Amyotrophic Lateral Sclerosis And Frontotemporal Dementia Research Articles

Neural stem cell homeostasis is affected in cortical organoids carrying a mutation in Angiogenin.

Mutations in Angiogenin (ANG) and TARDBP encoding the 43 kDa transactive response DNA binding protein (TDP-43) are associated with amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). ANG is neuroprotective and plays a role in stem cell dynamics in the haematopoietic system. We obtained skin fibroblasts from members of an ALS-FTD family, one with mutation in ANG, one with mutation in both TARDBP and ANG, and one with neither mutation. We reprogrammed these fibroblasts to induced pluripotent stem cells (iPSCs) and generated cortical organoids as well as induced stage-wise differentiation of the iPSCs to neurons. Using these two approaches we investigated the effects of FTD-associated mutations in ANG and TARDBP on neural precursor cells, neural differentiation, and response to stress. We observed striking neurodevelopmental defects such as abnormal and persistent rosettes in the organoids accompanied by increased self-renewal of neural precursor cells. There was also a propensity for differentiation to later-born neurons. In addition, cortical neurons showed increased susceptibility to stress, which is exacerbated in neurons carrying mutations in both ANG and TARDBP. The cortical organoids and neurons generated from patient-derived iPSCs carrying ANG and TARDBP gene variants recapitulate dysfunctions characteristic of frontotemporal lobar degeneration observed in FTD patients. These dysfunctions were ameliorated upon treatment with wild type ANG. In addition to its well-established role during the stress response of mature neurons, ANG also appears to play a role in neural progenitor dynamics. This has implications for neurogenesis and may indicate that subtle developmental defects play a role in disease susceptibility or onset. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Moderate intrinsic phenotypic alterations in C9orf72 ALS/FTD iPSC-microglia despite the presence of C9orf72 pathological features.

While motor and cortical neurons are affected in C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD), it remains largely unknown if and how non-neuronal cells induce or exacerbate neuronal damage. We differentiated C9orf72 ALS/FTD patient-derived induced pluripotent stem cells into microglia (iPSC-MG) and examined their intrinsic phenotypes. Similar to iPSC motor neurons, C9orf72 ALS/FTD iPSC-MG mono-cultures form G4C2 repeat RNA foci, exhibit reduced C9orf72 protein levels, and generate dipeptide repeat proteins. Healthy control and C9orf72 ALS/FTD iPSC-MG equally express microglial specific genes and perform microglial functions, including inflammatory cytokine release and phagocytosis of extracellular cargos, such as synthetic amyloid beta peptides and healthy human brain synaptoneurosomes. RNA sequencing analysis revealed select transcriptional changes of genes associated with neuroinflammation or neurodegeneration in diseased microglia yet no significant differentially expressed microglial-enriched genes. Moderate molecular and functional differences were observed in C9orf72 iPSC-MG mono-cultures despite the presence of C9orf72 pathological features suggesting that a diseased microenvironment may be required to induce phenotypic changes in microglial cells and the associated neuronal dysfunction seen in C9orf72 ALS/FTD neurodegeneration.

Impaired RNA Binding Does Not Prevent Histone Modification Changes in a FUS ALS/FTD Yeast Model.

Mutations in the RNA-binding protein FUS are linked to amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). FUS mutants mislocalize and aggregate in dying neurons. We previously established that FUS proteinopathy is linked to changes in the histone modification landscape in a yeast ALS/FTD model. Here, we examine whether FUS' RNA binding is necessary for this connection. We find that overexpression of a FUS mutant unable to bind RNA is still associated with reduced levels of H3S10ph, H3K14ac and H3K56ac. Hence, FUS' ability to bind RNA is not required in the mechanism connecting FUS proteinopathy to altered histone post-translational modifications.

Loss of TMEM106B exacerbates C9ALS/FTD DPR pathology by disrupting autophagosome maturation.

