The fidelity of transcription in human cells.

To determine the error rate of transcription in human cells, we analyzed the transcriptome of H1 human embryonic stem cells with a circle-sequencing approach that allows for high-fidelity sequencing of the transcriptome. These experiments identified approximately 100,000 errors distributed over every major RNA species in human cells. Our results indicate that different RNA species display different error rates, suggesting that human cells prioritize the fidelity of some RNAs over others. Cross-referencing the errors that we detected with various genetic and epigenetic features of the human genome revealed that the in vivo error rate in human cells changes along the length of a transcript and is further modified by genetic context, repetitive elements, epigenetic markers, and the speed of transcription. Our experiments further suggest that BRCA1, a DNA repair protein implicated in breast cancer, has a previously unknown role in the suppression of transcription errors. Finally, we analyzed the distribution of transcription errors in multiple tissues of a new mouse model and found that they occur preferentially in neurons, compared to other cell types. These observations lend additional weight to the idea that transcription errors play a key role in the progression of various neurological disorders, including Alzheimer's disease.

Tau filaments from amyotrophic lateral sclerosis/parkinsonism-dementia complex adopt the CTE fold.

The amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) of the island of Guam and the Kii peninsula of Japan is a fatal neurodegenerative disease of unknown cause that is characterized by the presence of abundant filamentous tau inclusions in brains and spinal cords. Here, we used electron cryo-microscopy to determine the structures of tau filaments from the cerebral cortex of three cases of ALS/PDC from Guam and eight cases from Kii, as well as from the spinal cord of two of the Guam cases. Tau filaments had the chronic traumatic encephalopathy (CTE) fold, with variable amounts of Type I and Type II filaments. Paired helical tau filaments were also found in three Kii cases and tau filaments with the corticobasal degeneration fold in one Kii case. We identified a new Type III CTE tau filament, where protofilaments pack against each other in an antiparallel fashion. ALS/PDC is the third known tauopathy with CTE-type filaments and abundant tau inclusions in cortical layers II/III, the others being CTE and subacute sclerosing panencephalitis. Because these tauopathies are believed to have environmental causes, our findings support the hypothesis that ALS/PDC is caused by exogenous factors.

Deciphering the Proteome Dynamics during Development of Neurons Derived from Induced Pluripotent Stem Cells.

Neuronal development is a complex multistep process that shapes neurons by progressing though several typical stages, including axon outgrowth, dendrite formation, and synaptogenesis. Knowledge of the mechanisms of neuronal development is mostly derived from the study of animal models. Advances in stem cell technology now enable us to generate neurons from human induced pluripotent stem cells (iPSCs). Here we provide a mass spectrometry-based quantitative proteomic signature of human iPSC-derived neurons, i.e., iPSC-derived induced glutamatergic neurons and iPSC-derived motor neurons, throughout neuronal differentiation. Tandem mass tag 10-plex labeling was carried out to perform proteomic profiling of cells at different time points. Our analysis reveals significant expression changes (FDR < 0.001) of several key proteins during the differentiation process, e.g., proteins involved in the Wnt and Notch signaling pathways. Overall, our data provide a rich resource of information on protein expression during human iPSC neuron differentiation.

TDP-43 controls lysosomal pathways thereby determining its own clearance and cytotoxicity.

TDP-43 is a nuclear RNA-binding protein whose cytoplasmic accumulation is the pathological hallmark of amyotrophic lateral sclerosis (ALS). For a better understanding of this devastating disorder at the molecular level, it is important to identify cellular pathways involved in the clearance of detrimental TDP-43. Using a yeast model system, we systematically analyzed to which extent TDP-43-triggered cytotoxicity is modulated by conserved lysosomal clearance pathways. We observed that the lysosomal fusion machinery and the endolysosomal pathway, which are crucial for proper lysosomal function, were pivotal for survival of cells exposed to TDP-43. Interestingly, TDP-43 itself interfered with these critical TDP-43 clearance pathways. In contrast, autophagy played a complex role in this process. It contributed to the degradation of TDP-43 in the absence of endolysosomal pathway activity, but its induction also enhanced cell death. Thus, TDP-43 interfered with lysosomal function and its own degradation via lysosomal pathways, and triggered lethal autophagy. We propose that these effects critically contribute to cellular dysfunction in TDP-43 proteinopathies.

