Stem cell-derived cranial and spinal motor neurons reveal proteostatic differences between ALS resistant and sensitive motor neurons.

In amyotrophic lateral sclerosis (ALS) spinal motor neurons (SpMN) progressively degenerate while a subset of cranial motor neurons (CrMN) are spared until late stages of the disease. Using a rapid and efficient protocol to differentiate mouse embryonic stem cells (ESC) to SpMNs and CrMNs, we now report that ESC-derived CrMNs accumulate less human (h)SOD1 and insoluble p62 than SpMNs over time. ESC-derived CrMNs have higher proteasome activity to degrade misfolded proteins and are intrinsically more resistant to chemically-induced proteostatic stress than SpMNs. Chemical and genetic activation of the proteasome rescues SpMN sensitivity to proteostatic stress. In agreement, the hSOD1 G93A mouse model reveals that ALS-resistant CrMNs accumulate less insoluble hSOD1 and p62-containing inclusions than SpMNs. Primary-derived ALS-resistant CrMNs are also more resistant than SpMNs to proteostatic stress. Thus, an ESC-based platform has identified a superior capacity to maintain a healthy proteome as a possible mechanism to resist ALS-induced neurodegeneration.

Rbfox Splicing Factors Promote Neuronal Maturation and Axon Initial Segment Assembly.

Neuronal maturation requires dramatic morphological and functional changes, but the molecular mechanisms governing this process are not well understood. Here, we studied the role of Rbfox1, Rbfox2, and Rbfox3 proteins, a family of tissue-specific splicing regulators mutated in multiple neurodevelopmental disorders. We generated Rbfox triple knockout (tKO) ventral spinal neurons to define a comprehensive network of alternative exons under Rbfox regulation and to investigate their functional importance in the developing neurons. Rbfox tKO neurons exhibit defects in alternative splicing of many cytoskeletal, membrane, and synaptic proteins, and display immature electrophysiological activity. The axon initial segment (AIS), a subcellular structure important for action potential initiation, is diminished upon Rbfox depletion. We identified an Rbfox-regulated splicing switch in ankyrin G, the AIS "interaction hub" protein, that regulates ankyrin G-beta spectrin affinity and AIS assembly. Our data show that the Rbfox-regulated splicing program plays a crucial role in structural and functional maturation of postmitotic neurons.

A cassette of N-terminal amino acids of histone H2B are required for efficient cell survival, DNA repair and Swi/Snf binding in UV irradiated yeast.

The highly charged histone N-terminal domains are engaged in inter- and intra-nucleosomal interactions, and contain a host of sites used for posttranslational modification. We have studied the effect of deleting residues 30-37 from the N-terminal domain of histone H2B in yeast cells, on nucleotide excision repair (NER) following UV irradiation, as these cells are quite sensitive to UV. We find that H2B Delta30-37 cells exhibit reduced NER efficiency at three specific chromatin loci: the transcriptionally active, RPB2 locus; the transcriptionally silenced, nucleosome-loaded HML locus; and the transcriptionally repressed, non-silenced, GAL10 locus. Nuclease digestion studies indicate that H2B Delta30-37 chromatin has increased nucleosome accessibility and/or nucleosome mobility. In addition, H2B Delta30-37 mutants acquire more DNA damage, compared to wt cells, following the same dose of UV radiation. Reducing the level of damage in H2B Delta30-37 cells to match that of wt cells restores the NER rate to wt levels in the RPB2 and GAL10 loci, but NER efficiency remains low in the silenced HML locus. Interestingly, recruitment of Snf5 to the HML locus is reduced in H2B Delta30-37 cells and more transient following UV irradiation. This may reflect a lower binding affinity of the SWI/SNF complex to H2B Delta30-37 nucleosomes.