Disruption to protein homeostasis caused by lysosomal dysfunction and associated impairment of autophagy is a prominent pathology in amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). The most common genetic cause of ALS/FTD is a G4C2 hexanucleotide repeat expansion in C9orf72 (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of G4C2 repeat transcripts gives rise to dipeptide repeat (DPR) proteins that have been shown to be toxic and may contribute to disease etiology. Genetic variants in TMEM106B have been associated with frontotemporal lobar degeneration with TDP-43 pathology and disease progression in C9ALS/FTD. TMEM106B encodes a lysosomal transmembrane protein of unknown function that is involved in various aspects of lysosomal biology. How TMEM106B variants affect C9ALS/FTD is not well understood but has been linked to changes in TMEM106B protein levels. Here, we investigated TMEM106B function in the context of C9ALS/FTD DPR pathology. We report that knockdown of TMEM106B expression exacerbates the accumulation of C9ALS/FTD-associated cytotoxic DPR proteins in cell models expressing RAN-translated or AUG-driven DPRs as well as in C9ALS/FTD-derived iAstrocytes with an endogenous G4C2 expansion by impairing autophagy. Loss of TMEM106B caused a block late in autophagy by disrupting autophagosome to autolysosome maturation which coincided with impaired lysosomal acidification, reduced cathepsin activity, and juxtanuclear clustering of lysosomes. Lysosomal clustering required Rab7A and coincided with reduced Arl8b-mediated anterograde transport of lysosomes to the cell periphery. Increasing Arl8b activity in TMEM106B-deficient cells not only restored the distribution of lysosomes, but also fully rescued autophagy and DPR protein accumulation. Thus, we identified a novel function of TMEM106B in autophagosome maturation via Arl8b. Our findings indicate that TMEM106B variants may modify C9ALS/FTD by regulating autophagic clearance of DPR proteins. Caution should therefore be taken when considering modifying TMEM106B expression levels as a therapeutic approach in ALS/FTD.

Nuclear import receptors are recruited by FG-nucleoporins to rescue hallmarks of TDP-43 proteinopathy.

Cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) disease spectrum, causing both nuclear loss-of-function and cytoplasmic toxic gain-of-function phenotypes. While TDP-43 proteinopathy has been associated with defects in nucleocytoplasmic transport, this process is still poorly understood. Here we study the role of karyopherin-β1 (KPNB1) and other nuclear import receptors in regulating TDP-43 pathology. We used immunostaining, immunoprecipitation, biochemical and toxicity assays in cell lines, primary neuron and organotypic mouse brain slice cultures, to determine the impact of KPNB1 on the solubility, localization, and toxicity of pathological TDP-43 constructs. Postmortem patient brain and spinal cord tissue was stained to assess KPNB1 colocalization with TDP-43 inclusions. Turbidity assays were employed to study the dissolution and prevention of aggregation of recombinant TDP-43 fibrils in vitro. Fly models of TDP-43 proteinopathy were used to determine the effect of KPNB1 on their neurodegenerative phenotype in vivo. We discovered that several members of the nuclear import receptor protein family can reduce the formation of pathological TDP-43 aggregates. Using KPNB1 as a model, we found that its activity depends on the prion-like C-terminal region of TDP-43, which mediates the co-aggregation with phenylalanine and glycine-rich nucleoporins (FG-Nups) such as Nup62. KPNB1 is recruited into these co-aggregates where it acts as a molecular chaperone that reverses aberrant phase transition of Nup62 and TDP-43. These findings are supported by the discovery that Nup62 and KPNB1 are also sequestered into pathological TDP-43 aggregates in ALS/FTD postmortem CNS tissue, and by the identification of the fly ortholog of KPNB1 as a strong protective modifier in Drosophila models of TDP-43 proteinopathy. Our results show that KPNB1 can rescue all hallmarks of TDP-43 pathology, by restoring its solubility and nuclear localization, and reducing neurodegeneration in cellular and animal models of ALS/FTD. Our findings suggest a novel NLS-independent mechanism where, analogous to its canonical role in dissolving the diffusion barrier formed by FG-Nups in the nuclear pore, KPNB1 is recruited into TDP-43/FG-Nup co-aggregates present in TDP-43 proteinopathies and therapeutically reverses their deleterious phase transition and mislocalization, mitigating neurodegeneration.

