Comprehensive evaluation of human-derived anti-poly-GA antibodies in cellular and animal models of C9orf72 disease

Hexanucleotide G4C2 repeat expansions in the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Dipeptide repeat proteins (DPRs) generated by translation of repeat-containing RNAs show toxic effects in vivo as well as in vitro and are key targets for therapeutic intervention. We generated human antibodies that bind DPRs with high affinity and specificity. Anti-GA antibodies engaged extra- and intra-cellular poly-GA and reduced aggregate formation in a poly-GA overexpressing human cell line. However, antibody treatment in human neuronal cultures synthesizing exogenous poly-GA resulted in the formation of large extracellular immune complexes and did not affect accumulation of intracellular poly-GA aggregates. Treatment with antibodies was also shown to directly alter the morphological and biochemical properties of poly-GA and to shift poly-GA/antibody complexes to more rapidly sedimenting ones. These alterations were not observed with poly-GP and have important implications for accurate measurement of poly-GA levels including the need to evaluate all centrifugation fractions and disrupt the interaction between treatment antibodies and poly-GA by denaturation. Targeting poly-GA and poly-GP in two mouse models expressing G4C2 repeats by systemic antibody delivery for up to 16 mo was well-tolerated and led to measurable brain penetration of antibodies. Long-term treatment with anti-GA antibodies produced improvement in an open-field movement test in aged C9orf72450 mice. However, chronic administration of anti-GA antibodies in AAV-(G4C2)149 mice was associated with increased levels of poly-GA detected by immunoassay and did not significantly reduce poly-GA aggregates or alleviate disease progression in this model. Keywords: C9orf72; amyotrophic lateral sclerosis; dipeptide repeat proteins; frontotemporal dementia; immunotherap

Comprehensive preclinical evaluation of human-derived anti-poly-GA antibodies in cellular and animal models of C9ORF72 disease

Hexanucleotide G4C2 repeat expansions in the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Dipeptide repeat proteins (DPRs) generated by translation of repeat-containing RNAs show toxic effects in vivo as well as in vitro and are key targets for therapeutic intervention. We generated human antibodies that bind DPRs with high affinity and specificity. Anti-GA antibodies engaged extra- and intracellular poly-GA and reduced aggregate formation in a poly-GA over-expressing human cell line. However, antibody treatment in human neuronal cultures synthesizing exogenous poly-GA resulted in the formation of large extracellular immune complexes and did not affect accumulation of intracellular poly-GA aggregates. Treatment with antibodies was also shown to directly alter the morphological and biochemical properties of poly-GA and to shift poly-GA/antibody complexes to more rapidly sedimenting ones. These alterations were not observed with poly-GP and have important implications for accurate measurement of poly-GA levels including the need to evaluate all centrifugation fractions and disrupt the interaction between treatment antibodies and poly-GA by denaturation. Targeting poly-GA and poly-GP in two mouse models expressing G4C2 repeats by systemic antibody delivery for up to 16 months was well-tolerated and led to measurable brain penetration of antibodies. Long term treatment with anti-GA antibodies produced improvement in an open field movement test in aged C9ORF72450 mice. However, chronic administration of anti-GA antibodies in AAV-(G4C2)149 mice was associated with increased levels of poly-GA detected by immunoassay and did not significantly reduce poly-GA aggregates or alleviate disease progression in this model. Significance Immunotherapy has been proposed for neurodegenerative disorders including Alzheimer’s or Parkinson’s diseases. Recent reports using antibodies against poly-GA or active immunization suggested similar immunotherapy in ALS/FTD caused by repeat expansion in the C9ORF72 gene (1, 2). Here, we systematically characterized human antibodies against multiple DPR species and tested the biological effects of antibodies targeting poly-GA in different cellular and mouse models. Target engagement was shown in three independent cellular models. Anti-GA antibodies reduced the number of intracellular poly-GA aggregates in human T98G cells but not in cultured human neurons. Whereas chronic anti-GA treatment in BAC C9ORF72450 mice did not impact poly-GA levels and modestly improved one behavioral phenotype, poly-GA levels detected by immunoassays were increased and disease progression was unaltered in AAV-(G4C2)149 mice

Functional and dynamic polymerization of the ALS-linked protein TDP-43 antagonizes its pathologic aggregation

