Targeting the TDP-43 low complexity domain blocks spreading of pathology in a mouse model of ALS/FTD.

Abnormal cytoplasmic localization and accumulation of pathological transactive response DNA binding protein of 43 kDa (TDP-43) underlies several devastating diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP). A key element is the correlation between disease progression and spatio-temporal propagation of TDP-43-mediated pathology in the central nervous system. Several lines of evidence support the concept of templated aggregation and cell to cell spreading of pathological TDP-43. To further investigate this mechanism in vivo, we explored the efficacy of capturing and masking the seeding-competent region of extracellular TDP-43 species. For this, we generated a novel monoclonal antibody (mAb), ACI-6677, that targets the pathogenic protease-resistant amyloid core of TDP-43. ACI-6677 has a picomolar binding affinity for TDP-43 and is capable of binding to all C-terminal TDP-43 fragments. In vitro, ACI-6677 inhibited TDP-43 aggregation and boosted removal of pathological TDP-43 aggregates by phagocytosis. When injecting FTLD-TDP brain extracts unilaterally in the CamKIIa-hTDP-43NLSm mouse model, ACI-6677 significantly limited the induction of phosphorylated TDP-43 (pTDP-43) inclusions. Strikingly, on the contralateral side, the mAb significantly prevented pTDP-43 inclusion appearance exemplifying blocking of the spreading process. Taken together, these data demonstrate for the first time that an immunotherapy targeting the protease-resistant amyloid core of TDP-43 has the potential to restrict spreading, substantially slowing or stopping progression of disease.

The Effect of Dipeptide Repeat Proteins on FUS/TDP43-RNA Condensation in C9orf72 ALS/FTD.

Condensation of RNA binding proteins (RBPs) with RNA is essential for cellular function. The most common familial cause of the diseases ALS and FTD is C9orf72 repeat expansion disorders that produce dipeptide repeat proteins (DPRs). We explore the hypothesis that DPRs disrupt the native condensation behavior of RBPs and RNA through molecular interactions resulting in toxicity. FUS and TDP43 are two RBPs known to be affected in ALS/FTD. We use our previously developed 1-bead-per-amino acid and a newly developed 3-bead-per-nucleotide molecular dynamics model to explore ternary phase diagrams of FUS/TDP43-RNA-DPR systems. We show that the most toxic arginine containing DPRs (R-DPRs) can disrupt the RBP condensates through cation-π interactions and can strongly sequester RNA through electrostatic interactions. The native droplet morphologies are already modified at small additions of R-DPRs leading to non-native FUS/TDP43-encapsulated condensates with a marbled RNA/DPR core.

NEMF mutations in mice illustrate how Importin-ß specific nuclear transport defects recapitulate neurodegenerative disease hallmarks.

Pathological disruption of Nucleocytoplasmic Transport (NCT), such as the mis-localization of nuclear pore complex proteins (Nups), nuclear transport receptors, Ran-GTPase, and RanGAP1, are seen in both animal models and in familial and sporadic forms of amyotrophic lateral sclerosis (ALS), frontal temporal dementia and frontal temporal lobar degeneration (FTD\FTLD), and Alzheimer's and Alzheimer's Related Dementias (AD/ADRD). However, the question of whether these alterations represent a primary cause, or a downstream consequence of disease is unclear, and what upstream factors may account for these defects are unknown. Here, we report four key findings that shed light on the upstream causal role of Importin-ß-specific nuclear transport defects in disease onset. First, taking advantage of two novel mouse models of NEMF neurodegeneration (NemfR86S and NemfR487G) that recapitulate many cellular and biochemical aspects of neurodegenerative diseases, we find an Importin-ß-specific nuclear import block. Second, we observe cytoplasmic mis-localization and aggregation of multiple proteins implicated in the pathogenesis of ALS/FTD and AD/ADRD, including TDP43, Importin-ß, RanGap1, and Ran. These findings are further supported by a pathological interaction between Importin-ß and the mutant NEMFR86S protein in cytoplasmic accumulations. Third, we identify similar transcriptional dysregulation in key genes associated with neurodegenerative disease. Lastly, we show that even transient pharmaceutical inhibition of Importin-ß in both mouse and human neuronal and non-neuronal cells induces key proteinopathies and transcriptional alterations seen in our mouse models and in neurodegeneration. Our convergent results between mouse and human neuronal and non-neuronal cellular biology provide mechanistic evidence that many of the mis-localized proteins and dysregulated transcriptional events seen in multiple neurodegenerative diseases may in fact arise primarily from a primary upstream defect in Importin- ß nuclear import. These findings have critical implications for investigating how sporadic forms of neurodegeneration may arise from presently unidentified genetic and environmental perturbations in Importin-ß function.

