Precision proteoform design for 4R tau isoform selective templated aggregation.

Prion-like spread of disease-specific tau conformers is a hallmark of all tauopathies. A 19-residue probe peptide containing a P301L mutation and spanning the R2/R3 splice junction of tau folds and stacks into seeding-competent fibrils and induces aggregation of 4R, but not 3R tau. These tau peptide fibrils propagate aggregated intracellular tau over multiple generations, have a high ß-sheet content, a colocalized lipid signal, and adopt a well-defined U-shaped fold found in 4R tauopathy brain-derived fibrils. Fully atomistic replica exchange molecular dynamics (MD) simulations were used to compute the free energy landscapes of the conformational ensemble of the peptide monomers. These identified an aggregation-prohibiting ß-hairpin structure and an aggregation-competent U-fold unique to 4R tauopathy fibrils. Guided by MD simulations, we identified that the N-terminal-flanking residues to PHF6, which slightly vary between 4R and 3R isoforms, modulate seeding. Strikingly, when a single amino acid switch at position 305 replaced the serine of 4R tau with a lysine from the corresponding position in the first repeat of 3R tau, the seeding induced by the 19-residue peptide was markedly reduced. Conversely, a 4R tau mimic with three repeats, prepared by replacing those amino acids in the first repeat with those amino acids uniquely present in the second repeat, recovered aggregation when exposed to the 19-residue peptide. These peptide fibrils function as partial prions to recruit naive 4R tau-ten times the length of the peptide-and serve as a critical template for 4R tauopathy propagation. These results hint at opportunities for tau isoform-specific therapeutic interventions.

Precision Proteoform Design for 4R Tau Isoform Selective Templated Aggregation.

Prion-like spread of disease-specific tau conformers is a hallmark of all tauopathies. A 19-residue probe peptide containing a P301L mutation and spanning the R2/R3 splice junction of tau, folds and stacks into seeding-competent fibrils and induces aggregation of 4R, but not 3R tau. These tau peptide fibrils propagate aggregated intracellular tau over multiple generations, have a high ß-sheet content, a colocalized lipid signal, and adopt a well-defined U-shaped fold found in 4R tauopathy brain-derived fibrils. Fully atomistic replica exchange molecular dynamics (MD) simulations were used to compute the free energy landscapes of the conformational ensemble of the peptide monomers. These identified an aggregation-prohibiting ß-hairpin structure and an aggregation-competent U-fold unique to 4R tauopathy fibrils. Guided by MD simulations, we identified that the N-terminal-flanking residues to PHF6, which slightly vary between 4R and 3R isoforms, modulate seeding. Strikingly, when a single amino acid switch at position 305 replaced the serine of 4R tau with a lysine from the corresponding position in the first repeat of 3R tau, the seeding induced by the 19-residue peptide was markedly reduced. Conversely, a 4R tau mimic with three repeats, prepared by replacing those amino acids in the first repeat with those amino acids uniquely present in the second repeat, recovered aggregation when exposed to the 19-residue peptide. These peptide fibrils function as partial prions to recruit naïve 4R tau-ten times the length of the peptide-and serve as a critical template for 4R tauopathy propagation. These results hint at opportunities for tau isoform-specific therapeutic interventions.

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.

Mesoporous silica particles are phagocytosed by microglia and induce a mild inflammatory response in vitro.

Aim: Mesoporous silica particles (MSPs) are broadly used drug delivery carriers. In this study, the authors analyzed the responses to MSPs of astrocytes and microglia, the two main cellular players in neuroinflammation. Materials & methods: Primary murine cortical mixed glial cultures were treated with rhodamine B-labeled MSPs. Results: MSPs are avidly internalized by microglial cells and remain inside the cells for at least 14 days. Despite this, MSPs do not affect glial cell viability or morphology, basal metabolic activity or oxidative stress. MSPs also do not affect mRNA levels of key proinflammatory genes; however, in combination with lipopolysaccharide, they significantly increase extracellular IL-1ß levels. Conclusion: These results suggest that MSPs could be novel tools for specific drug delivery to microglial cells.

Mesoporous silica particles (MSPs) are broadly used drug delivery carriers. In this study, the authors analyzed the responses of two types of brain cells, astrocytes and microglia, to MSPs. Mouse astrocytes and microglia were kept alive in cultures and were treated with MSPs that were labeled with a red fluorescent agent to facilitate visualization under the microscope. MSPs are avidly internalized by microglial cells and remain inside the cells for at least 14 days. Despite this, MSPs do not affect glial cell viability or morphology, basal metabolic activity or oxidative stress. When given alone, MSPs do not affect mRNA levels of key proinflammatory genes. However, MSPs given in combination with lipopolysaccharide, a strong proinflammatory agent, significantly increase extracellular levels of IL-1ß, one of the proinflammatory mediators studied. These results suggest that MSPs could be novel tools for specific drug delivery to microglial cells.

Development of a simple and versatile in vitro method for production, stimulation, and analysis of bioengineered muscle.

In recent years, 3D in vitro modeling of human skeletal muscle has emerged as a subject of increasing interest, due to its applicability in basic studies or screening platforms. These models strive to recapitulate key features of muscle architecture and function, such as cell alignment, maturation, and contractility in response to different stimuli. To this end, it is required to culture cells in biomimetic hydrogels suspended between two anchors. Currently available protocols are often complex to produce, have a high rate of breakage, or are not adapted to imaging and stimulation. Therefore, we sought to develop a simplified and reliable protocol, which still enabled versatility in the study of muscle function. In our method, we have used human immortalized myoblasts cultured in a hydrogel composed of MatrigelTM and fibrinogen, to create muscle strips suspended between two VELCROTM anchors. The resulting muscle constructs show a differentiated phenotype and contractile activity in response to electrical, chemical and optical stimulation. This activity is analyzed by two alternative methods, namely contraction analysis and calcium analysis with Fluo-4 AM. In all, our protocol provides an optimized version of previously published methods, enabling individual imaging of muscle bundles and straightforward analysis of muscle response with standard image analysis software. This system provides a start-to-finish guide on how to produce, validate, stimulate, and analyze bioengineered muscle. This ensures that the system can be quickly established by researchers with varying degrees of expertise, while maintaining reliability and similarity to native muscle.

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.

Corrigendum: Capacity for Seeding and Spreading of Argyrophilic Grain Disease in a Wild-Type Murine Model; Comparisons With Primary Age-Related Tauopathy.

[This corrects the article DOI: 10.3389/fnmol.2020.00101.].

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.