APP antisense oligonucleotides reduce Aß aggregation and rescue endolysosomal dysfunction in Alzheimer's disease.

APP gene dosage is strongly associated with Alzheimer's disease (AD) pathogenesis. Genomic duplication of the APP locus leads to autosomal dominant early-onset AD. Individuals with Down syndrome (trisomy of chromosome 21) harbor 3 copies of the APP gene and invariably develop progressive AD with highly characteristic neuropathological features. Restoring expression of APP to the equivalent of that of two gene copies, or lower, is a rational therapeutic strategy, as it would restore physiological levels of neuronal APP protein without the potentially deleterious consequences of inadvertently inducing loss of APP function. Here we find that antisense oligonucleotides (ASOs) targeting APP are an effective approach to reduce APP protein levels and rescue endolysosome and autophagy dysfunction in APP duplication human induced pluripotent stem cell (hiPSC)-derived cortical neurons. Importantly, using ultrasensitive single-aggregate imaging techniques, we show that APP targeting ASOs significantly reduce both intracellular and extracellular Aß-containing aggregates. Our results highlight the potential of APP ASOs as a therapeutic approach for forms of AD caused by duplication of the APP gene, including monogenic AD and AD related to Down syndrome.

4R tau drives endolysosomal and autophagy dysfunction in frontotemporal dementia.

Dysfunction of the neuronal endolysosome and macroautophagy/autophagy pathway is emerging as an important pathogenic mechanism in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). The VCP (valosin-containing protein) gene is of significant relevance, directly implicated in both FTD and ALS. In our recent study, we used patient-derived stem cells to study the effects of VCP mutations on the endolysosome and autophagy system in human cortical excitatory neurons. We found that VCP mutations cause an abnormal accumulation of enlarged endosomes and lysosomes, accompanied by reduced autophagy flux. VCP mutations also lead to the spatial dissociation of intra-nuclear RNA-binding proteins, FUS and SFPQ, which correlates with alternative splicing of the MAPT pre-mRNA and increased tau phosphorylation. Importantly, we found that an increase in the 4R-tau isoform is sufficient to drive toxic changes in healthy human cortical excitatory neurons, including tau hyperphosphorylation, endolysosomal dysfunction, lysosomal membrane rupture, endoplasmic reticulum stress, and apoptosis. Together, our data suggest that endolysosomal and autophagy dysfunction could represent a convergent pathogenic "design principle" shared by both FTD and ALS.

Elevated 4R tau contributes to endolysosomal dysfunction and neurodegeneration in VCP-related frontotemporal dementia.

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are two incurable neurodegenerative diseases that exist on a clinical, genetic and pathological spectrum. The VCP gene is highly relevant, being directly implicated in both FTD and ALS. Here, we investigate the effects of VCP mutations on the cellular homoeostasis of human induced pluripotent stem cell-derived cortical neurons, focusing on endolysosomal biology and tau pathology. We found that VCP mutations cause abnormal accumulation of enlarged endolysosomes accompanied by impaired interaction between two nuclear RNA binding proteins: fused in sarcoma (FUS) and splicing factor, proline- and glutamine-rich (SFPQ) in human cortical neurons. The spatial dissociation of intranuclear FUS and SFPQ correlates with alternative splicing of the MAPT pre-mRNA and increased tau phosphorylation. Importantly, we show that inducing 4R tau expression using antisense oligonucleotide technology is sufficient to drive neurodegeneration in control human neurons, which phenocopies VCP-mutant neurons. In summary, our findings demonstrate that tau hyperphosphorylation, endolysosomal dysfunction, lysosomal membrane rupture, endoplasmic reticulum stress and apoptosis are driven by a pathogenic increase in 4R tau.

