RNA-binding properties orchestrate TDP-43 homeostasis through condensate formation in vivo.

Insoluble cytoplasmic aggregate formation of the RNA-binding protein TDP-43 is a major hallmark of neurodegenerative diseases including Amyotrophic Lateral Sclerosis. TDP-43 localizes predominantly in the nucleus, arranging itself into dynamic condensates through liquid-liquid phase separation (LLPS). Mutations and post-translational modifications can alter the condensation properties of TDP-43, contributing to the transition of liquid-like biomolecular condensates into solid-like aggregates. However, to date it has been a challenge to study the dynamics of this process in vivo. We demonstrate through live imaging that human TDP-43 undergoes nuclear condensation in spinal motor neurons in a living animal. RNA-binding deficiencies as well as post-translational modifications can lead to aberrant condensation and altered TDP-43 compartmentalization. Single-molecule tracking revealed an altered mobility profile for RNA-binding deficient TDP-43. Overall, these results provide a critically needed in vivo characterization of TDP-43 condensation, demonstrate phase separation as an important regulatory mechanism of TDP-43 accessibility, and identify a molecular mechanism of how functional TDP-43 can be regulated.

Identification of phosphorylated tau protein interactors in progressive supranuclear palsy (PSP) reveals networks involved in protein degradation, stress response, cytoskeletal dynamics, metabolic processes, and neurotransmission.

Progressive supranuclear palsy (PSP) is a late-onset neurodegenerative disease defined pathologically by the presence of insoluble phosphorylated-Tau (p-Tau) in neurons and glia. Identifying co-aggregating proteins within p-Tau inclusions may reveal important insights into processes affected by the aggregation of Tau. We used a proteomic approach, which combines antibody-mediated biotinylation and mass spectrometry (MS) to identify proteins proximal to p-Tau in PSP. Using this proof-of-concept workflow for identifying interacting proteins of interest, we characterized proteins proximal to p-Tau in PSP cases, identifying >84% of previously identified interaction partners of Tau and known modifiers of Tau aggregation, while 19 novel proteins not previously found associated with Tau were identified. Furthermore, our data also identified confidently assigned phosphorylation sites that have been previously reported on p-Tau. Additionally, using ingenuity pathway analysis (IPA) and human RNA-seq datasets, we identified proteins previously associated with neurological disorders and pathways involved in protein degradation, stress responses, cytoskeletal dynamics, metabolism, and neurotransmission. Together, our study demonstrates the utility of biotinylation by antibody recognition (BAR) approach to answer a fundamental question to rapidly identify proteins in proximity to p-Tau from post-mortem tissue. The application of this workflow opens up the opportunity to identify novel protein targets to give us insight into the biological process at the onset and progression of tauopathies.

Aurora kinase B regulates axonal outgrowth and regeneration in the spinal motor neurons of developing zebrafish.

Aurora kinase B (AurkB) is a serine/threonine protein kinase with a well-characterised role in orchestrating cell division and cytokinesis, and is prominently expressed in healthy proliferating and cancerous cells. However, the role of AurkB in differentiated and non-dividing cells has not been extensively explored. Previously, we have described a significant upregulation of AurkB expression in cultured cortical neurons following an experimental axonal transection. This is somewhat surprising, as AurkB expression is generally associated only with dividing cells Frangini et al. (Mol Cell 51:647-661, 2013); Hegarat et al. (J Cell Biol 195:1103-1113, 2011); Lu et al. (J Biol Chem 283:31785-31790, 2008); Trakala et al. (Cell Cycle 12:1030-1041, 2014). Herein, we present the first description of a role for AurkB in terminally differentiated neurons. AurkB was prominently expressed within post-mitotic neurons of the zebrafish brain and spinal cord. The expression of AurkB varied during the development of the zebrafish spinal motor neurons. Utilising pharmacological and genetic manipulation to impair AurkB activity resulted in truncation and aberrant motor axon morphology, while overexpression of AurkB resulted in extended axonal outgrowth. Further pharmacological inhibition of AurkB activity in regenerating axons delayed their recovery following UV laser-mediated injury. Collectively, these results suggest a hitherto unreported role of AurkB in regulating neuronal development and axonal outgrowth.

Extracellular Interactions of Alpha-Synuclein in Multiple System Atrophy.

