ANXIETY-RELATED BEHAVIORS

Our laboratory previously developed a novel neuropathic and inflammatory facial pain model for mice referred to as the Trigeminal Inflammatory Compression (TIC) model. Rather than inducing whole nerve ischemia and neuronal loss, this injury induces only slight peripheral nerve demyelination triggering long-term mechanical allodynia and cold hypersensitivity on the ipsilateral whisker pad. The aim of the present study is to further characterize the phenotype of the TIC injury model using specific behavioral assays (i.e. light-dark box, open field exploratory activity, and elevated plus maze) to explore pain- and anxiety-like behaviors associated with this model. Our findings determined that the TIC injury produces hypersensitivity 100% of the time after surgery that persists at least 21 weeks post injury (until the animals are euthanized). Three receptive field sensitivity pattern variations in mice with TIC injury are specified. Animals with TIC injury begin displaying anxiety-like behavior in the light-dark box preference and open field exploratory tests at week eight post injury as compared to sham and naïve animals. Panic anxiety-like behavior was shown in the elevated plus maze in mice with TIC injury if the test was preceded with acoustic startle. Thus, in addition to mechanical and cold hypersensitivity, the present study identified significant anxiety-like behaviors in mice with TIC injury resembling the clinical symptomatology and psychosocial impairments of patients with chronic facial pain. Overall, the TIC injury models chronicity, reproducibility, and reliability in producing pain- and anxiety-like behaviors demonstrate its usefulness as a chronic neuropathic facial pain model.

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.

Combination Drug Therapy of Pioglitazone and D-cycloserine Attenuates Chronic Orofacial Neuropathic Pain and Anxiety by Improving Mitochondrial Function Following Trigeminal Nerve Injury

Objectives: The study aim was to determine how peripheral trigeminal nerve injury affects mitochondrial respiration and to test efficacy of combined treatment with 2 Federal Drug Administration approved drugs with potential for improving mitochondrial bioenergetics, pain and anxiety–related behaviors in a chronic orofacial neuropathic pain mouse model. Methods: Efficacy of (R)-(+)-4-amino-3-isoxazolidinone (D-cycloserine, DCS), an N-Methyl-D-aspartate antagonist/agonist, and Pioglitazone (PIO), a selective agonist of nuclear receptor peroxisome proliferator-activated receptor gamma was investigate in the trigeminal inflammatory compression (TIC) neuropathic nerve injury mouse model. Combined low doses of these drugs (80 mg/kg DCS and 100 mg/kg PIO) were given as a single bolus or daily for 7 days post-TIC to test ability to attenuate neuropathic nociceptive and associated cognitive dependent anxiety behaviors. In addition, beneficial effects of the DCS/PIO drug combination were explored ex vivo in isolated cortex/brainstem mitochondria at 28 weeks post-TIC. Results: The DCS/PIO combination not only attenuated orofacial neuropathic pain and anxiety–related behaviors associated with trigeminal nerve injury, but it also improved mitochondrial bioenergetics. Discussion: The DCS/PIO combination uncoupled mitochondrial respiration in the TIC model to improve cortical mitochondrial dysfunction, as well as reduced nociceptive and anxiety behaviors present in mice with centralized chronic neuropathic nerve injury. Combining these drugs could be a beneficial treatment for patients with depression, anxiety, or other psychological conditions due to their chronic pain status.

Pparγ Agonists Attenuate Trigeminal Neuropathic Pain

Objectives: The aim of this study is to investigate the role of peroxisome proliferator-activated receptor-gamma isoform (PPAR&ggr;), in trigeminal neuropathic pain utilizing a novel mouse trigeminal inflammatory compression (TIC) injury model. Results: The study determined that the PPAR&ggr; nuclear receptor plays a significant role in trigeminal nociception transmission, evidenced by: 1) Intense PPAR&ggr; immunoreactivity is expressed 3 weeks after TIC nerve injury in the spinal trigeminal caudalis, the termination site of trigeminal nociceptive nerve fibers. 2) Systemic administration of a PPAR&ggr; agonist, pioglitazone (PIO), attenuates whisker pad mechanical allodynia at doses of 300 mg/kg i.p. and 600 mg/kg p.o. 3) Administration of a PPAR&ggr; antagonist, GW9662 (30 mg/kg i.p.), prior to providing the optimal dose of PIO (300 mg/kg i.p.) blocked the analgesic effect of PIO. Discussion: This is the first study localizing PPAR&ggr; immunoreactivity throughout the brainstem trigeminal sensory spinal nucleus (spV) and its increase three weeks after TIC nerve injury. This is also the first study to demonstrate that activation of PPAR&ggr; attenuates trigeminal hypersensitivity in the mouse TIC nerve injury model. The findings presented here suggest the possibility of utilizing the FDA approved diabetic treatment drug, PIO, as a new therapeutic that targets PPAR&ggr; for treatment of patients suffering from orofacial neuropathic pain.

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.