The ALS/FTD-related C9orf72 hexanucleotide repeat expansion forms RNA condensates through multimolecular G-quadruplexes.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases that exist on a clinico-pathogenetic spectrum, designated ALS/FTD. The most common genetic cause of ALS/FTD is expansion of the intronic hexanucleotide repeat (GGGGCC)n in C9orf72. Here, we investigate the formation of nucleic acid secondary structures in these expansion repeats, and their role in generating condensates characteristic of ALS/FTD. We observe significant aggregation of the hexanucleotide sequence (GGGGCC)n, which we associate to the formation of multimolecular G-quadruplexes (mG4s) by using a range of biophysical techniques. Exposing the condensates to G4-unfolding conditions leads to prompt disassembly, highlighting the key role of mG4-formation in the condensation process. We further validate the biological relevance of our findings by detecting an increased prevalence of G4-structures in C9orf72 mutant human motor neurons when compared to healthy motor neurons by staining with a G4-selective fluorescent probe, revealing signal in putative condensates. Our findings strongly suggest that RNA G-rich repetitive sequences can form protein-free condensates sustained by multimolecular G-quadruplexes, highlighting their potential relevance as therapeutic targets for C9orf72 mutation-related ALS/FTD.

Nonsense-mediated mRNA decay in neuronal physiology and neurodegeneration.

The processes of mRNA export from the nucleus and subsequent mRNA translation in the cytoplasm are of particular relevance in eukaryotic cells. In highly polarised cells such as neurons, finely-tuned molecular regulation of these processes serves to safeguard the spatiotemporal fidelity of gene expression. Nonsense-mediated mRNA decay (NMD) is a cytoplasmic translation-dependent quality control process that regulates gene expression in a wide range of scenarios in the nervous system, including neurodevelopment, learning, and memory formation. Moreover, NMD dysregulation has been implicated in a broad range of neurodevelopmental and neurodegenerative disorders. We discuss how NMD and related aspects of mRNA translation regulate key neuronal functions and, in particular, we focus on evidence implicating these processes in the molecular pathogenesis of neurodegeneration. Finally, we discuss the therapeutic potential and challenges of targeting mRNA translation and NMD across the spectrum of largely untreatable neurological diseases.

Amplifying the Heat Shock Response Ameliorates ALS and FTD Pathology in Mouse and Human Models.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are now known as parts of a disease spectrum with common pathological features and genetic causes. However, as both conditions are clinically heterogeneous, patient groups may be phenotypically similar but pathogenically and genetically variable. Despite numerous clinical trials, there remains no effective therapy for these conditions, which, in part, may be due to challenges of therapy development in a heterogeneous patient population. Disruption to protein homeostasis is a key feature of different forms of ALS and FTD. Targeting the endogenous protein chaperone system, the heat shock response (HSR) may, therefore, be a potential therapeutic approach. We conducted a preclinical study of a known pharmacological amplifier of the HSR, called arimoclomol, in mice with a mutation in valosin-containing protein (VCP) which causes both ALS and FTD in patients. We demonstrate that amplification of the HSR ameliorates the ALS/FTD-like phenotype in the spinal cord and brain of mutant VCP mice and prevents neuronal loss, replicating our earlier findings in the SOD1 mouse model of ALS. Moreover, in human cell models, we demonstrate improvements in pathology upon arimoclomol treatment in mutant VCP patient fibroblasts and iPSC-derived motor neurons. Our findings suggest that targeting of the HSR may have therapeutic potential, not only in non-SOD1 ALS, but also for the treatment of FTD.

Nucleocytoplasmic mRNA redistribution accompanies RNA binding protein mislocalization in ALS motor neurons and is restored by VCP ATPase inhibition.

Amyotrophic lateral sclerosis (ALS) is characterized by nucleocytoplasmic mislocalization of the RNA-binding protein (RBP) TDP-43. However, emerging evidence suggests more widespread mRNA and protein mislocalization. Here, we employed nucleocytoplasmic fractionation, RNA sequencing, and mass spectrometry to investigate the localization of mRNA and protein in induced pluripotent stem cell-derived motor neurons (iPSMNs) from ALS patients with TARDBP and VCP mutations. ALS mutant iPSMNs exhibited extensive nucleocytoplasmic mRNA redistribution, RBP mislocalization, and splicing alterations. Mislocalized proteins exhibited a greater affinity for redistributed transcripts, suggesting a link between RBP mislocalization and mRNA redistribution. Notably, treatment with ML240, a VCP ATPase inhibitor, partially restored mRNA and protein localization in ALS mutant iPSMNs. ML240 induced changes in the VCP interactome and lysosomal localization and reduced oxidative stress and DNA damage. These findings emphasize the link between RBP mislocalization and mRNA redistribution in ALS motor neurons and highlight the therapeutic potential of VCP inhibition.