Correction to: Somatic mutations in neurons during aging and neurodegeneration.

In the original article, the panels "Brain organoids" and "Transgenics" were included in Fig. 5 without permission.

Metalloprotease-mediated cleavage of PlexinD1 and its sequestration to actin rods in the motoneuron disease spinal muscular atrophy (SMA).

Cytoskeletal rearrangement during axon growth is mediated by guidance receptors and their ligands which act either as repellent, attractant or both. Regulation of the actin cytoskeleton is disturbed in Spinal Muscular Atrophy (SMA), a devastating neurodegenerative disease affecting mainly motoneurons, but receptor-ligand interactions leading to the dysregulation causing SMA are poorly understood. In this study, we analysed the role of the guidance receptor PlexinD1 in SMA pathogenesis. We showed that PlexinD1 is cleaved by metalloproteases in SMA and that this cleavage switches its function from an attractant to repellent. Moreover, we found that the PlexinD1 cleavage product binds to actin rods, pathological aggregate-like structures which had so far been described for age-related neurodegenerative diseases. Our data suggest a novel disease mechanism for SMA involving formation of actin rods as a molecular sink for a cleaved PlexinD1 fragment leading to dysregulation of receptor signaling.

Somatic mutations in neurons during aging and neurodegeneration.

The nervous system is composed of a large variety of neurons with a diverse array of morphological and functional properties. This heterogeneity is essential for the construction and maintenance of a distinct set of neural networks with unique characteristics. Accumulating evidence now indicates that neurons do not only differ at a functional level, but also at the genomic level. These genomic discrepancies seem to be the result of somatic mutations that emerge in nervous tissue during development and aging. Ultimately, these mutations bring about a genetically heterogeneous population of neurons, a phenomenon that is commonly referred to as "somatic brain mosaicism". Improved understanding of the development and consequences of somatic brain mosaicism is crucial to understand the impact of somatic mutations on neuronal function in human aging and disease. Here, we highlight a number of topics related to somatic brain mosaicism, including some early experimental evidence for somatic mutations in post-mitotic neurons of the hypothalamo-neurohypophyseal system. We propose that age-related somatic mutations are particularly interesting, because aging is a major risk factor for a variety of neuronal diseases, including Alzheimer's disease. We highlight potential links between somatic mutations and the development of these diseases and argue that recent advances in single-cell genomics and in vivo physiology have now finally made it possible to dissect the origins and consequences of neuronal mutations in unprecedented detail.

Selective Transgenic Expression of Mutant Ubiquitin in Purkinje Cell Stripes in the Cerebellum.

The ubiquitin-proteasome system (UPS) is one of the major mechanisms for protein breakdown in cells, targeting proteins for degradation by enzymatically conjugating them to ubiquitin molecules. Intracellular accumulation of ubiquitin-B+1 (UBB+1), a frameshift mutant of ubiquitin-B, is indicative of a dysfunctional UPS and has been implicated in several disorders, including neurodegenerative disease. UBB+1-expressing transgenic mice display widespread labeling for UBB+1 in brain and exhibit behavioral deficits. Here, we show that UBB+1 is specifically expressed in a subset of parasagittal stripes of Purkinje cells in the cerebellar cortex of a UBB+1-expressing mouse model. This expression pattern is reminiscent of that of the constitutively expressed Purkinje cell antigen HSP25, a small heat shock protein with neuroprotective properties.

Comparative interactomics analysis of different ALS-associated proteins identifies converging molecular pathways.

