Loss of TDP-43 oligomerization or RNA binding elicits distinct aggregation patterns

Aggregation of the RNA-binding protein TAR DNA-binding protein 43 (TDP-43) is the key neuropathological feature of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). In physiological conditions, TDP-43 is predominantly nuclear, forms oligomers, and is contained in biomolecular condensates assembled by liquid-liquid phase separation (LLPS). In disease, TDP-43 forms cytoplasmic or intranuclear inclusions. How TDP-43 transitions from physiological to pathological states remains poorly understood. Using a variety of cellular systems to express structure-based TDP-43 variants, including human neurons and cell lines with near-physiological expression levels, we show that oligomerization and RNA binding govern TDP-43 stability, splicing functionality, LLPS, and subcellular localization. Importantly, our data reveal that TDP-43 oligomerization is modulated by RNA binding. By mimicking the impaired proteasomal activity observed in ALS/FTLD patients, we found that monomeric TDP-43 forms inclusions in the cytoplasm, whereas its RNA binding-deficient counterpart aggregated in the nucleus. These differentially localized aggregates emerged via distinct pathways: LLPS-driven aggregation in the nucleus and aggresome-dependent inclusion formation in the cytoplasm. Therefore, our work unravels the origins of heterogeneous pathological species reminiscent of those occurring in TDP-43 proteinopathy patients

Granulovacuolar degeneration bodies:red alert for neurons with MAPT/tau pathology

In Alzheimer disease patients, MAPT/tau pathology and granulovacuolar degeneration bodies (GVBs) co-occur in the same brain regions and in the same cells. The interdependence of these neuropathological hallmarks and the identity of GVBs have long been elusive. Recently, we showed that MAPT pathology causes GVB formation in neurons in vivo and in vitro. Using these novel GVB models, we identified GVBs as lysosomal structures at the convergence of the endo- and autolysosomal pathways. Here, the possible functional consequences of neuronal GVB formation are discussed

Tau: a phase in the crowd

From the management of microtubules to the production of pathological species: liquid–liquid phase separation may tune the behavior of the protein tau in health and neurodegenerative disease. In this issue of The EMBO Journal, Hochmair et al (2022) demystify important aspects of tau condensate compilation

Untangling the origin and function of granulovacuolar degeneration bodies in neurodegenerative proteinopathies

In the brains of tauopathy patients, tau pathology coincides with the presence of granulovacuolar degeneration bodies (GVBs) both at the regional and cellular level. Recently, it was shown that intracellular tau pathology causes GVB formation in experimental models thus explaining the strong correlation between these neuropathological hallmarks in the human brain. These novel models of GVB formation provide opportunities for future research into GVB biology, but also urge reevaluation of previous post-mortem observations. Here, we review neuropathological data on GVBs in tauopathies and other neurodegenerative proteinopathies. We discuss the possibility that intracellular aggregates composed of proteins other than tau are also able to induce GVB formation. Furthermore, the potential mechanisms of GVB formation and the downstream functional implications hereof are outlined in view of the current available data. In addition, we provide guidelines for the identification of GVBs in tissue and cell models that will help to facilitate and streamline research towards the elucidation of the role of these enigmatic and understudied structures in neurodegeneration

Granulovacuolar degeneration bodies are independently induced by tau and α-synuclein pathology