Transcriptome-wide RNA binding analysis of C9orf72 poly(PR) dipeptides.

An intronic GGGGCC repeat expansion in C9orf72 is a common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeats are transcribed in both sense and antisense directions to generate distinct dipeptide repeat proteins, of which poly(GA), poly(GR), and poly(PR) have been implicated in contributing to neurodegeneration. Poly(PR) binding to RNA may contribute to toxicity, but analysis of poly(PR)-RNA binding on a transcriptome-wide scale has not yet been carried out. We therefore performed crosslinking and immunoprecipitation (CLIP) analysis in human cells to identify the RNA binding sites of poly(PR). We found that poly(PR) binds to nearly 600 RNAs, with the sequence GAAGA enriched at the binding sites. In vitro experiments showed that poly(GAAGA) RNA binds poly(PR) with higher affinity than control RNA and induces the phase separation of poly(PR) into condensates. These data indicate that poly(PR) preferentially binds to poly(GAAGA)-containing RNAs, which may have physiological consequences.

Functional roles of reactive astrocytes in neuroinflammation and neurodegeneration.

Despite advances in uncovering the mechanisms that underlie neuroinflammation and neurodegenerative disease, therapies that prevent neuronal loss remain elusive. Targeting of disease-defining markers in conditions such as Alzheimer disease (amyloid-ß and tau) or Parkinson disease (α-synuclein) has been met with limited success, suggesting that these proteins do not act in isolation but form part of a pathological network. This network could involve phenotypic alteration of multiple cell types in the CNS, including astrocytes, which have a major neurosupportive, homeostatic role in the healthy CNS but adopt reactive states under acute or chronic adverse conditions. Transcriptomic studies in human patients and disease models have revealed the co-existence of many putative reactive sub-states of astrocytes. Inter-disease and even intra-disease heterogeneity of reactive astrocytic sub-states are well established, but the extent to which specific sub-states are shared across different diseases is unclear. In this Review, we highlight how single-cell and single-nuclei RNA sequencing and other 'omics' technologies can enable the functional characterization of defined reactive astrocyte states in various pathological scenarios. We provide an integrated perspective, advocating cross-modal validation of key findings to define functionally important sub-states of astrocytes and their triggers as tractable therapeutic targets with cross-disease relevance.

Modeling the Inflammatory Response of Traumatic Brain Injury Using Human Induced Pluripotent Stem Cell Derived Microglia.

The neuroinflammatory response after traumatic brain injury (TBI) is implicated as a key mediator of secondary injury in both the acute and chronic periods after primary injury. Microglia are the key innate immune cell in the central nervous system, responding to injury with the release of cytokines and chemokines. In this context, we aimed to characterize the downstream cytokine response of human induced pluripotent stem cell (iPSC)-derived microglia when stimulated with five separate cytokines identified after human TBI. The iPSC-derived microglia were exposed to interleukin (IL)-1ß, IL-4, IL-6, IL-10, and tumor necrosis factor (TNF) in the concentration ranges identified in clinical TBI studies. The downstream cytokine response was measured against a panel of 37 separate cytokines over a 72h time-course. The secretome revealed concentration-, time- and combined concentration and time-dependent downstream responses. TNF appeared to be the strongest inducer of downstream cytokine changes (51), followed by IL-1ß (26) and IL-4 (19). IL-10 (11) and IL-6 (10) produced fewer responses. We also compare these responses with our previous studies of iPSC-derived neuronal and astrocyte cultures and the in vivo human TBI cytokine response. Notably, we found microglial culture to induce both a wider range of downstream cytokine responses and a greater fold change in concentration for those downstream responses, compared with astrocyte and neuronal cultures. In summary, we present a dataset for human microglial cytokine responses specific to the secretome found in the clinical context of TBI. This reductionist approach complements our previous datasets for astrocyte and neuronal responses and will provide a platform to enable future studies to unravel the complex neuroinflammatory network activated after TBI.