Tubulin deacetylase NDST3 modulates lysosomal acidification: Implications in neurological diseases.

Neurological diseases (NDs), featured by progressive dysfunctions of the nervous system, have become a growing burden for the aging populations. N-Deacetylase and N-sulfotransferase 3 (NDST3) is known to catalyze deacetylation and N-sulfation on disaccharide substrates. Recently, NDST3 is identified as a novel deacetylase for tubulin, and its newly recognized role in modulating microtubule acetylation and lysosomal acidification provides fresh insights into ND therapeutic approaches using NDST3 as a target. Microtubule acetylation and lysosomal acidification have been reported to be critical for activities in neurons, implying that the regulators of these two biological processes, such as the previously known microtubule deacetylases, histone deacetylase 6 (HDAC6) and sirtuin 2 (SIRT2), could play important roles in various NDs. Aberrant NDST3 expression or tubulin acetylation has been observed in an increasing number of NDs, including amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD), schizophrenia and bipolar disorder, Alzheimer's disease (AD), and Parkinson's disease (PD), suggesting that NDST3 is a key player in the pathogenesis of NDs and may serve as a target for development of new treatment of NDs.

An interaction between synapsin and C9orf72 regulates excitatory synapses and is impaired in ALS/FTD

Dysfunction and degeneration of synapses is a common feature of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). A GGGGCC hexanucleotide repeat expansion in the C9ORF72 gene is the main genetic cause of ALS/FTD (C9ALS/FTD). The repeat expansion leads to reduced expression of the C9orf72 protein. How C9orf72 haploinsufficiency contributes to disease has not been resolved. Here we identify the synapsin family of synaptic vesicle proteins, the most abundant group of synaptic phosphoproteins, as novel interactors of C9orf72 at synapses and show that C9orf72 plays a cell-autonomous role in the regulation of excitatory synapses. We mapped the interaction of C9orf72 and synapsin to the N-terminal longin domain of C9orf72 and the conserved C domain of synapsin, and show interaction of the endogenous proteins in synapses. Functionally, C9orf72 deficiency reduced the number of excitatory synapses and decreased synapsin levels at remaining synapses in vitro in hippocampal neuron cultures and in vivo in the hippocampal mossy fibre system of C9orf72 knockout mice. Consistent with synaptic dysfunction, electrophysiological recordings identified impaired excitatory neurotransmission and network function in hippocampal neuron cultures with reduced C9orf72 expression, which correlated with a severe depletion of synaptic vesicles from excitatory synapses in the hippocampus of C9orf72 knockout mice. Finally, neuropathological analysis of post-mortem sections of C9ALS/FTD patient hippocampus with C9orf72 haploinsufficiency revealed a marked reduction in synapsin, indicating that disruption of the interaction between C9orf72 and synapsin may contribute to ALS/FTD pathobiology. Thus, our data show that C9orf72 plays a cell-autonomous role in the regulation of neurotransmission at excitatory synapses by interaction with synapsin and modulation of synaptic vesicle pools, and identify a novel role for C9orf72 haploinsufficiency in synaptic dysfunction in C9ALS/FTD.

Multi-omics profiling identifies a deregulated FUS-MAP1B axis in ALS/FTD-associated UBQLN2 mutants.

