Summary A hexanucleotide repeat expansion (HRE), (GGGGCC)n, in C9orf72 is the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we identify a molecular mechanism by which structural polymorphism of the HRE leads to ALS/FTD pathology and defects. The HRE forms DNA and RNA G-quadruplexes with distinct structures and promotes RNA•DNA hybrids (R-loops). The structural polymorphism causes a repeat length-dependent accumulation of transcripts aborted in the HRE region. These transcribed repeats bind to ribonucleoproteins in a conformationdependent manner. Specifically, nucleolin (NCL), an essential nucleolar protein, preferentially binds the HRE G-quadruplex, and patient cells show evidence of nucleolar stress. Our results demonstrate that distinct C9orf72 HRE structural polymorphism at both DNA and RNA levels initiates molecular cascades leading to ALS/FTD pathologies, and provide the basis for a mechanistic model for repeat-associated neurodegenerative diseases.

TAR DNA-binding protein 43 (TDP-43) plays a key role in the neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. The nature of the TDP-43-mediated neurotoxicity associated with these diseases is not yet understood. Here, we have established transgenic Caenorhabditis elegans models that express human TDP-43 variants in the nervous system, including the full-length wild-type (WT) and mutant proteins and a pathologic C-terminal fragment. The C. elegans models developed severe locomotor defects associated with the aggregation of TDP-43 in neurons. In comparison to parallel Cu/Zn superoxide dismutase worm models, transgenic full-length TDP-43, including the WT protein, was highly neurotoxic. In addition, TDP-43 demonstrated an unusually high tendency to aggregate, a property intrinsic to the WT protein. The C-terminal 25 kDa fragment of TDP-43 was unstable but remarkably aggregation-prone. Distinct disulfide-linked TDP-43 dimers and oligomers were detected. In C. elegans, the neurotoxicity and the protein aggregation of TDP-43 were regulated by environmental temperature and heat shock transcriptional factor 1, indicating that a deficiency in protein quality control is a risk factor for TDP-43 proteinopathy. Furthermore, the neurotoxicity and the protein aggregation of TDP-43 can be significantly attenuated by a deficiency in the insulin/insulin-like growth factor 1 (IGF-1) signaling in C. elegans and mammalian cells. These results suggest that protein misfolding underlies the aging-dependent neurodegeneration associated with TDP-43 and that the insulin/IGF-1 signaling may be a target for therapies.

The acquisition and maintenance of final neuronal identity depends in part upon the implementation of fate-specification programs in postmitotic neurons; however, the mechanisms involved remain unclear. In the developing spinal cord, retinoic acid (RA) signaling pathways specify the columnar and divisional identities of postmitotic motoneurons (MNs). Here we show that RA signals induce expression of the NET transcriptional regulator Nolz1 in differentiated chick MNs, where it regulates the progressive specification of prospective Lim3-negative motor columns. Nolz1 controls the initial formation of forelimb and thoracic Lim3-negative motor columns by downregulating Lim3 expression and maintaining the expression of key homeodomain proteins necessary for MN identity and survival. At forelimb levels, Nolz1 specifies lateral motor column (LMC) identity by inducing the expression of the postmitotic LMC determinant Hoxc6, and implements the partial specification of lateral LMC identity through Lim1 induction. The specificity of Nolz1 function depends upon distinct repressor activities that require, in part, the modulatory activity of Grg5, an atypical member of the Gro-TLE family of co-repressors. Thus, RA signals regulate diverse events in MN subtype specification by inducing the expression of a key transcriptional regulator that controls multiple developmental pathways via functionally distinct repressor complexes.

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