Pavan Krishnamurthy

Anesthesia leads to tau hyperphosphorylation through inhibition of phosphatase activity by hypothermia.

Postoperative cognitive dysfunction, confusion, and delirium are common after general anesthesia in the elderly, with symptoms persisting for months or years in some patients. Even middle-aged patients are likely to have postoperative cognitive dysfunction for months after surgery, and Alzheimers disease (AD) patients appear to be particularly at risk of deterioration after anesthesia. Several investigators have thus examined whether general anesthesia is associated with AD, with some studies suggesting that exposure to anesthetics may increase the risk of AD. However, little is known on the biochemical consequences of anesthesia on pathogenic pathways in vivo. Here, we investigated the effect of anesthesia on tau phosphorylation and amyloid precursor protein (APP) metabolism in mouse brain. We found that, regardless of the anesthetic used, anesthesia induced rapid and massive hyperphosphorylation of tau, rapid and prolonged hypothermia, inhibition of Ser/Thr PP2A (protein phosphatase 2A), but no changes in APP metabolism or Aβ (β-amyloid peptide) accumulation. Reestablishing normothermia during anesthesia completely rescued tau phosphorylation to normal levels. Our results indicate that changes in tau phosphorylation were not a result of anesthesia per se, but a consequence of anesthesia-induced hypothermia, which led to inhibition of phosphatase activity and subsequent hyperphosphorylation of tau. These findings call for careful monitoring of core temperature during anesthesia in laboratory animals to avoid artifactual elevation of protein phosphorylation. Furthermore, a thorough examination of the effect of anesthesia-induced hypothermia on the risk and progression of AD is warranted.

Anesthesia-Induced Hyperphosphorylation Detaches 3-Repeat Tau from Microtubules without Affecting Their Stability In Vivo

In Alzheimers disease, tau is hyperphosphorylated, which is thought to detach it from microtubules (MTs), induce MT destabilization, and promote aggregation. Using a previously described in vivo model, we investigated whether hyperphosphorylation impacts tau function in wild-type and transgenic mice. We found that after anesthesia-induced hypothermia, MT-free tau was hyperphosphorylated, which impaired its ability to bind MTs and promote MT assembly. MT-bound tau was more resistant to hyperphosphorylation compared with free tau and tau did not dissociate from MTs in wild-type mice. However, 3-repeat tau detached from MT in the transgenic mice. Surprisingly, dissociation of tau from MTs did not lead to overt depolymerization of tubulin, and there was no collapse, or disturbance of axonal MT networks. These results indicate that, in vivo, a subpopulation of tau bound to MTs does not easily dissociate under conditions that extensively phosphorylate tau. Tau remaining on the MTs under these conditions is sufficient to maintain MT network integrity.

Tau as a therapeutic target for Alzheimer's disease.

Neurofibrillary tangles (NFTs) are one of the pathological hallmarks of Alzheimers disease (AD) and are primarily composed of aggregates of hyperphosphorylated forms of the microtubule associated protein tau. It is likely that an imbalance of kinase and phosphatase activities leads to the abnormal phosphorylation of tau and subsequent aggregation. The wide ranging therapeutic approaches that are being developed include to inhibit tau kinases, to enhance phosphatase activity, to promote microtubule stability, and to reduce tau aggregate formation and/or enhance their clearance with small molecule drugs or by immunotherapeutic means. Most of these promising approaches are still in preclinical development whilst some have progressed to Phase II clinical trials. By pursuing these lines of study, a viable therapy for AD and related tauopathies may be obtained.

Mechanistic Studies of Antibody-Mediated Clearance of Tau Aggregates Using an ex vivo Brain Slice Model.