Stress granule formation helps to mitigate neurodegeneration.

Cellular stress pathways that inhibit translation initiation lead to transient formation of cytoplasmic RNA/protein complexes known as stress granules. Many of the proteins found within stress granules and the dynamics of stress granule formation and dissolution are implicated in neurodegenerative disease. Whether stress granule formation is protective or harmful in neurodegenerative conditions is not known. To address this, we took advantage of the alphavirus protein nsP3, which selectively binds dimers of the central stress granule nucleator protein G3BP and markedly reduces stress granule formation without directly impacting the protein translational inhibitory pathways that trigger stress granule formation. In Drosophila and rodent neurons, reducing stress granule formation with nsP3 had modest impacts on lifespan even in the setting of serial stress pathway induction. In contrast, reducing stress granule formation in models of ataxia, amyotrophic lateral sclerosis and frontotemporal dementia largely exacerbated disease phenotypes. These data support a model whereby stress granules mitigate, rather than promote, neurodegenerative cascades.

Lysosomal TBK1 responds to amino acid availability to relieve Rab7-dependent mTORC1 inhibition.

Lysosomes play a pivotal role in coordinating macromolecule degradation and regulating cell growth and metabolism. Despite substantial progress in identifying lysosomal signaling proteins, understanding the pathways that synchronize lysosome functions with changing cellular demands remains incomplete. This study uncovers a role for TANK-binding kinase 1 (TBK1), well known for its role in innate immunity and organelle quality control, in modulating lysosomal responsiveness to nutrients. Specifically, we identify a pool of TBK1 that is recruited to lysosomes in response to elevated amino acid levels. This lysosomal TBK1 phosphorylates Rab7 on serine 72. This is critical for alleviating Rab7-mediated inhibition of amino acid-dependent mTORC1 activation. Furthermore, a TBK1 mutant (E696K) associated with amyotrophic lateral sclerosis and frontotemporal dementia constitutively accumulates at lysosomes, resulting in elevated Rab7 phosphorylation and increased mTORC1 activation. This data establishes the lysosome as a site of amino acid regulated TBK1 signaling that is crucial for efficient mTORC1 activation. This lysosomal pool of TBK1 has broader implications for lysosome homeostasis, and its dysregulation could contribute to the pathogenesis of ALS-FTD.

An anti-sortilin affibody-peptide fusion inhibits sortilin-mediated progranulin degradation.

Heterozygous loss-of-function mutations in the GRN gene are a common cause of frontotemporal dementia. Such mutations lead to decreased plasma and cerebrospinal fluid levels of progranulin (PGRN), a neurotrophic factor with lysosomal functions. Sortilin is a negative regulator of extracellular PGRN levels and has shown promise as a therapeutic target for frontotemporal dementia, enabling increased extracellular PGRN levels through inhibition of sortilin-mediated PGRN degradation. Here we report the development of a high-affinity sortilin-binding affibody-peptide fusion construct capable of increasing extracellular PGRN levels in vitro. By genetic fusion of a sortilin-binding affibody generated through phage display and a peptide derived from the progranulin C-terminus, an affinity protein (A3-PGRNC15*) with 185-pM affinity for sortilin was obtained. Treating PGRN-secreting and sortilin-expressing human glioblastoma U-251 cells with the fusion protein increased extracellular PGRN levels up to 2.5-fold, with an EC50 value of 1.3 nM. Our results introduce A3-PGRNC15* as a promising new agent with therapeutic potential for the treatment of frontotemporal dementia. Furthermore, the work highlights means to increase binding affinity through synergistic contribution from two orthogonal polypeptide units.

MAPT mutations associated with familial tauopathies lead to formation of conformationally distinct oligomers that have cross-seeding ability.