Direct m6A recognition by IMP1 underlays an alternative model of target selection for non-canonical methyl-readers.

m6A methylation provides an essential layer of regulation in organismal development, and is aberrant in a range of cancers and neuro-pathologies. The information encoded by m6A methylation is integrated into existing RNA regulatory networks by RNA binding proteins that recognise methylated sites, the m6A readers. m6A readers include a well-characterised class of dedicated proteins, the YTH proteins, as well as a broader group of multi-functional regulators where recognition of m6A is only partially understood. Molecular insight in this recognition is essential to build a mechanistic understanding of global m6A regulation. In this study, we show that the reader IMP1 recognises the m6A using a dedicated hydrophobic platform that assembles on the methyl moiety, creating a stable high-affinity interaction. This recognition is conserved across evolution and independent from the underlying sequence context but is layered upon the strong sequence specificity of IMP1 for GGAC RNA. This leads us to propose a concept for m6A regulation where methylation plays a context-dependent role in the recognition of selected IMP1 targets that is dependent on the cellular concentration of available IMP1, differing from that observed for the YTH proteins.

Integrated transcriptome landscape of ALS identifies genome instability linked to TDP-43 pathology.

Amyotrophic Lateral Sclerosis (ALS) causes motor neuron degeneration, with 97% of cases exhibiting TDP-43 proteinopathy. Elucidating pathomechanisms has been hampered by disease heterogeneity and difficulties accessing motor neurons. Human induced pluripotent stem cell-derived motor neurons (iPSMNs) offer a solution; however, studies have typically been limited to underpowered cohorts. Here, we present a comprehensive compendium of 429 iPSMNs from 15 datasets, and 271 post-mortem spinal cord samples. Using reproducible bioinformatic workflows, we identify robust upregulation of p53 signalling in ALS in both iPSMNs and post-mortem spinal cord. p53 activation is greatest with C9orf72 repeat expansions but is weakest with SOD1 and FUS mutations. TDP-43 depletion potentiates p53 activation in both post-mortem neuronal nuclei and cell culture, thereby functionally linking p53 activation with TDP-43 depletion. ALS iPSMNs and post-mortem tissue display enrichment of splicing alterations, somatic mutations, and gene fusions, possibly contributing to the DNA damage response.

Molecular mechanisms of amyotrophic lateral sclerosis as broad therapeutic targets for gene therapy applications utilizing adeno-associated viral vectors.

Despite the devastating clinical outcome of the neurodegenerative disease, amyotrophic lateral sclerosis (ALS), its etiology remains mysterious. Approximately 90% of ALS is characterized as sporadic, signifying that the patient has no family history of the disease. The development of an impactful disease modifying therapy across the ALS spectrum has remained out of grasp, largely due to the poorly understood mechanisms of disease onset and progression. Currently, ALS is invariably fatal and rapidly progressive. It is hypothesized that multiple factors can lead to the development of ALS, however, treatments are often focused on targeting specific familial forms of the disease (10% of total cases). There is a strong need to develop disease modifying treatments for ALS that can be effective across the full ALS spectrum of familial and sporadic cases. Although the onset of disease varies significantly between patients, there are general disease mechanisms and progressions that can be seen broadly across ALS patients. Therefore, this review explores the targeting of these widespread disease mechanisms as possible areas for therapeutic intervention to treat ALS broadly. In particular, this review will focus on targeting mechanisms of defective protein homeostasis and RNA processing, which are both increasingly recognized as design principles of ALS pathogenesis. Additionally, this review will explore the benefits of gene therapy as an approach to treating ALS, specifically focusing on the use of adeno-associated virus (AAV) as a vector for gene delivery to the CNS and recent advances in the field.

Protein aggregation and calcium dysregulation are hallmarks of familial Parkinson's disease in midbrain dopaminergic neurons.