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease with no effective treatment available. An increasing number of genetic causes of ALS are being identified, but how these genetic defects lead to motor neuron degeneration and to which extent they affect common cellular pathways remains incompletely understood. To address these questions, we performed an interactomic analysis to identify binding partners of wild-type (WT) and ALS-associated mutant versions of ATXN2, C9orf72, FUS, OPTN, TDP-43 and UBQLN2 in neuronal cells. This analysis identified several known but also many novel binding partners of these proteins. Interactomes of WT and mutant ALS proteins were very similar except for OPTN and UBQLN2, in which mutations caused loss or gain of protein interactions. Several of the identified interactomes showed a high degree of overlap: shared binding partners of ATXN2, FUS and TDP-43 had roles in RNA metabolism; OPTN- and UBQLN2-interacting proteins were related to protein degradation and protein transport, and C9orf72 interactors function in mitochondria. To confirm that this overlap is important for ALS pathogenesis, we studied fragile X mental retardation protein (FMRP), one of the common interactors of ATXN2, FUS and TDP-43, in more detail in in vitro and in vivo model systems for FUS ALS. FMRP localized to mutant FUS-containing aggregates in spinal motor neurons and bound endogenous FUS in a direct and RNA-sensitive manner. Furthermore, defects in synaptic FMRP mRNA target expression, neuromuscular junction integrity, and motor behavior caused by mutant FUS in zebrafish embryos, could be rescued by exogenous FMRP expression. Together, these results show that interactomics analysis can provide crucial insight into ALS disease mechanisms and they link FMRP to motor neuron dysfunction caused by FUS mutations.

Tau Filaments from Amyotrophic Lateral Sclerosis/Parkinsonism-Dementia Complex (ALS/PDC) adopt the CTE Fold.

The amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) of the island of Guam and the Kii peninsula of Japan is a fatal neurodegenerative disease of unknown cause that is characterised by the presence of abundant filamentous tau inclusions in brains and spinal cords. Here we used electron cryo-microscopy (cryo-EM) to determine the structures of tau filaments from the cerebral cortex of three cases of ALS/PDC from Guam and eight cases from Kii, as well as from the spinal cord of two of the Guam cases. Tau filaments had the chronic traumatic encephalopathy (CTE) fold, with variable amounts of Type I and Type II filaments. Paired helical tau filaments were also found in two Kii cases. We also identified a novel Type III CTE tau filament, where protofilaments pack against each other in an anti-parallel fashion. ALS/PDC is the third known tauopathy with CTE-type filaments and abundant tau inclusions in cortical layers II/III, the others being CTE and subacute sclerosing panencephalitis. Because these tauopathies are believed to have environmental causes, our findings support the hypothesis that ALS/PDC is caused by exogenous factors.

Evolutionary conservation of the fidelity of transcription.

Accurate transcription is required for the faithful expression of genetic information. However, relatively little is known about the molecular mechanisms that control the fidelity of transcription, or the conservation of these mechanisms across the tree of life. To address these issues, we measured the error rate of transcription in five organisms of increasing complexity and found that the error rate of RNA polymerase II ranges from 2.9 × 10-6 ± 1.9 × 10-7/bp in yeast to 4.0 × 10-6 ± 5.2 × 10-7/bp in worms, 5.69 × 10-6 ± 8.2 × 10-7/bp in flies, 4.9 × 10-6 ± 3.6 × 10-7/bp in mouse cells and 4.7 × 10-6 ± 9.9 × 10-8/bp in human cells. These error rates were modified by various factors including aging, mutagen treatment and gene modifications. For example, the deletion or modification of several related genes increased the error rate substantially in both yeast and human cells. This research highlights the evolutionary conservation of factors that control the fidelity of transcription. Additionally, these experiments provide a reasonable estimate of the error rate of transcription in human cells and identify disease alleles in a subunit of RNA polymerase II that display error-prone transcription. Finally, we provide evidence suggesting that the error rate and spectrum of transcription co-evolved with our genetic code.

Altered ubiquitin signaling induces Alzheimer's disease-like hallmarks in a three-dimensional human neural cell culture model.