Ubiquilin-2 (UBQLN2) is a ubiquitin-binding protein that shuttles ubiquitinated proteins to proteasomal and autophagic degradation. UBQLN2 mutations are genetically linked to the neurodegenerative disorders amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). However, it remains elusive how UBQLN2 mutations cause ALS/FTD. Here, we systematically examined proteomic and transcriptomic changes in patient-derived lymphoblasts and CRISPR/Cas9-engineered HeLa cells carrying ALS/FTD UBQLN2 mutations. This analysis revealed a strong up-regulation of the microtubule-associated protein 1B (MAP1B) which was also observed in UBQLN2 knockout cells and primary rodent neurons depleted of UBQLN2, suggesting that a UBQLN2 loss-of-function mechanism is responsible for the elevated MAP1B levels. Consistent with MAP1B's role in microtubule binding, we detected an increase in total and acetylated tubulin. Furthermore, we uncovered that UBQLN2 mutations result in decreased phosphorylation of MAP1B and of the ALS/FTD-linked fused in sarcoma (FUS) protein at S439 which is critical for regulating FUS-RNA binding and MAP1B protein abundance. Together, our findings point to a deregulated UBQLN2-FUS-MAP1B axis that may link protein homeostasis, RNA metabolism, and cytoskeleton dynamics, three molecular pathomechanisms of ALS/FTD.

A mouse model with widespread expression of the C9orf72-linked glycine\u2013arginine dipeptide displays non-lethal ALS/FTD-like phenotypes

Translation of the hexanucleotide G4C2 expansion associated with C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) produces five different dipeptide repeat protein (DPR) species that can confer toxicity. There is yet much to learn about the contribution of a single DPR to disease pathogenesis. We show here that a short repeat length is sufficient for the DPR poly-GR to confer neurotoxicity in vitro, a phenomenon previously unobserved. This toxicity is also reported in vivo in our novel knock-in mouse model characterized by widespread central nervous system (CNS) expression of the short-length poly-GR. We observe sex-specific chronic ALS/FTD-like phenotypes in these mice, including mild motor neuron loss, but no TDP-43 mis-localization, as well as motor and cognitive impairments. We suggest that this model can serve as the foundation for phenotypic exacerbation through second-hit forms of stress.

Editors' Note: Phenotypic Variability in ALS-FTD and Effect on Survival

Dr. Ahmed et al. examined initial presentations and cognitive and behavioral profiles in 59 patients with amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD) and 39 patients with behavioral variant frontotemporal dementia (bvFTD) and compared their MRI brain atrophy patterns and survival trajectories. They found that initial motor presentation in ALS-FTD was associated with shorter survival than those with a cognitive presentation, despite similar overall cognitive deficits. Patients with ALS-FTD also showed reduced emotional processing as well as greater motor cortex and dorsal lateral prefrontal cortex atrophy than those with bvFTD, with motor cortex atrophy, in turn, associated with a 1.5-fold reduction in survival. In response, Dr. Abe cites a case report of a patient with ALS and dementia who presented with disturbances in emotion processing and behavior, with such early behavioral changes also noted in other studies of ALS-FTD. Dr. Abe suggests that functional neuroimaging studies may help further elucidate pathophysiologic processes in these cases, citing a case of a patient with ALS-FTD and prominent bulbar palsy and memory loss but preserved personality who had hypoperfusion in the central and prefrontal cortex, but not in the dorsolateral prefrontal cortex. Responding to these comments, the authors agree that ALS-FTD can present with cognitive and behavioral changes. They note that their study results suggest that survival in ALS-FTD may be mediated by motor function and other critical physiologic aspects. They also note that patients with ALS-FTD and an initial cognitive presentation had more prominent language changes, but contrary to the conventional perception of bvFTD as having more deficits in emotional processing, patients with ALS-FTD had greater deficits in this domain in their study. They agree that functional neuroimaging, in combination with clinical, structural, and pathologic evaluation, can help improve our understanding of these diseases. This exchange demonstrates how modern phenotypic studies are challenging previous assumptions about distinctions between different subtypes of FTD and ALS-FTD and elucidating new discoveries about these conditions. Dr. Ahmed et al. examined initial presentations and cognitive and behavioral profiles in 59 patients with amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD) and 39 patients with behavioral variant frontotemporal dementia (bvFTD) and compared their MRI brain atrophy patterns and survival trajectories. They found that initial motor presentation in ALS-FTD was associated with shorter survival than those with a cognitive presentation, despite similar overall cognitive deficits. Patients with ALS-FTD also showed reduced emotional processing as well as greater motor cortex and dorsal lateral prefrontal cortex atrophy than those with bvFTD, with motor cortex atrophy, in turn, associated with a 1.5-fold reduction in survival. In response, Dr. Abe cites a case report of a patient with ALS and dementia who presented with disturbances in emotion processing and behavior, with such early behavioral changes also noted in other studies of ALS-FTD. Dr. Abe suggests that functional neuroimaging studies may help further elucidate pathophysiologic processes in these cases, citing a case of a patient with ALS-FTD and prominent bulbar palsy and memory loss but preserved personality who had hypoperfusion in the central and prefrontal cortex, but not in the dorsolateral prefrontal cortex. Responding to these comments, the authors agree that ALS-FTD can present with cognitive and behavioral changes. They note that their study results suggest that survival in ALS-FTD may be mediated by motor function and other critical physiologic aspects. They also note that patients with ALS-FTD and an initial cognitive presentation had more prominent language changes, but contrary to the conventional perception of bvFTD as having more deficits in emotional processing, patients with ALS-FTD had greater deficits in this domain in their study. They agree that functional neuroimaging, in combination with clinical, structural, and pathologic evaluation, can help improve our understanding of these diseases. This exchange demonstrates how modern phenotypic studies are challenging previous assumptions about distinctions between different subtypes of FTD and ALS-FTD and elucidating new discoveries about these conditions.