Recent studies have shown that immunotherapy clears amyloid beta (Aβ) plaques and reduces Aβ levels in mouse models of Alzheimer’s disease (AD), as well as in AD patients. Tangle pathology is also relevant for the neurodegeneration in AD, and our studies have shown that active immunization with an AD related phospho-tau peptide reduces aggregated tau within the brain and slows the progression of tauopathy-induced behavioral impairments. Thus, clearance of neurofibrillary tangles and/or their precursors may reduce synaptic and neuronal loss associated with AD and other tauopathies. So far the mechanisms involved in antibody-mediated clearance of tau pathology are yet to be elucidated. In this study we have used a mouse brain slice model to examine the uptake and localization of FITC labeled anti-tau antibodies. Confocal microscopy analysis showed that the FITC labeled anti-tau antibody co-stained with phosphorylated tau, had a perinuclear appearance and co-localized with markers of the endosomal/lysosomal pathway. Additionally, tau and FITC–IgG were found together in an enriched lysosome fraction. In summary, antibody-mediated clearance of intracellular tau aggregates appears to occur via the lysosomal pathway.

Tau pathology induces loss of GABAergic interneurons leading to altered synaptic plasticity and behavioral impairments

BackgroundTau is a microtubule stabilizing protein and is mainly expressed in neurons. Tau aggregation into oligomers and tangles is considered an important pathological event in tauopathies, such as frontotemporal dementia (FTD) and Alzheimer’s disease (AD). Tauopathies are also associated with deficits in synaptic plasticity such as long-term potentiation (LTP), but the specific role of tau in the manifestation of these deficiencies is not well-understood. We examined long lasting forms of synaptic plasticity in JNPL3 (BL6) mice expressing mutant tau that is identified in some inherited FTDs.ResultsWe found that aged (>12 months) JNPL3 (BL6) mice exhibit enhanced hippocampal late-phase (L-LTP), while young JNPL3 (BL6) mice (age 6 months) displayed normal L-LTP. This enhanced L-LTP in aged JNPL3 (BL6) mice was rescued with the GABAAR agonist, zolpidem, suggesting a loss of GABAergic function. Indeed, we found that mutant mice displayed a reduction in hippocampal GABAergic interneurons. Finally, we also found that expression of mutant tau led to severe sensorimotor-gating and hippocampus-dependent memory deficits in the aged JNPL3 (BL6) mice.ConclusionsWe show for the first time that hippocampal GABAergic function is impaired by pathological tau protein, leading to altered synaptic plasticity and severe memory deficits. Increased understanding of the molecular mechanisms underlying the synaptic failure in AD and FTD is critical to identifying targets for therapies to restore cognitive deficiencies associated with tauopathies.

Antibody-Derived In Vivo Imaging of Tau Pathology

Antibodies or their derivatives as imaging probes for pathological tau protein have great potential, but have not been well studied. In particular, smaller, single-chain-variable antibody fragments (scFvs) are attractive for detecting tau lesions in live subjects. Here, we generated libraries of scFvs and identified numerous phospho-tau-selective scFvs. Peripheral injection of one of these scFvs consistently resulted in a strong in vivo brain signal in transgenic tauopathy mice, but not in wild-type or amyloid-β plaque mice. The parent tau antibody provided similar results, albeit with a weaker signal intensity. The imaging signal correlated very well with colocalization of the probe with intraneuronal tau aggregates. Both were associated with markers of endosomes, autophagosomes, and lysosomes, suggesting their interaction in these degradation pathways. Such specific antibody-derived imaging probes have great potential as diagnostic markers for Alzheimers disease and related tauopathies.

Non-invasive, in vivo monitoring of neuronal transport impairment in a mouse model of tauopathy using MEMRI

The impairment of axonal transport by overexpression or hyperphosphorylation of tau is well documented for in vitro conditions; however, only a few studies on this phenomenon have been conducted in vivo, using invasive procedures, and with contradictory results. Here we used the non-invasive, Manganese-Enhanced Magnetic Resonance Imaging technique (MEMRI), to study for the first time a pure model of tauopathy, the JNPL3 transgenic mouse line, which overexpresses a mutated (P301L) form of the human tau protein. We show progressive impairment in neuronal transport as tauopathy advances. These findings are further supported by a significant correlation between the severity of the impairment in neuronal transport assessed by MEMRI, and the degree of abnormal tau assessed by histology. Unlike conventional techniques that focus on axonal transport measurement, MEMRI can provide a global analysis of neuronal transport, i.e. from dendrites to axons and at the macroscopic scale of fiber tracts. Neuronal transport impairment has been shown to be a key pathogenic process in Alzheimers disease and numerous other neurodegenerative disorders. Hence, MEMRI provides a promising set of functional biomarkers to be used during preclinical trials to facilitate the selection of new drugs aimed at restoring neuronal transport in neurodegenerative diseases.