Alzheimer's disease (AD) is characterized by toxic protein accumulation in the brain. Ubiquitination is essential for protein clearance in cells, making altered ubiquitin signaling crucial in AD development. A defective variant, ubiquitin B + 1 (UBB+1), created by a non-hereditary RNA frameshift mutation, is found in all AD patient brains post-mortem. We now detect UBB+1 in human brains during early AD stages. Our study employs a 3D neural culture platform derived from human neural progenitors, demonstrating that UBB+1 alone induces extracellular amyloid-ß (Aß) deposits and insoluble hyperphosphorylated tau aggregates. UBB+1 competes with ubiquitin for binding to the deubiquitinating enzyme UCHL1, leading to elevated levels of amyloid precursor protein (APP), secreted Aß peptides, and Aß build-up. Crucially, silencing UBB+1 expression impedes the emergence of AD hallmarks in this model system. Our findings highlight the significance of ubiquitin signalling as a variable contributing to AD pathology and present a nonclinical platform for testing potential therapeutics.

Spinal Muscular Atrophy Patient iPSC-Derived Motor Neurons Display Altered Proteomes at Early Stages of Differentiation.

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder characterized by loss of motor neurons (MN) in the spinal cord leading to progressive muscle atrophy and weakness. SMA is caused by mutations in the survival motor neuron 1 (SMN1) gene, resulting in reduced levels of survival motor neuron (SMN) protein. The mechanisms that link SMN deficiency to selective motor neuron dysfunction in SMA remain largely unknown. We present here, for the first time, a comprehensive quantitative TMT-10plex proteomics analysis that covers the development of induced pluripotent stem cell-derived MNs from both healthy individuals and SMA patients. We show that the proteomes of SMA samples segregate from controls already at early stages of neuronal differentiation. The altered proteomic signature in SMA MNs is associated with mRNA splicing, ribonucleoprotein biogenesis, organelle organization, cellular biogenesis, and metabolic processes. We highlight several known SMN-binding partners and evaluate their expression changes during MN differentiation. In addition, we compared our study to human and mouse in vivo proteomic studies revealing distinct and similar signatures. Altogether, our work provides a comprehensive resource of molecular events during early stages of MN differentiation, containing potentially therapeutically interesting protein expression profiles for SMA.

Expression Profile of Long Non-Coding RNAs during Early Postnatal Development of Mouse Spinal Cord.

Long non-coding RNAs (lncRNAs) are a diverse class of transcripts that are >200 nucleotides long and lack significant protein-coding potential. LncRNAs are emerging as major regulators of gene expression networks in various physiological and pathological processes. Interestingly, many lncRNAs show tissue-specific expression, for example, in the nervous system. Although lncRNAs have been suggested to play key roles in the brain, most functions of neural lncRNAs remain poorly understood. In order to provide a catalog of lncRNA changes that occur in spinal cord during early postnatal development, RNA from mouse spinal cord was sequenced at different time points in the first week after birth (postnatal day 1 and postnatal day 7). Two hundred and ninty-six differentially expressed lncRNAs (FDR < 0.05) were identified in the resulting dataset. Altered transcripts were associated with several biological processes including myelination, neural differentiation, and glial cell development. PCR validation confirmed differential expression of select lncRNAs (i.e., Cerox1, lncOL3, Neat1, and Sox2ot). Additionally, analysis of circular RNAs (circRNAs), another class of non-coding RNA with regulatory potency, pointed out a number of circRNAs associated with spinal cord development. These data can be used as a resource for future studies on transcriptional changes during early postnatal nervous system development and studies of disorders that affect the spinal cord, e.g., spinal muscular atrophy.

Expression of Mutant Ubiquitin and Proteostasis Impairment in Kii Amyotrophic Lateral Sclerosis/Parkinsonism-Dementia Complex Brains.