C9orf72-associated arginine-rich dipeptide repeats induce RNA-dependent nuclear accumulation of Staufen in neurons.

RNA-binding proteins (RBPs) play essential roles in diverse cellular processes through post-transcriptional regulation of RNAs. The subcellular localization of RBPs is thus under tight control, the breakdown of which is associated with aberrant cytoplasmic accumulation of nuclear RBPs such as TDP-43 and FUS, well-known pathological markers for amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). Here, we report in Drosophila model for ALS/FTD that nuclear accumulation of a cytoplasmic RBP Staufen may be a new pathological feature. We found that in Drosophila C4da neurons expressing PR36, one of the arginine-rich dipeptide repeat proteins (DPRs), Staufen accumulated in the nucleus in Importin- and RNA-dependent manner. Notably, expressing Staufen with exogenous NLS—but not with mutated endogenous NLS—potentiated PR-induced dendritic defect, suggesting that nuclear-accumulated Staufen can enhance PR toxicity. PR36 expression increased Fibrillarin staining in the nucleolus, which was enhanced by heterozygous mutation of stau (stau+/−), a gene that codes Staufen. Furthermore, knockdown of fib, which codes Fibrillarin, exacerbated retinal degeneration mediated by PR toxicity, suggesting that increased amount of Fibrillarin by stau+/− is protective. stau+/− also reduced the amount of PR-induced nuclear-accumulated Staufen and mitigated retinal degeneration and rescued viability of flies expressing PR36. Taken together, our data show that nuclear accumulation of Staufen in neurons may be an important pathological feature contributing to the pathogenesis of ALS/FTD.

TDP-43 and PINK1 mediate CHCHD10S59L mutation\u2013induced defects in Drosophila and in vitro

Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) can cause amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). However, the underlying mechanisms are unclear. Here, we generate CHCH10S59L-mutant Drosophila melanogaster and HeLa cell lines to model CHCHD10-associated ALS-FTD. The CHCHD10S59L mutation results in cell toxicity in several tissues and mitochondrial defects. CHCHD10S59L independently affects the TDP-43 and PINK1 pathways. CHCHD10S59L expression increases TDP-43 insolubility and mitochondrial translocation. Blocking TDP-43 mitochondrial translocation with a peptide inhibitor reduced CHCHD10S59L-mediated toxicity. While genetic and pharmacological modulation of PINK1 expression and activity of its substrates rescues and mitigates the CHCHD10S59L-induced phenotypes and mitochondrial defects, respectively, in both Drosophila and HeLa cells. Our findings suggest that CHCHD10S59L-induced TDP-43 mitochondrial translocation and chronic activation of PINK1-mediated pathways result in dominant toxicity, providing a mechanistic insight into the CHCHD10 mutations associated with ALS-FTD.

