In Vivo Validation of a Small Molecule Inhibitor of Tau Self-Association in htau Mice.

Tau oligomers have been shown to transmit tau pathology from diseased neurons to healthy neurons through seeding, tau misfolding, and aggregation that is thought to play an influential role in the progression of Alzheimer's disease (AD) and related tauopathies. To develop a small molecule therapeutic for AD and related tauopathies, we have developed in vitro and cellular assays to select molecules inhibiting the first step in tau aggregation, the self-association of tau into oligomers. In vivo validation studies of an optimized lead compound were independently performed in the htau mouse model of tauopathy that expresses the human isoforms of tau without inherited tauopathy mutations that are irrelevant to AD. Treated mice did not show any adverse events related to the compound. The lead compound significantly reduced the level of self-associated tau and total and phosphorylated insoluble tau aggregates. The dose response was linear with respect to levels of compound in the brain. A confirmatory study was performed with male htau mice that gave consistent results. The results validated our screening approach by showing that targeting tau self-association can inhibit the entire tau aggregation pathway by using the selected and optimized lead compound whose activity translated from in vitro and cellular assays to an in vivo model of tau aggregation.

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 ß-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.

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 (Aß) plaques and reduces Aß 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.

Therapeutic applications of antibodies in non-infectious neurodegenerative diseases.

Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease, Huntington's disease (HD) or amyotrophic lateral sclerosis (ALS) are all characterised histologically by the presence of deposits of misfolded proteins, tau and amyloid-ß, 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 body's 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.

Distinct patterns of tau-dependent process formation in mammalian cell lines.

Tau is a microtubule-associated protein involved in axonal elongation and central to the pathogenesis of a number of neurodegenerative conditions. To better establish the contribution of the cellular context to tau-dependent microtubule organization, we compared the phenotypes resulting from heterologous tau expression in different mammalian cell lines after disruption of the actin cytoskeleton. After cytochalasin D treatment, tau-expressing CHO cells display one or two long neurite-like extensions whereas cells transfected with MAP2c developed multiple shorter processes. By contrast, under the same conditions, tau-transfected PtK2 cells elaborate microtubule bundles forming numerous processes. These results suggest that cell-specific factors are involved in tau-dependent microtubule organization, a notion that could facilitate functional assessment of tau abnormalities associated with neurodegenerative disease.

Mutant (R406W) human tau is hyperphosphorylated and does not efficiently bind microtubules in a neuronal cortical cell model.

Frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17) is an autosomal dominant neurodegenerative disorder caused by mutations in the gene that encodes for tau, a microtubule-binding protein. Neuropathologically the disease is characterized by extensive neuronal loss in the frontal and temporal lobes and the filamentous accumulation of hyperphosphorylated tau. The R406W missense mutation was originally described in an American and a Dutch family. Although R406W tau is hyperphosphorylated in FTDP-17 cases, R406W tau expressed in cell model systems has not shown increased phosphorylation. The purpose of this study was to establish a neuronal model system in which the phosphorylation of R406W tau is increased and thus more representative of the in vivo situation. To accomplish this goal immortalized mouse cortical cells that express low levels of endogenous tau were stably transfected with human wild type or R406W tau. In this neuronal model R406W tau was more highly phosphorylated at numerous epitopes and showed decreased microtubule binding compared with wild type tau, an effect that could be reversed by dephosphorylation. In addition the expression of R406W tau in the cortical cells resulted in increased cell death as compared with wild type tau-expressing cells when the cells were exposed to an apoptotic stressor. These results indicate that in an appropriate cellular context R406W tau is hyperphosphorylated, which leads to decreased microtubule binding. Furthermore, expression of R406W tau sensitized cells to apoptotic stress, which may contribute to the neuronal cell loss that occurs in this FTDP-17 tauopathy.

Cdk5 phosphorylates p53 and regulates its activity.

Cyclin dependent kinase 5 (Cdk5) is a proline-direct protein kinase that is most active in the CNS, and has been implicated as a contributing factor in certain neurodegenerative diseases. Further, there is evidence to suggest that Cdk5 may facilitate the progression of apoptosis. However, the mechanisms involved have not been elucidated. The tumor suppressor protein p53, a transcription factor that is regulated by phosphorylation, increases the expression of genes that control growth arrest or cell death. To understand how Cdk5 could facilitate apoptosis, the effects of Cdk5 on p53 activity were examined. In the present study it is shown that in apoptotic PC12 cells the levels of p53 and Cdk5 increase concomitantly. Further, Cdk5/p25 effectively phosphorylates recombinant p53 in vitro. Transient transfection of Cdk5/p25 into cells results in an increase in p53 levels, as well as the expression of the p53-responsive genes p21 and Bax. Furthermore, evidence is provided that increased Cdk5 activity increases p53 transcriptional activity significantly, suggesting that p53 is modulated in situ by Cdk5. This is the first demonstration that p53 is a substrate of Cdk5, and that Cdk5 can modulate p53 levels and activity.