Multiple system atrophy, characterized by atypical Parkinsonism, results from central nervous system (CNS) cell loss and dysfunction linked to aggregates of the normally pre-synaptic α-synuclein protein. Mostly cytoplasmic pathological α-synuclein inclusion bodies occur predominantly in oligodendrocytes in affected brain regions and there is evidence that α-synuclein released by neurons is taken up preferentially by oligodendrocytes. However, extracellular α-synuclein has also been shown to interact with other neural cell types, including astrocytes and microglia, as well as extracellular factors, mediating neuroinflammation, cell-to-cell spread and other aspects of pathogenesis. Here, we review the current evidence for how α-synuclein present in the extracellular milieu may act at the cell surface to drive components of disease progression. A more detailed understanding of the important extracellular interactions of α-synuclein with neuronal and non-neuronal cell types both in the brain and periphery may provide new therapeutic targets to modulate the disease process.

Potential Modes of Intercellular α-Synuclein Transmission.

Intracellular aggregates of the α-synuclein protein result in cell loss and dysfunction in Parkinson's disease and atypical Parkinsonism, such as multiple system atrophy and dementia with Lewy bodies. Each of these neurodegenerative conditions, known collectively as α-synucleinopathies, may be characterized by a different suite of molecular triggers that initiate pathogenesis. The mechanisms whereby α-synuclein aggregates mediate cytotoxicity also remain to be fully elucidated. However, recent studies have implicated the cell-to-cell spread of α-synuclein as the major mode of disease propagation between brain regions during disease progression. Here, we review the current evidence for different modes of α-synuclein cellular release, movement and uptake, including exocytosis, exosomes, tunneling nanotubes, glymphatic flow and endocytosis. A more detailed understanding of the major modes by which α-synuclein pathology spreads throughout the brain may provide new targets for therapies that halt the progression of disease.

Microglia morphophysiological diversity and its implications for the CNS.

Microglia are mononuclear phagocytes of mesodermal origin that migrate to the central nervous system (CNS) during the early stages of embryonic development. After colonizing the CNS, they proliferate and remain able to self-renew throughout life, maintaining the number of microglia around 5-12% of the cells in the CNS parenchyma. They are considered to play key roles in development, homeostasis and innate immunity of the CNS. Microglia are exceptionally diverse in their morphological characteristics, actively modifying the shape of their processes and soma in response to different stimuli. This broad morphological spectrum of microglia responses is considered to be closely correlated to their diverse range of functions in health and disease. However, the morphophysiological attributes of microglia, and the structural and functional features of microglia-neuron interactions, remain largely unknown. Here, we assess the current knowledge of the diverse microglial morphologies, with a focus on the correlation between microglial shape and function. We also outline some of the current challenges, opportunities, and future directions that will help us to tackle unanswered questions about microglia, and to continue unravelling the mysteries of microglia, in all its shapes.

In vivo Validation of Bimolecular Fluorescence Complementation (BiFC) to Investigate Aggregate Formation in Amyotrophic Lateral Sclerosis (ALS).

Amyotrophic lateral sclerosis (ALS) is a form of motor neuron disease (MND) that is characterized by the progressive loss of motor neurons within the spinal cord, brainstem, and motor cortex. Although ALS clinically manifests as a heterogeneous disease, with varying disease onset and survival, a unifying feature is the presence of ubiquitinated cytoplasmic protein inclusion aggregates containing TDP-43. However, the precise mechanisms linking protein inclusions and aggregation to neuronal loss are currently poorly understood. Bimolecular fluorescence complementation (BiFC) takes advantage of the association of fluorophore fragments (non-fluorescent on their own) that are attached to an aggregation-prone protein of interest. Interaction of the proteins of interest allows for the fluorescent reporter protein to fold into its native state and emit a fluorescent signal. Here, we combined the power of BiFC with the advantages of the zebrafish system to validate, optimize, and visualize the formation of ALS-linked aggregates in real time in a vertebrate model. We further provide in vivo validation of the selectivity of this technique and demonstrate reduced spontaneous self-assembly of the non-fluorescent fragments in vivo by introducing a fluorophore mutation. Additionally, we report preliminary findings on the dynamic aggregation of the ALS-linked hallmark proteins Fus and TDP-43 in their corresponding nuclear and cytoplasmic compartments using BiFC. Overall, our data demonstrates the suitability of this BiFC approach to study and characterize ALS-linked aggregate formation in vivo. Importantly, the same principle can be applied in the context of other neurodegenerative diseases and has therefore critical implications to advance our understanding of pathologies that underlie aberrant protein aggregation.

Extracellular Alpha-Synuclein Promotes a Neuroinhibitory Secretory Phenotype in Astrocytes.

