Protein aggregation is the main hallmark of neurodegenerative diseases. Many proteins found in pathological inclusions are known to undergo liquid-liquid phase separation, a reversible process of ...molecular self-assembly. Emerging evidence supports the hypothesis that aberrant phase separation behavior may serve as a trigger of protein aggregation in neurodegeneration, and efforts to understand and control the underlying mechanisms are underway. Here, we review similarities and differences among four main proteins, α-synuclein, FUS, tau, and TDP-43, which are found aggregated in different diseases and were independently shown to phase separate. We discuss future directions in the field that will help shed light on the molecular mechanisms of aggregation and neurodegeneration.
Protein aggregation and dysfunction of the ubiquitin-proteasome system are hallmarks of many neurodegenerative diseases. Here, we address the elusive link between these phenomena by employing ...cryo-electron tomography to dissect the molecular architecture of protein aggregates within intact neurons at high resolution. We focus on the poly-Gly-Ala (poly-GA) aggregates resulting from aberrant translation of an expanded GGGGCC repeat in C9orf72, the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. We find that poly-GA aggregates consist of densely packed twisted ribbons that recruit numerous 26S proteasome complexes, while other macromolecules are largely excluded. Proximity to poly-GA ribbons stabilizes a transient substrate-processing conformation of the 26S proteasome, suggesting stalled degradation. Thus, poly-GA aggregates may compromise neuronal proteostasis by driving the accumulation and functional impairment of a large fraction of cellular proteasomes.
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•Neuronal C9orf72 poly-GA aggregates were analyzed by cryoelectron tomography•Poly-GA aggregates in neurons consist of planar twisted ribbons•Poly-GA aggregates recruit proteasomes while excluding other large macromolecules•Interactions with poly-GA aggregates lead to proteasome stalling
Neuronal poly-GA aggregates linked to amyotrophic lateral sclerosis and frontotemporal dementia selectively sequester proteasomes.
Mutations disrupting the nuclear localization of the RNA-binding protein FUS characterize a subset of amyotrophic lateral sclerosis patients (ALS-FUS). FUS regulates nuclear RNAs, but its role at the ...synapse is poorly understood. Using super-resolution imaging we determined that the localization of FUS within synapses occurs predominantly near the vesicle reserve pool of presynaptic sites. Using CLIP-seq on synaptoneurosomes, we identified synaptic FUS RNA targets, encoding proteins associated with synapse organization and plasticity. Significant increase of synaptic FUS during early disease in a mouse model of ALS was accompanied by alterations in density and size of GABAergic synapses. mRNAs abnormally accumulated at the synapses of 6-month-old ALS-FUS mice were enriched for FUS targets and correlated with those depicting increased short-term mRNA stability via binding primarily on multiple exonic sites. Our study indicates that synaptic FUS accumulation in early disease leads to synaptic impairment, potentially representing an initial trigger of neurodegeneration.
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.
Synopsis
Loss of TDP‐43 oligomerization and RNA binding promotes diverse TDP‐43 pathologies providing insight into the origins of heterogeneous pathological species occurring in human disease.
TDP‐43 oligomerization and RNA binding govern its stability, splicing regulation, LLPS, and subcellular localization.
TDP‐43 oligomerization is modulated by RNA binding.
Monomeric TDP‐43 forms inclusions in the cytoplasm, whereas its RNA binding‐deficient counterpart aggregates in the nucleus.
TDP‐43 aggregates emerge via distinct pathways: LLPS‐driven aggregation in the nucleus and aggresome‐dependent inclusion formation in the cytoplasm.
Our work unravels the origins of heterogeneous TDP‐43 inclusions reminiscent of those occurring in TDP‐43 proteinopathy patients.
Loss of TDP‐43 oligomerization and RNA binding promotes diverse TDP‐43 pathologies providing insights into the origins of heterogeneous pathological species occurring in human disease.
The most frequent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia is a G
4
C
2
repeat expansion in the
C9orf72
gene. This expansion gives rise to translation of aggregating ...dipeptide repeat (DPR) proteins, including poly-GA as the most abundant species. However, gain of toxic function effects have been attributed to either the DPRs or the pathological G
4
C
2
RNA. Here, we analyzed in a cellular model the relative toxicity of DPRs and RNA. Cytoplasmic poly-GA aggregates, generated in the absence of G
4
C
2
RNA, interfered with nucleocytoplasmic protein transport, but had little effect on cell viability. In contrast, nuclear poly-GA was more toxic, impairing nucleolar protein quality control and protein biosynthesis. Production of the G
4
C
2
RNA strongly reduced viability independent of DPR translation and caused pronounced inhibition of nuclear mRNA export and protein biogenesis. Thus, while the toxic effects of G
4
C
2
RNA predominate in the cellular model used, DPRs exert additive effects that may contribute to pathology.