Background: Granulovacuolar degeneration bodies (GVBs) are intracellular vesicular structures that commonly accompany pathological tau accumulations in neurons of patients with tauopathies. Recently, we developed the first model for GVBs in primary neurons, that requires exogenous tau seeds to elicit tau aggregation. This model allowed the identification of GVBs as proteolytically active lysosomes induced by tau pathology. GVBs selectively accumulate cargo in a dense core, that shows differential and inconsistent immunopositivity for (phosphorylated) tau epitopes. Despite the strong evidence connecting GVBs to tau pathology, these structures have been reported in neurons without apparent pathology in brain tissue of tauopathy patients. Additionally, GVBs and putative GVBs have also been reported in the brain of patients with non-tau proteinopathies. Here, we investigated the connection between pathological protein assemblies and GVBs in more detail. Methods: This study combined newly developed primary neuron models for tau and α-synuclein pathology with observations in human brain tissue from tauopathy and Parkinson’s disease patients. Immunolabeling and imaging techniques were employed for extensive characterisation of pathological proteins and GVBs. Quantitative data were obtained by high-content automated microscopy as well as single-cell analysis of confocal images. Results: Employing a novel seed-independent neuronal tau/GVB model, we show that in the context of tauopathy, GVBs are inseparably associated with the presence of cytosolic pathological tau and that intracellular tau aggregation precedes GVB formation, strengthening the causal relationship between pathological accumulation of tau and GVBs. We also report that GVBs are inseparably associated with pathological tau at the single-cell level in the hippocampus of tauopathy patients. Paradoxically, we demonstrate the presence of GVBs in the substantia nigra of Parkinson’s disease patients and in a primary neuron model for α-synuclein pathology. GVBs in this newly developed α-synuclein/GVB model are induced in the absence of cytosolic pathological tau accumulations. GVBs in the context of tau or α-synuclein pathology showed similar immunoreactivity for different phosphorylated tau epitopes. The phosphorylated tau immunoreactivity signature of GVBs is therefore independent of the presence of cytosolic tau pathology. Conclusion: Our data identify the emergence of GVBs as a more generalised response to cytosolic protein pathology

Activation of the unfolded protein response and granulovacuolar degeneration are not common features of human prion pathology

Human prion diseases are fatal neurodegenerative disorders with a genetic, sporadic or infectiously acquired aetiology. Neuropathologically, human prion diseases are characterized by deposition of misfolded prion protein and neuronal loss. In post-mortem brain tissue from patients with other neurodegenerative diseases characterized by protein misfolding, including Alzheimer’s disease (AD) and frontotemporal lobar degeneration with tau pathology (FTLD-tau), increased activation of the unfolded protein response (UPR) has been observed. The UPR is a cellular stress response that copes with the presence of misfolded proteins. Recent studies have indicated that UPR activation is also involved in experimental models of prion disease and have suggested intervention in the UPR as a therapeutic strategy. On the other hand, it was previously shown that the active form of the UPR stress sensor dsRNA-activated protein kinase-like ER kinase (PERK) is not increased in post-mortem brain tissue samples from human prion disease cases. In the present study, we assessed the active form of another UPR stress sensor, inositol-requiring enzyme 1α (IRE1α), in human post-mortem frontal cortex of a large cohort of sporadic, inherited and acquired prion disease patients (n = 47) and non-neurological controls. Immunoreactivity for phosphorylated IRE1α was not increased in prion disease cases compared with non-neurological controls. In addition, immunoreactivity for phosphorylated PERK was unaltered in human prion disease cases included in the current cohort. Moreover, no difference in the extent of granulovacuolar degeneration, a pathological feature associated with the presence of UPR activation markers, was detected. Our data indicate that, in contrast to AD and primary tauopathies, activation of the UPR is not a common feature of human prion pathology

FTLD‐TDP assemblies seed neoaggregates with subtype‐specific features via a prion‐like cascade

Morphologically distinct TDP-43 aggregates occur in clinically different FTLD-TDP subtypes, yet the mechanism of their emergence and contribution to clinical heterogeneity are poorly understood. Several lines of evidence suggest that pathological TDP-43 follows a prion-like cascade, but the molecular determinants of this process remain unknown. We use advanced microscopy techniques to compare the seeding properties of pathological FTLD-TDP-A and FTLD-TDP-C aggregates. Upon inoculation of patient-derived aggregates in cells, FTLD-TDP-A seeds amplify in a template-dependent fashion, triggering neoaggregation more efficiently than those extracted from FTLD-TDP-C patients, correlating with the respective disease progression rates. Neoaggregates are sequentially phosphorylated with N-to-C directionality and with subtype-specific timelines. The resulting FTLD-TDP-A neoaggregates are large and contain densely packed fibrils, reminiscent of the pure compacted fibrils present within cytoplasmic inclusions in postmortem brains. In contrast, FTLD-TDP-C dystrophic neurites show less dense fibrils mixed with cellular components, and their respective neoaggregates are small, amorphous protein accumulations. These cellular seeding models replicate aspects of the patient pathological diversity and will be a useful tool in the quest for subtype-specific therapeutics