Towards a Mechanistic Model of Tau-Mediated Pathology in Tauopathies: What Can We Learn from Cell-Based In Vitro Assays?

Tauopathies are a group of neurodegenerative diseases characterized by the hyperphosphorylation and deposition of tau proteins in the brain. In Alzheimer's disease, and other related tauopathies, the pattern of tau deposition follows a stereotypical progression between anatomically connected brain regions. Increasing evidence suggests that tau behaves in a prion-like manner, and that seeding and spreading of pathological tau drive progressive neurodegeneration. Although several advances have been made in recent years, the exact cellular and molecular mechanisms involved remain largely unknown. Since there are no effective therapies for any tauopathy, there is a growing need for reliable experimental models that would provide us with better knowledge and understanding of their etiology and identify novel molecular targets. In this review, we will summarize the development of cellular models for modeling tau pathology. We will discuss their different applications and contributions to our current understanding of the prion-like nature of pathological tau

Capacity for seeding and spreading of argyrophilic grain disease in a wild-type murine model; comparisons with primary age-related tauopathy

Argyrophilic grain disease (AGD) is a common 4R-tauopathy, causing or contributing to cognitive impairment in the elderly. AGD is characterized neuropathologically by pre-tangles in neurons, dendritic swellings called grains, threads, thorn-shaped astrocytes, and coiled bodies in oligodendrocytes in the limbic system. AGD has a characteristic pattern progressively involving the entorhinal cortex, amygdala, hippocampus, dentate gyrus, presubiculum, subiculum, hypothalamic nuclei, temporal cortex, and neocortex and brainstem, thus suggesting that argyrophilic grain pathology is a natural model of tau propagation. One series of WT mice was unilaterally inoculated in the hippocampus with sarkosyl-insoluble and sarkosyl-soluble fractions from 'pure' AGD at the age of 3 or 7/12 months and killed 3 or 7 months later. Abnormal hyper-phosphorylated tau deposits were found in ipsilateral hippocampal neurons, grains (dots) in the hippocampus, and threads, dots and coiled bodies in the fimbria, as well as the ipsilateral and contralateral corpus callosum. The extension of lesions was wider in animals surviving 7 months compared with those surviving 3 months. Astrocytic inclusions were not observed at any time. Tau deposits were mainly composed of 4Rtau, but also 3Rtau. For comparative purposes, another series of WT mice was inoculated with sarkosyl-insoluble fractions from primary age-related tauopathy (PART), a pure neuronal neurofibrillary tangle 3Rtau + 4Rtau tauopathy involving the deep temporal cortex and limbic system. Abnormal hyper-phosphorylated tau deposits were found in neurons in the ipsilateral hippocampus, coiled bodies and threads in the fimbria, and the ipsilateral and contralateral corpus callosum, which extended with time along the anterior-posterior axis and distant regions such as hypothalamic nuclei and nuclei of the septum when comparing mice surviving 7 months with mice surviving 3 months. Astrocytic inclusions were not observed. Tau deposits were mainly composed of 4Rtau and 3Rtau. These results show the capacity for seeding and spreading of AGD tau and PART tau in the brain of WT mouse, and suggest that characteristics of host tau, in addition to those of inoculated tau, are key to identifying commonalities and differences between human tauopathies and corresponding murine models

Neuromuscular activity induces paracrine signaling and triggers axonal regrowth after injury in microfluidic lab‐on‐chip devices

Peripheral nerve injuries, including motor neuron axonal injury, often lead to functional impairments. Current therapies are mostly limited to surgical intervention after lesion, yet these interventions have limited success in restoring functionality. Current activity‐based therapies after axonal injuries are based on trial‐error approaches in which the details of the underlying cellular and molecular processes are largely unknown. Here we show the effects of the modulation of both neuronal and muscular activity with optogenetic approaches to assess the regenerative capacity of cultured motor neuron (MN) after lesion in a compartmentalized microfluidic‐assisted axotomy device. With increased neuronal activity, we observed an increase in the ratio of regrowing axons after injury in our peripheral‐injury model. Moreover, increasing muscular activity induces the liberation of leukemia inhibitory factor and glial cell line‐derived neurotrophic factor in a paracrine fashion that in turn triggers axonal regrowth of lesioned MN in our 3D hydrogel cultures. The relevance of our findings as well as the novel approaches used in this study could be useful not only after axotomy events but also in diseases affecting MN survival