Kii amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) is a progressive neurodegenerative disorder that is endemic to the Kii peninsula of Japan. The disorder is clinically characterized by a variable combination of parkinsonism, dementia, and motor neuron symptoms. Despite extensive investigations, the etiology and pathogenesis of ALS/PDC remain unclear. At the neuropathological level, Kii ALS/PDC is characterized by neuronal loss and tau-dominant polyproteinopathy. Here, we report the accumulation of several proteins involved in protein homeostasis pathways, that is, the ubiquitin-proteasome system and the autophagy-lysosome pathway, in postmortem brain tissue from a number of Kii ALS/PDC cases (n = 4). Of particular interest is the presence of a mutant ubiquitin protein (UBB+1), which is indicative of disrupted ubiquitin homeostasis. The findings suggest that abnormal protein aggregation is linked to impaired protein homeostasis pathways in Kii ALS/PDC.

Activation of the Unfolded Protein Response and Proteostasis Disturbance in Parkinsonism-Dementia of Guam.

Guam parkinsonism-dementia (G-PD) is a progressive and fatal neurodegenerative disorder among the native inhabitants of the Mariana Islands that manifests clinically with parkinsonism as well as dementia. Neuropathologically, G-PD is characterized by abundant neurofibrillary tangles composed of hyperphosphorylated tau, marked deposition of transactive response DNA-binding protein 43 kDa (TDP-43), and neuronal loss. The mechanisms that underlie neurodegeneration in G-PD are poorly understood. Here, we report that the unfolded protein response (UPR) is activated in G-PD brains. Specifically, we show that the endoplasmic reticulum (ER) chaperone binding immunoglobulin protein/glucose-regulated protein 78 kDa and phosphorylated (activated) ER stress sensor protein kinase RNA-like ER kinase accumulate in G-PD brains. Furthermore, proteinaceous aggregates in G-PD brains are found to contain several proteins related to the ubiquitin-proteasome system (UPS) and the autophagy pathway, two major mechanisms for intracellular protein degradation. In particular, a mutant ubiquitin (UBB+1), whose presence is a marker for UPS dysfunction, is shown to accumulate in G-PD brains. We demonstrate that UBB+1 is a potent modifier of TDP-43 aggregation and cytotoxicity in vitro. Overall, these data suggest that UPR activation and intracellular proteolytic pathways are intimately connected with the accumulation of aggregated proteins in G-PD.

Modeling Brain Somatic Mosaicism With Cerebral Organoids, Including a Note on Mutant Microglia.

The brain is a genomic mosaic. Cell-to-cell genomic differences, which are the result of somatic mutations during development and aging, contribute to cellular diversity in the nervous system. This genomic diversity has important implications for nervous system development, function, and disease. Brain somatic mosaicism might contribute to individualized behavioral phenotypes and has been associated with several neuropsychiatric and neurodegenerative disorders. Therefore, understanding the causes and consequences of somatic mosaicism in neural circuits is of great interest. Recent advances in 3D cell culture technology have provided new means to study human organ development and various human pathologies in vitro. Cerebral organoids ("mini-brains") are pluripotent stem cell-derived 3D culture systems that recapitulate, to some extent, the developmental processes and organization of the developing human brain. Here, I discuss the application of these neural organoids for modeling brain somatic mosaicism in a lab dish. Special emphasis is given to the potential role of microglial mutations in the pathogenesis of neurodegenerative diseases.

Dysfunction of Protein Quality Control in Parkinsonism-Dementia Complex of Guam.

Guam parkinsonism-dementia complex (G-PDC) is an enigmatic neurodegenerative disease that is endemic to the Pacific island of Guam. G-PDC patients are clinically characterized by progressive cognitive impairment and parkinsonism. Neuropathologically, G-PDC is characterized by abundant neurofibrillary tangles, which are composed of hyperphosphorylated tau, marked deposition of 43-kDa TAR DNA-binding protein, and neuronal loss. Although both genetic and environmental factors have been implicated, the etiology and pathogenesis of G-PDC remain unknown. Recent neuropathological studies have provided new clues about the pathomechanisms involved in G-PDC. For example, deposition of abnormal components of the protein quality control system in brains of G-PDC patients indicates a role for proteostasis imbalance in the disease. This opens up promising avenues for new research on G-PDC and could have important implications for the study of other neurodegenerative disorders.