Therapeutic applications of antibodies in non-infectious neurodegenerative diseases.

Neurodegenerative diseases such as Alzheimers disease (AD), Parkinsons disease, Huntingtons disease (HD) or amyotrophic lateral sclerosis (ALS) are all characterised histologically by the presence of deposits of misfolded proteins, tau and amyloid-β, α-synuclein, huntingtin or superoxide dismutase, respectively. Currently, these illnesses do not have any disease modifying treatment options. A novel therapeutic strategy that is being pursued is immunomodulation, which is using the bodys immune system to target the self-proteins that are deposited. Most of these promising approaches are still in preclinical development while some have progressed to Phase III clinical trials. As new insights are gained, it is hoped that these immunotherapies will be effective tools at slowing the progression of these debilitating diseases.

Mechanistic studies of antibody mediated clearance of tau aggregates using an ex vivo brain slice model

neurodegeneration. This is an important area of investigation, which may lead to more timely and effective interventions. Methods: We investigated synaptic plasticity and the dendritic localization of human tau (htau) in the rTg4510 mouse model of tauopathy at an age (4.5M) when the mice are cognitively impaired but exhibit no overt synapse or neuron loss. Subsequently, we examined the dendritic localization of htau and synaptic function in transfected, dissociated rat hippocampal neurons. Finally, we studied surface AMPA receptors in neurons cultured from mice expressing htau. Results: We found higher concentrations of htau in the postsynaptic densities of impaired rTg4510 mice expressing htauP301L than intact rTg21221 mice expressing wild-type htauWT. This mislocalization of tau to dendritic spines also occurs to a greater extent in rat hippocampal neurons transfected with htauP301L than htauWT. Inhibiting proline-directed phosphorylation by substituting alanine at 14 serine and threonine residues blocked the mislocalization of htauP301L to dendritic spines. Conversely, pseudo-hyperphosphorylation of these 14 residues by substituting glutamate mimics htauP301L mislocalization. Spines containing htau showed decreased frequencies and amplitudes of miniature excitatory postsynaptic currents, and reduced levels of surface AMPA receptors. Conclusions: The earliest tau-related neurological deficits develop not from the loss of synapses or neurons, but rather as a result of synaptic dysfunction arising from the mislocalization of tau to dendritic spines, which is accompanied by a reduction in surface AMPA receptors.

Sex and Immunogen-Specific Benefits of Immunotherapy Targeting Islet Amyloid Polypeptide in Transgenic and Wild-Type Mice

Type 2 diabetes mellitus is characterized by the deposition of islet amyloid polypeptide (IAPP) as amyloid in islets, a process thought to be toxic to β-cells. To determine the feasibility of targeting these aggregates therapeutically, we vaccinated transgenic (Tg) mice that overexpress human IAPP and were fed a high-fat diet to promote their diabetic phenotype. Our findings indicate that prophylactic vaccination with IAPP and its derivative IAPP7-19-TT, protects wild-type female mice, but not males, from obesity-induced early mortality, and the derivative showed a strong trend for prolonging the lifespan of Tg females but not males. Furthermore, IAPP7-19-TT-immunized Tg females cleared a glucose bolus more efficiently than controls, while IAPP-immunized Tg females showed an impaired ability to clear a glucose bolus compared to their adjuvant injected Tg controls. Interestingly, IAPP or IAPP7-19-TT treatments had no effect on glucose clearance in Tg males. Overall, these beneficial effects of IAPP targeted immunization depend on Tg status, sex, and immunogen. Hence, future studies in this field should carefully consider these variables that clearly affect the therapeutic outcome. In conclusion, IAPP targeting immunotherapy may have benefits in patients with type 2 diabetes.