Differences in Tau Seeding in Newborn and Adult Wild-Type Mice

Alzheimer’s disease (AD) and other tauopathies are common neurodegenerative diseases in older adults; in contrast, abnormal tau deposition in neurons and glial cells occurs only exceptionally in children. Sarkosyl-insoluble fractions from sporadic AD (sAD) containing paired helical filaments (PHFs) were inoculated unilaterally into the thalamus in newborn and three-month-old wild-type C57BL/6 mice, which were killed at different intervals from 24 h to six months after inoculation. Tau-positive cells were scanty and practically disappeared at three months in mice inoculated at the age of a newborn. In contrast, large numbers of tau-positive cells, including neurons and oligodendrocytes, were found in the thalamus of mice inoculated at three months and killed at the ages of six months and nine months. Mice inoculated at the age of newborn and re-inoculated at the age of three months showed similar numbers and distribution of positive cells in the thalamus at six months and nine months. This study shows that (a) differences in tau seeding between newborn and young adults may be related to the ratios between 3Rtau and 4Rtau, and the shift to 4Rtau predominance in adults, together with the immaturity of connections in newborn mice, and (b) intracerebral inoculation of sAD PHFs in newborn mice does not protect from tau seeding following intracerebral inoculation of sAD PHFs in young/adult mice

Evaluation of tau seeding, spreading, and cytotoxicity using in vitro and in vivo models of tau pathology

[eng] Abnormal folding, hyperphosphorylation, aggregation, and subsequent deposition of the microtubule-associated protein tau, is the hallmark of a group of devastating neurodegenerative diseases known as tauopathies, including Alzheimer’s disease. One striking aspect of Alzheimer’s disease is that the presence of tau-related lesions in the brain occurs in a systematic, sequential manner, maintaining a predictable distribution pattern between synaptically connected neurons that varies very little among individuals. Increasing evidence suggests that the progression of tau pathology in the diseased brain behaves like a prion. The “prion-like” hypothesis suggests that “pathological” tau engages in self-seeded fibrillization and propagates through cell-to-cell spreading. However, despite intensive research, the cellular and molecular mechanisms involved and the pathological processes linking neuronal death and tau dysfunction are not fully understood. Although Alzheimer’s disease was first described in 1906 and has an increasing prevalence in the aging population, there is currently no treatment to prevent or cure this or any other tauopathy. Progress limitations are partially explained by the lack of appropriate models to study human tauopathies. Indeed, tau-targeting therapies that had demonstrated an improvement in the pathology in several models (i.e., in vitro and in vivo) were unable to produce positive results in clinical trials. These incongruences could be related to the fact that most experimental models rely on the over-expression of mutated tau species and the use of recombinant tau fibrils, which do not reproduce the sporadic nature of most human tauopathies. Through this doctoral thesis, we examined various aspects of tau pathology, including tau seeding, spreading, and cytotoxicity, by implementing experimental approaches that better mimicked sporadic tauopathies. Nevertheless, at the beginning of this work, the reliability of the only commercially available cell line designed to be used as a cell-based assay to detect and report proteopathic seeding in biological samples was questioned in one publication. Given that this cell-based assay was central to the validation of the samples employed in this thesis, we conducted a thorough characterization of this cell line, known as the Tau biosensor cell line, and its ability to produce fluorescent tau aggregates. Our results show that the Tau biosensor cell line is a reliable cell-based assay that forms amyloid-like inclusions upon the addition of extracellular seed- competent tau species. Next, we investigated the impact of extracellular seed-competent tau on the neuronal activity of primary cortical cultures derived from wild-type mice. We established an experimental setup that included microfluidic devices and calcium imaging, which allowed us to specifically treat the axons with tau, as well as monitor changes in spontaneous neural activity in a time-course manner. Although we demonstrate that cortical neurons in our microfluidic platforms display typical patterns of neuronal network activity, we do not detect changes after treating them with seed-competent tau. We then investigated how the presence of various extracellular seed-competent tau may affect neural metabolism (i.e., as an indicator of cellular viability) also in primary cortical cultures. Nevertheless, similar to what we observed in the analysis of neuronal activity, within the course of 10 days, no differences between tau-treated and untreated cells are found. Finally, recent evidence suggests that the cellular prion protein is involved in the pathology of other prion-like proteins, such as amyloid-β and α-synuclein; however, much less is known about its role in tauopathies. We inoculated human Alzheimer’s disease-derived samples into the hippocampus of transgenic mouse models with different expressions of the cellular prion protein. We found that all mice, regardless of their genotype, have similar profiles of tau-related lesions in their brains. Hence, our findings indicate that the cellular prion protein does not have a paramount role in the onset, seeding, or spreading of tau pathology. Taken together, our work underscores the need for more pathologically relevant models to study certain aspects of sporadic human tauopathies, which could lead to the development of effective therapeutic strategies