The microtubule associated protein, tau, is implicated in a multitude of neurodegenerative disorders that are collectively termed as tauopathies. These disorders are characterized by the presence of tau aggregates within the brain of afflicted individuals. Mutations within the MAPT gene that encodes the tau protein form the genetic backdrop for familial forms of tauopathies, such as frontotemporal dementia (FTD), but the molecular consequences of such alterations and their pathological effects are unclear. We sought to investigate the conformational properties of the aggregates of three tau mutants: A152T, P301L, and R406W, all implicated within FTD, and compare them to those of the native form (WT-Tau 2N4R). Our immunochemical analysis reveals that mutants and WT tau oligomers exhibit similar affinity for conformation-specific antibodies but have distinct morphology and secondary structure. Additionally, these oligomers possess different dye-binding properties and varying sensitivity to proteolytic processing. These results point to conformational variety among them. We then tested the ability of the mutant oligomers to cross-seed the aggregation of WT tau monomer. Using similar array of experiments, we found that cross-seeding with mutant aggregates leads to the formation of conformationally unique WT oligomers. The results discussed in this paper provide a novel perspective on the structural properties of oligomeric forms of WT tau 2N4R and its mutant, along with shedding some light on their cross-seeding behavior.

Poly-GR Impairs PRMT1-Mediated Arginine Methylation of Disease-Linked RNA-Binding Proteins by Acting as a Substrate Sink.

Dipeptide repeat proteins (DPRs) are aberrant protein species found in C9orf72-linked amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two neurodegenerative diseases characterized by the cytoplasmic mislocalization and aggregation of RNA-binding proteins (RBPs). In particular, arginine (R)-rich DPRs (poly-GR and poly-PR) have been suggested to promiscuously interact with multiple cellular proteins and thereby exert high cytotoxicity. Components of the protein arginine methylation machinery have been identified as modulators of DPR toxicity and/or potential cellular interactors of R-rich DPRs; however, the molecular details and consequences of such an interaction are currently not well understood. Here, we demonstrate that several members of the family of protein arginine methyltransferases (PRMTs) can directly interact with R-rich DPRs in vitro and in the cytosol. In vitro, R-rich DPRs reduce solubility and promote phase separation of PRMT1, the main enzyme responsible for asymmetric arginine-dimethylation (ADMA) in mammalian cells, in a concentration- and length-dependent manner. Moreover, we demonstrate that poly-GR interferes more efficiently than poly-PR with PRMT1-mediated arginine methylation of RBPs such as hnRNPA3. We additionally show by two alternative approaches that poly-GR itself is a substrate for PRMT1-mediated arginine dimethylation. We propose that poly-GR may act as a direct competitor for arginine methylation of cellular PRMT1 targets, such as disease-linked RBPs.

Poly-GR repeats associated with ALS/FTD gene C9ORF72 impair translation elongation and induce a ribotoxic stress response in neurons.

Hexanucleotide repeat expansion in the C9ORF72 gene is the most frequent inherited cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The expansion results in multiple dipeptide repeat proteins, among which arginine-rich poly-GR proteins are highly toxic to neurons and decrease the rate of protein synthesis. We investigated whether the effect on protein synthesis contributes to neuronal dysfunction and degeneration. We found that the expression of poly-GR proteins inhibited global translation by perturbing translation elongation. In iPSC-differentiated neurons, the translation of transcripts with relatively slow elongation rates was further slowed, and stalled, by poly-GR. Elongation stalling increased ribosome collisions and induced a ribotoxic stress response (RSR) mediated by ZAKα that increased the phosphorylation of the kinase p38 and promoted cell death. Knockdown of ZAKα or pharmacological inhibition of p38 ameliorated poly-GR-induced toxicity and improved the survival of iPSC-derived neurons from patients with C9ORF72-ALS/FTD. Our findings suggest that targeting the RSR may be neuroprotective in patients with ALS/FTD caused by repeat expansion in C9ORF72.

Valosin-Containing Protein (VCP)/p97 Oligomerization.

Valosin-containing protein (VCP), also known as p97, is an evolutionarily conserved AAA+ ATPase essential for cellular homeostasis. Cooperating with different sets of cofactors, VCP is involved in multiple cellular processes through either the ubiquitin-proteasome system (UPS) or the autophagy/lysosomal route. Pathogenic mutations frequently found at the interface between the NTD domain and D1 ATPase domain have been shown to cause malfunction of VCP, leading to degenerative disorders including the inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD), amyotrophic lateral sclerosis (ALS), and cancers. Therefore, VCP has been considered as a potential therapeutic target for neurodegeneration and cancer. Most of previous studies found VCP predominantly exists and functions as a hexamer, which unfolds and extracts ubiquitinated substrates from protein complexes for degradation. However, recent studies have characterized a new VCP dodecameric state and revealed a controlling mechanism of VCP oligomeric states mediated by the D2 domain nucleotide occupancy. Here, we summarize our recent knowledge on VCP oligomerization, regulation, and potential implications of VCP in cellular function and pathogenic progression.