Corticospinal Tract and Related Grey Matter Morphometric Shape Analysis in ALS Phenotypes: A Fractal Dimension Study

A pathological hallmark of amyotrophic lateral sclerosis (ALS) is corticospinal tract (CST) degeneration resulting in upper motor neuron (UMN) dysfunction. No quantitative test is available to easily assess UMN pathways. Brain neuroimaging in ALS promises to potentially change this through identifying biomarkers of UMN dysfunction that may accelerate diagnosis and track disease progression. Fractal dimension (FD) has successfully been used to quantify brain grey matter (GM) and white matter (WM) shape complexity in various neurological disorders. Therefore, we investigated CST and whole brain GM and WM morphometric changes using FD analyses in ALS patients with different phenotypes. We hypothesized that FD would detect differences between ALS patients and neurologic controls and even between the ALS subgroups. Neuroimaging was performed in neurologic controls (n = 14), and ALS patients (n = 75). ALS patients were assigned into four groups based on their clinical or radiographic phenotypes. FD values were estimated for brain WM and GM structures. Patients with ALS and frontotemporal dementia (ALS-FTD) showed significantly higher CST FD values and lower primary motor and sensory cortex GM FD values compared to other ALS groups. No other group of ALS patients revealed significant FD value changes when compared to neurologic controls or with other ALS patient groups. These findings support a more severe disease process in ALS-FTD patients compared to other ALS patient groups. FD value measures may be a sensitive index to evaluate GM and WM (including CST) degeneration in ALS patients.

Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning and reduces neurodegeneration.

mRNA transport in neurons requires formation of transport granules containing many protein components, and subsequent alterations in phosphorylation status can release transcripts for translation. Further, mutations in a structurally disordered domain of the transport granule protein hnRNPA2 increase its aggregation and cause hereditary proteinopathy of neurons, myocytes, and bone. We examine in vitro hnRNPA2 granule component phase separation, partitioning specificity, assembly/disassembly, and the link to neurodegeneration. Transport granule components hnRNPF and ch‐TOG interact weakly with hnRNPA2 yet partition specifically into liquid phase droplets with the low complexity domain (LC) of hnRNPA2, but not FUS LC. In vitro hnRNPA2 tyrosine phosphorylation reduces hnRNPA2 phase separation, prevents partitioning of hnRNPF and ch‐TOG into hnRNPA2 LC droplets, and decreases aggregation of hnRNPA2 disease variants. The expression of chimeric hnRNPA2 D290V in Caenorhabditis elegans results in stress‐induced glutamatergic neurodegeneration; this neurodegeneration is rescued by loss of tdp‐1, suggesting gain‐of‐function toxicity. The expression of Fyn, a tyrosine kinase that phosphorylates hnRNPA2, reduces neurodegeneration associated with chimeric hnRNPA2 D290V. These data suggest a model where phosphorylation alters LC interaction specificity, aggregation, and toxicity.

Systematic review and meta-analysis of human transcriptomics reveals neuroinflammation, deficient energy metabolism, and proteostasis failure across neurodegeneration

Neurodegenerative disorders such as Alzheimer's disease (AD), Lewy body diseases (LBD), and the amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD) spectrum are defined by the accumulation of specific misfolded protein aggregates. However, the mechanisms by which each proteinopathy leads to neurodegeneration remain elusive. We hypothesized that there is a common “pan-neurodegenerative” gene expression signature driving pathophysiology across these clinically and pathologically diverse proteinopathies. To test this hypothesis, we performed a systematic review of human CNS transcriptomics datasets from AD, LBD, and ALS-FTD patients and age-matched controls in the Gene Expression Omnibus (GEO) and ArrayExpress databases, followed by consistent processing of each dataset, meta-analysis, pathway enrichment, and overlap analyses. After applying pre-specified eligibility criteria and stringent data pre-processing, a total of 2600 samples from 26 AD, 21 LBD, and 13 ALS-FTD datasets were included in the meta-analysis. The pan-neurodegenerative gene signature is characterized by an upregulation of innate immunity, cytoskeleton, and transcription and RNA processing genes, and a downregulation of the mitochondrial electron transport chain. Pathway enrichment analyses also revealed the upregulation of neuroinflammation (including Toll-like receptor, TNF, and NFκB signaling) and phagocytosis, and the downregulation of mitochondrial oxidative phosphorylation, lysosomal acidification, and ubiquitin-proteasome pathways. Our findings suggest that neuroinflammation and a failure in both neuronal energy metabolism and protein degradation systems are consistent features underlying neurodegenerative diseases, despite differences in the extent of neuronal loss and brain regions involved.