Mutations in the SNCA gene cause autosomal dominant Parkinson's disease (PD), with loss of dopaminergic neurons in the substantia nigra, and aggregation of α-synuclein. The sequence of molecular events that proceed from an SNCA mutation during development, to end-stage pathology is unknown. Utilising human-induced pluripotent stem cells (hiPSCs), we resolved the temporal sequence of SNCA-induced pathophysiological events in order to discover early, and likely causative, events. Our small molecule-based protocol generates highly enriched midbrain dopaminergic (mDA) neurons: molecular identity was confirmed using single-cell RNA sequencing and proteomics, and functional identity was established through dopamine synthesis, and measures of electrophysiological activity. At the earliest stage of differentiation, prior to maturation to mDA neurons, we demonstrate the formation of small ß-sheet-rich oligomeric aggregates, in SNCA-mutant cultures. Aggregation persists and progresses, ultimately resulting in the accumulation of phosphorylated α-synuclein aggregates. Impaired intracellular calcium signalling, increased basal calcium, and impairments in mitochondrial calcium handling occurred early at day 34-41 post differentiation. Once midbrain identity fully developed, at day 48-62 post differentiation, SNCA-mutant neurons exhibited mitochondrial dysfunction, oxidative stress, lysosomal swelling and increased autophagy. Ultimately these multiple cellular stresses lead to abnormal excitability, altered neuronal activity, and cell death. Our differentiation paradigm generates an efficient model for studying disease mechanisms in PD and highlights that protein misfolding to generate intraneuronal oligomers is one of the earliest critical events driving disease in human neurons, rather than a late-stage hallmark of the disease.

Physiological intron retaining transcripts in the cytoplasm abound during human motor neurogenesis.

Intron retention (IR) is now recognized as a dominant splicing event during motor neuron (MN) development; however, the role and regulation of intron-retaining transcripts (IRTs) localized to the cytoplasm remain particularly understudied. Here we show that IR is a physiological process that is spatiotemporally regulated during MN lineage restriction and that IRTs in the cytoplasm are detected in as many as 13% (n = 2297) of the genes expressed during this process. We identify a major class of cytoplasmic IRTs that are not associated with reduced expression of their own genes but instead show a high capacity for RNA-binding protein and miRNA occupancy. Finally, we show that ALS-causing VCP mutations lead to a selective increase in cytoplasmic abundance of this particular class of IRTs, which in turn temporally coincides with an increase in the nuclear expression level of predicted miRNA target genes. Altogether, our study identifies a previously unrecognized class of cytoplasmic intronic sequences with potential regulatory function beyond gene expression.

The role of astrocytes in prion-like mechanisms of neurodegeneration.

Accumulating evidence suggests that neurodegenerative diseases are not merely neuronal in nature but comprise multicellular involvement, with astrocytes emerging as key players. The pathomechanisms of several neurodegenerative diseases involve the deposition of misfolded protein aggregates in neurons that have characteristic prion-like behaviours such as template-directed seeding, intercellular propagation, distinct conformational strains and protein-mediated toxicity. The role of astrocytes in dealing with these pathological prion-like protein aggregates and whether their responses either protect from or conspire with the disease process is currently unclear. Here we review the existing literature implicating astrocytes in multiple neurodegenerative proteinopathies with a focus on prion-like behaviour in this context.

Axonal Length Determines Distinct Homeostatic Phenotypes in Human iPSC Derived Motor Neurons on a Bioengineered Platform.

Stem cell-based experimental platforms for neuroscience can effectively model key mechanistic aspects of human development and disease. However, conventional culture systems often overlook the engineering constraints that cells face in vivo. This is particularly relevant for neurons covering long range connections such as spinal motor neurons (MNs). Their axons extend up to 1m in length and require a complex interplay of mechanisms to maintain cellular homeostasis. However, shorter axons in conventional cultures may not faithfully capture important aspects of their longer counterparts. Here this issue is directly addressed by establishing a bioengineered platform to assemble arrays of human axons ranging from micrometers to centimeters, which allows systematic investigation of the effects of length on human axonas for the first time. This approach reveales a link between length and metabolism in human MNs in vitro, where axons above a "threshold" size induce specific molecular adaptations in cytoskeleton composition, functional properties, local translation, and mitochondrial homeostasis. The findings specifically demonstrate the existence of a length-dependent mechanism that switches homeostatic processes within human MNs. The findings have critical implications for in vitro modeling of several neurodegenerative disorders and reinforce the importance of modeling cell shape and biophysical constraints with fidelity and precision in vitro.