Accumulation of abnormally phosphorylated TDP-43 (pTDP-43) is the main pathology in affected neurons of people with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). ...Morphological diversity and neuroanatomical distribution of pTDP-43 accumulations allowed classification of FTLD cases into at least four subtypes, which are correlated with clinical presentations and genetic causes. To understand the molecular basis of this heterogeneity, we developed SarkoSpin, a new method for biochemical isolation of pathological TDP-43. By combining SarkoSpin with mass spectrometry, we revealed proteins beyond TDP-43 that become abnormally insoluble in a disease subtype-specific manner. We show that pTDP-43 extracted from brain forms stable assemblies of distinct densities and morphologies that are associated with disease subtypes. Importantly, biochemically extracted pTDP-43 assemblies showed differential neurotoxicity and seeding that were correlated with disease duration of FTLD subjects. Our data are consistent with the notion that disease heterogeneity could originate from alternate pathological TDP-43 conformations, which are reminiscent of prion strains.
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.
Synopsis
Pathological TDP‐43 derived from FTLD patient brains triggers de novo aggregation of physiological TDP‐43 in host cells via a prion‐like cascade. Different subtypes of FTLD‐TDP show distinct organization of TDP‐43 aggregates in patient brains, different seeding potencies, neoaggregate structures and C‐terminal serine phosphorylation timelines.
Pathological TDP‐43 triggers template‐dependent aggregation and amplification of neoaggregates
FTLD‐TDP‐A and FTLD‐TDP‐C aggregates have different seeding potency (high vs. low) and follow distinct timelines of C‐terminal serine phosphorylation
Neoaggregates triggered by FTLD‐TDP‐A and FTLD‐TDP‐C display different aggregation profiles (fibrillar vs. amorphous) resembling the original aggregates in patient brains (densely vs. loosely packed fibrils)
Pathological TDP‐43 derived from FTLD patient brains triggers de novo aggregation of physiological TDP‐43 in host cells via a prion‐like cascade. Different subtypes of FTLD‐TDP show distinct organization of TDP‐43 aggregates in patient brains, different seeding potencies, neoaggregate structures and C‐terminal serine phosphorylation timelines.
Altered cellular localization and pathologic aggregation of RNA binding proteins (RPBs) containing low complexity regions (LCRs) is a hallmark of neurodegenerative diseases such as amyotrophic ...lateral sclerosis (ALS) and frontotemporal dementia (FTD). Given the importance of RBPs in maintaining a healthy RNA homeostasis, a common mechanism in disease progression is the loss of RNA-related cellular functions. In this review, we summarize and discuss the knowledge gained in the recent years on the molecular mechanisms of TDP-43 proteinopathies that comprise a set of neurodegenerative diseases characterized by the mislocalization and aggregation of the RNA-binding protein TDP-43. Based on biophysical, biochemical and
data, we highlight pathways that are misregulated early in disease and contribute to its progression, thereby representing attractive therapeutic targets.
Human cellular models of neurodegeneration require reproducibility and longevity, which is necessary for simulating age-dependent diseases. Such systems are particularly needed for TDP-43 ...proteinopathies
, which involve human-specific mechanisms
that cannot be directly studied in animal models. Here, to explore the emergence and consequences of TDP-43 pathologies, we generated induced pluripotent stem cell-derived, colony morphology neural stem cells (iCoMoNSCs) via manual selection of neural precursors
. Single-cell transcriptomics and comparison to independent neural stem cells
showed that iCoMoNSCs are uniquely homogenous and self-renewing. Differentiated iCoMoNSCs formed a self-organized multicellular system consisting of synaptically connected and electrophysiologically active neurons, which matured into long-lived functional networks (which we designate iNets). Neuronal and glial maturation in iNets was similar to that of cortical organoids
. Overexpression of wild-type TDP-43 in a minority of neurons within iNets led to progressive fragmentation and aggregation of the protein, resulting in a partial loss of function and neurotoxicity. Single-cell transcriptomics revealed a novel set of misregulated RNA targets in TDP-43-overexpressing neurons and in patients with TDP-43 proteinopathies exhibiting a loss of nuclear TDP-43. The strongest misregulated target encoded the synaptic protein NPTX2, the levels of which are controlled by TDP-43 binding on its 3' untranslated region. When NPTX2 was overexpressed in iNets, it exhibited neurotoxicity, whereas correcting NPTX2 misregulation partially rescued neurons from TDP-43-induced neurodegeneration. Notably, NPTX2 was consistently misaccumulated in neurons from patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration with TDP-43 pathology. Our work directly links TDP-43 misregulation and NPTX2 accumulation, thereby revealing a TDP-43-dependent pathway of neurotoxicity.
The Cu(+) pump ATP7B plays an irreplaceable role in the elimination of excess Cu(+) by the hepatocyte into the bile. The trafficking and site of action of ATP7B are subjects of controversy. One ...current proposal is that an increase in intracellular Cu(+) results in the translocation of ATP7B to the lysosomes and excretion of excess Cu(+) through lysosomal-mediated exocytosis at the bile canaliculus. Here, we show that ATP7B is transported from the trans-Golgi network (TGN) to the bile canaliculus by basolateral sorting and endocytosis, and microtubule-mediated transcytosis through the subapical compartment. Trafficking ATP7B is not incorporated into lysosomes, and addition of Cu(+) does not cause relocalization of lysosomes and the appearance of lysosome markers in the bile canaliculus. Our data reveal the pathway of the Cu(+)-mediated transport of ATP7B from the TGN to the bile canaliculus and indicates that the bile canaliculus is the primary site of ATP7B action in the elimination of excess Cu(.)
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