G4C2 Repeat RNA Initiates a POM121-Mediated Reduction in Specific Nucleoporins in C9orf72 ALS/FTD

Through mechanisms that remain poorly defined, defects in nucleocytoplasmic transport and accumulations of specific nuclear-pore-complex-associated proteins have been reported in multiple neurodegenerative diseases, including C9orf72 Amyotrophic Lateral Sclerosis and Frontotemporal Dementia (ALS/FTD). Using super-resolution structured illumination microscopy, we have explored the mechanism by which nucleoporins are altered in nuclei isolated from C9orf72 induced pluripotent stem-cell-derived neurons (iPSNs). Of the 23 nucleoporins evaluated, we observed a reduction in a subset of 8, including key components of the nuclear pore complex scaffold and the transmembrane nucleoporin POM121. Reduction in POM121 appears to initiate a decrease in the expression of seven additional nucleoporins, ultimately affecting the localization of Ran GTPase and subsequent cellular toxicity in C9orf72 iPSNs. Collectively, our data suggest that the expression of expanded C9orf72 ALS/FTD repeat RNA alone affects nuclear POM121 expression in the initiation of a pathological cascade affecting nucleoporin levels within neuronal nuclei and ultimately downstream neuronal survival.

Targeted DNA methylation of neurodegenerative disease genes via homology directed repair.

DNA methyltransferases (DNMTs) are thought to be involved in the cellular response to DNA damage, thus linking DNA repair mechanisms with DNA methylation. In this study we present Homology Assisted Repair Dependent Epigenetic eNgineering (HARDEN), a novel method of targeted DNA methylation that utilizes endogenous DNA double strand break repair pathways. This method allows for stable targeted DNA methylation through the process of homology directed repair (HDR) via an in vitro methylated exogenous repair template. We demonstrate that HARDEN can be applied to the neurodegenerative disease genes C9orf72 and APP, and methylation can be induced via HDR with both single and double stranded methylated repair templates. HARDEN allows for higher targeted DNA methylation levels than a dCas9-DNMT3a fusion protein construct at C9orf72, and genome-wide methylation analysis reveals no significant off-target methylation changes when inducing methylation via HARDEN, whereas the dCas9-DNMT3a fusion construct causes global off-target methylation. HARDEN is applied to generate a patient derived iPSC model of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) that recapitulates DNA methylation patterns seen in patients, demonstrating that DNA methylation of the 5′ regulatory region directly reduces C9orf72 expression and increases histone H3K9 tri-methylation levels.

Small-Molecule Modulation of TDP-43 Recruitment to Stress Granules Prevents Persistent TDP-43 Accumulation in ALS/FTD

Stress granules (SGs) form during cellular stress and are implicated in neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). To yield insights into the role of SGs in pathophysiology, we performed a high-content screen to identify small molecules that alter SG properties in proliferative cells and human iPSC-derived motor neurons (iPS-MNs). One major class of active molecules contained extended planar aromatic moieties, suggesting a potential to intercalate in nucleic acids. Accordingly, we show that several hit compounds can prevent the RNA-dependent recruitment of the ALS-associated RNA-binding proteins (RBPs) TDP-43, FUS, and HNRNPA2B1 into SGs. We further demonstrate that transient SG formation contributes to persistent accumulation of TDP-43 into cytoplasmic puncta and that our hit compounds can reduce this accumulation in iPS-MNs from ALS patients. We propose that compounds with planar moieties represent a promising starting point to develop small-molecule therapeutics for treating ALS/FTD.