Multiple system atrophy (MSA) and dementia with Lewy bodies (DLB) are α-synucleinopathies that exhibit widespread astrogliosis as a component of the neuroinflammatory response. Munc18, a protein critical to vesicle exocytosis, was previously found to strongly mark morphologically activated astrocytes in brain tissue of MSA patients. Immunofluorescence of MSA, DLB and normal brain tissue sections was combined with cell culture and co-culture experiments to investigate the relationship between extracellular α-synuclein and the transition to a secretory astrocyte phenotype. Increased Munc18-positive vesicles were resolved in activated astrocytes in MSA and DLB tissue compared to controls, and they were also significantly upregulated in the human 1321N1 astrocytoma cell line upon treatment with α-synuclein, with parallel increases in GFAP expression and IL-6 secretion. In co-culture experiments, rat primary astrocytes pretreated with α-synuclein inhibited the growth of neurites of co-cultured primary rat neurons and upregulated chondroitin sulphate proteoglycan. Taken together, these results indicate that the secretory machinery is significantly upregulated in the astrocyte response to extracellular α-synuclein and may participate in the release of neuroinhibitory and proinflammatory factors in α-synucleinopathies.

Switching on Endogenous Metal Binding Proteins in Parkinson's Disease.

The formation of cytotoxic intracellular protein aggregates is a pathological signature of multiple neurodegenerative diseases. The principle aggregating protein in Parkinson's disease (PD) and atypical Parkinson's diseases is α-synuclein (α-syn), which occurs in neural cytoplasmic inclusions. Several factors have been found to trigger α-syn aggregation, including raised calcium, iron, and copper. Transcriptional inducers have been explored to upregulate expression of endogenous metal-binding proteins as a potential neuroprotective strategy. The vitamin-D analogue, calcipotriol, induced increased expression of the neuronal vitamin D-dependent calcium-binding protein, calbindin-D28k, and this significantly decreased the occurrence of α-syn aggregates in cells with transiently raised intracellular free Ca, thereby increasing viability. More recently, the induction of endogenous expression of the Zn and Cu binding protein, metallothionein, by the glucocorticoid analogue, dexamethasone, gave a specific reduction in Cu-dependent α-syn aggregates. Fe accumulation has long been associated with PD. Intracellularly, Fe is regulated by interactions between the Fe storage protein ferritin and Fe transporters, such as poly(C)-binding protein 1. Analysis of the transcriptional regulation of Fe binding proteins may reveal potential inducers that could modulate Fe homoeostasis in disease. The current review highlights recent studies that suggest that transcriptional inducers may have potential as novel mechanism-based drugs against metal overload in PD.

Real-time visualization of oxidative stress-mediated neurodegeneration of individual spinal motor neurons in vivo.

Generation of reactive oxygen species (ROS) has been shown to be important for many physiological processes, ranging from cell differentiation to apoptosis. With the development of the genetically encoded photosensitiser KillerRed (KR) it is now possible to efficiently produce ROS dose-dependently in a specific cell type upon green light illumination. Zebrafish are the ideal vertebrate animal model for these optogenetic methods because of their transparency and efficient transgenesis. Here we describe a zebrafish model that expresses membrane-targeted KR selectively in motor neurons. We show that KR-activated neurons in the spinal cord undergo stress and cell death after induction of ROS. Using single-cell resolution and time-lapse confocal imaging, we selectively induced neurodegeneration in KR-expressing neurons leading to characteristic signs of apoptosis and cell death. We furthermore illustrate a targeted microglia response to the induction site as part of a physiological response within the zebrafish spinal cord. Our data demonstrate the successful implementation of KR mediated ROS toxicity in motor neurons in vivo and has important implications for studying the effects of ROS in a variety of conditions within the central nervous system, including aging and age-related neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis.

Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy.

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and cell-cell interactions in the CNS in real-time. Previously, studying these cell-specific responses after injury often required complicated setups or the transfer of cells or animals into different, non-physiological environments, confounding immediate and short-term analysis. For example, drug-mediated ablation approaches often lack the specificity that is required to study single-cell responses and immediate cell-cell interactions. Similarly, while high-power pulsed laser ablation approaches provide very good control and tissue penetration, they require specialized equipment that can complicate real-time visualization of cellular responses. The refined UV laser ablation approach described here allows researchers to stress or kill an individual cell in a dose- and time-dependent manner using a conventional confocal microscope equipped with a 405-nm laser. The method was applied to selectively ablate a single neuron within a dense network of surrounding cells in the zebrafish spinal cord. This approach revealed a dose-dependent response of the ablated neurons, causing the fragmentation of cellular bodies and anterograde degeneration along the axon within minutes to hours. This method allows researchers to study the fate of an individual dying cell and, importantly, the instant response of cells-such as microglia and astrocytes-surrounding the ablation site.