Astrocytes display cell autonomous and diverse early reactive states in familial amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a rapidly progressive and fatal disease. Although astrocytes are increasingly recognized contributors to the underlying pathogenesis, the cellular autonomy and uniformity of astrocyte reactive transformation in different genetic forms of amyotrophic lateral sclerosis remain unresolved. Here we systematically examine these issues by using highly enriched and human induced pluripotent stem cell-derived astrocytes from patients with VCP and SOD1 mutations. We show that VCP mutant astrocytes undergo cell-autonomous reactive transformation characterized by increased expression of complement component 3 (C3) in addition to several characteristic gene expression changes. We then demonstrate that isochronic SOD1 mutant astrocytes also undergo a cell-autonomous reactive transformation, but that this is molecularly distinct from VCP mutant astrocytes. This is shown through transcriptome-wide analyses, identifying divergent gene expression profiles and activation of different key transcription factors in SOD1 and VCP mutant human induced pluripotent stem cell-derived astrocytes. Finally, we show functional differences in the basal cytokine secretome between VCP and SOD1 mutant human induced pluripotent stem cell-derived astrocytes. Our data therefore reveal that reactive transformation can occur cell autonomously in human amyotrophic lateral sclerosis astrocytes and with a striking degree of early molecular and functional heterogeneity when comparing different disease-causing mutations. These insights may be important when considering astrocyte reactivity as a putative therapeutic target in familial amyotrophic lateral sclerosis.

Image-based deep learning reveals the responses of human motor neurons to stress and VCP-related ALS.

AIMS: Although morphological attributes of cells and their substructures are recognised readouts of physiological or pathophysiological states, these have been relatively understudied in amyotrophic lateral sclerosis (ALS) research. METHODS: In this study, we integrate multichannel fluorescence high-content microscopy data with deep learning imaging methods to reveal-directly from unsegmented images-novel neurite-associated morphological perturbations associated with (ALS-causing) VCP-mutant human motor neurons (MNs). RESULTS: Surprisingly, we reveal that previously unrecognised disease-relevant information is withheld in broadly used and often considered 'generic' biological markers of nuclei (DAPI) and neurons ( ß III-tubulin). Additionally, we identify changes within the information content of ALS-related RNA binding protein (RBP) immunofluorescence imaging that is captured in VCP-mutant MN cultures. Furthermore, by analysing MN cultures exposed to different extrinsic stressors, we show that heat stress recapitulates key aspects of ALS. CONCLUSIONS: Our study therefore reveals disease-relevant information contained in a range of both generic and more specific fluorescent markers and establishes the use of image-based deep learning methods for rapid, automated and unbiased identification of biological hypotheses.

Elevated 4R-tau in astrocytes from asymptomatic carriers of the MAPT 10+16 intronic mutation.

The microtubule-associated protein tau gene (MAPT) 10+16 intronic mutation causes frontotemporal lobar degeneration (FTLD) by increasing expression of four-repeat (4R)-tau isoforms. We investigated the potential role for astrocytes in the pathogenesis of FTLD by studying the expression of 4R-tau. We derived astrocytes and neurons from induced pluripotent stem cells from two asymptomatic 10+16 carriers which, compared to controls, showed persistently increased 4R:3R-tau transcript and protein ratios in both cell types. However, beyond 300 days culture, 10+16 neurons showed less marked increase of this 4R:3R-tau transcript ratio compared to astrocytes. Interestingly, throughout maturation, both 10+16 carriers consistently displayed different 4R:3R-tau transcript and protein ratios. These elevated levels of 4R-tau in astrocytes implicate glial cells in the pathogenic process and also suggests a cell-type-specific regulation and may inform and help on treatment of pre-clinical tauopathies.