Robustness and Vulnerability of the Autoregulatory System That Maintains Nuclear TDP-43 Levels: A Trade-off Hypothesis for ALS Pathology Based on in Silico Data.

Abnormal accumulation of TAR DNA-binding protein 43 (TDP-43) in the cytoplasm and its disappearance from the nucleus are pathological features of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) and are directly involved in the pathogenesis of these conditions. TDP-43 is an essential nuclear protein that readily aggregates in a concentration-dependent manner. Therefore, cells must strictly maintain an appropriate amount of nuclear TDP-43. In one relevant maintenance mechanism, TDP-43 binds to its pre-mRNA and promotes alternative splicing, resulting in mRNA degradation via nonsense-mediated mRNA decay. The level of nuclear TDP-43 is tightly regulated by these mechanisms, which control the amount of mRNA that may be translated. Based on the results of previous experiments, we developed an in silico model that mimics the intracellular dynamics of TDP-43 and examined TDP-43 metabolism under various conditions. We discovered an inherent trade-off in this mechanism between transcriptional redundancy, which maintains the robustness of TDP-43 metabolism, and vulnerability to specific interfering factors. These factors include an increased tendency of TDP-43 to aggregate, impaired nuclear-cytoplasmic TDP-43 transport, and a decreased efficiency of degrading abnormal proteins, all of which are functional abnormalities related to the gene that causes familial ALS/FTD. When these conditions continue at a certain intensity, the vulnerability of the autoregulatory machinery becomes apparent over time, and transcriptional redundancy enters a vicious cycle that ultimately results in TDP-43 pathology. The results obtained using this in silico model reveal the difference in TDP-43 metabolism between normal and disease states. Furthermore, using this model, we simulated the effect of a decrease in TDP-43 transcription and found that this decrease improved TDP-43 pathology and suppressed the abnormal propagation of TDP-43. Therefore, we propose a potential therapeutic strategy to suppress transcriptional redundancy, which is the driving force of the pathological condition caused by the specific factors described above, in patients with ALS presenting with TDP-43 pathology. An ALS animal model exhibiting TDP-43 pathology without overexpression of exogenous TDP-43 should be developed to investigate the effect of alleviating the transcriptional redundancy of TARDBP.

Thermodynamic and spectroscopic investigations of TMPyP4 association with guanine- and cytosine-rich DNA and RNA repeats of C9orf72

BackgroundAn expansion of the hexanucleotide repeat (GGGGCC)n·(GGCCCC)n in the C9orf72 promoter has been shown to be the cause of Amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). The C9orf72 repeat can form four-stranded structures; the cationic porphyrin (TMPyP4) binds and distorts these structures. MethodsIsothermal titration calorimetry (ITC), and circular dichroism (CD) were used to study the binding of TMPyP4 to the C-rich and G-rich DNA and RNA oligos containing the hexanucleotide repeat at pH 7.5 and 0.1 M K+. ResultsThe CD spectra of G-rich DNA and RNA TMPyP4 complexes showed features of antiparallel and parallel G-quadruplexes, respectively. The shoulder at 260 nm in the CD spectrum becomes more intense upon formation of complexes between TMPyP4 and the C-rich DNA. The peak at 290 nm becomes more intense in the c-rich RNA molecules, suggesting induction of an i-motif structure. The ITC data showed that TMPyP4 binds at two independent sites for all DNA and RNA molecules. ConclusionsFor DNA, the data are consistent with TMPyP4 stacking on the terminal tetrads and intercalation. For RNA, the thermodynamics of the two binding modes are consistent with groove binding and intercalation. In both cases, intercalation is the weaker binding mode. These findings are considered with respect to the structural differences of the folded DNA and RNA molecules and the energetics of the processes that drive site-specific recognition by TMPyP4; these data will be helpful in efforts to optimize the specificity and affinity of the binding of porphyrin-like molecules.