TDP-43 is a primarily nuclear RNA-binding protein, whose abnormal phosphorylation and cytoplasmic aggregation characterizes affected neurons in patients with amyotrophic lateral sclerosis and frontotemporal dementia. Here, we report that physiological nuclear TDP-43 in mouse and human brain forms homo-oligomers that are resistant to cellular stress. Physiological TDP-43 oligomerization is mediated by its N-terminal domain, which can adopt dynamic, solenoid-like structures, as revealed by a 2.1 Å crystal structure in combination with nuclear magnetic resonance spectroscopy and electron microscopy. These head-to-tail TDP-43 oligomers are unique among known RNA-binding proteins and represent the functional form of the protein in vivo, since their destabilization results in loss of alternative splicing regulation of known neuronal RNA targets. Our findings indicate that N-terminal domain-driven oligomerization spatially separates the adjoining highly aggregation-prone, C-terminal low-complexity domains of consecutive TDP-43 monomers, thereby preventing low-complexity domain inter-molecular interactions and antagonizing the formation of pathologic aggregates.TDP-43 aggregation is observed in amyotrophic lateral sclerosis. Here the authors combine X-ray crystallography, nuclear magnetic resonance and electron microscopy studies and show that physiological oligomerization of TDP-43 is mediated through its N-terminal domain, which forms functional and dynamic oligomers antagonizing pathologic aggregation

GST pull-down assay with substituted forms of human ERH.

<p>Indicated FLAG-tagged ERH forms incubated with either GST-tagged fragment L7 of human PDIP46/SKAR (GST-PDIP46/SKAR[L7]) or GST-tagged fragment B of human Ciz1 (GST-Ciz1[B]) and detected by western blotting with anti-FLAG antibody followed by enhanced chemiluminescence reaction. PDIP46/SKAR does not interact with ERH H3A Q9A or ERH H3A Q9A E37A T51A, and Ciz1 does not interact with ERH E37A T51A or ERH H3A Q9A E37A T51A.</p

Recruitment of substituted forms of human ERH to nuclear speckles and replication foci in HeLa cells visualized by confocal microscopy.

<p>EGFP-tagged substituted forms of ERH expressed alone (top) or coexpressed with mCherry-tagged human PDIP46/SKAR (middle) or mCherry-tagged human Ciz1 (bottom). <b>A</b>. ERH T18A S24A localizes to the nucleus and is recruited both to nuclear speckles and to replication foci similarly to wild-type ERH. <b>B</b>. ERH H3A Q9A is present not only in the nucleus but also in the cytoplasm, shows diminished recruitment to nuclear speckles but still accumulates in replication foci. <b>C</b>. ERH E37A T51A localizes partly to the cytoplasm, is recruited to nuclear speckles, and shows very week accumulation in replication foci. <b>D</b>. ERH H3A Q9A E37A T51A is also present in the cytoplasm and recruited neither to nuclear speckles nor to replication foci.</p

Amino acid residues of human ERH critical for its recruitment to nuclear speckles and replication foci.

<p>Three-dimensional structure of a monomer of ERH was produced by UCSF Chimera package using coordinates from Protein Data Bank (2nmlA) [30]. Four β strands (β1, β2, β3 and β4), three α helices (α1, α2 and α3) and the N- and C-termini are indicated. Critical residues are shown with their side chains in color and are labeled using a single-letter code and position number in the polypeptide chain. Residues involved in the recruitment to nuclear speckles (in red) and replication foci (in green) are situated on the β sheet and near the loop between helices α1 and α2, respectively.</p

Hypertonic Stress Causes Cytoplasmic Translocation of Neuronal, but Not Astrocytic, FUS due to Impaired Transportin Function

International audienceThe primarily nuclear RNA-binding protein FUS (fused in sarcoma) forms pathological cytoplasmic inclusions in a subset of early-onset amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients. In response to cellular stress, FUS is recruited to cytoplasmic stress granules, which are hypothesized to act as precursors of pathological inclusions. We monitored the stress-induced nucleocytoplasmic shuttling of endogenous FUS in an ex vivo mouse CNS model and human neural networks. We found that hyperosmolar, but not oxidative, stress induced robust cytoplasmic translocation of neuronal FUS, with transient nuclear clearance and loss of function. Surprisingly, this reaction is independent of stress granule formation and the molecular pathways activated by hyperosmolarity. Instead, it represents a mechanism mediated by cytoplasmic redistribution of Transportin 1/2 and is potentiated by transcriptional inhibition. Importantly, astrocytes, which remain unaffected in ALS/FTD-FUS, are spared from this stress reaction that may signify the initial event in the development of FUS pathology