Impaired ribosome-associated quality control of C9orf72 arginine-rich dipeptide-repeat proteins.

Protein quality control pathways have evolved to ensure the fidelity of protein synthesis and efficiently clear potentially toxic protein species. Defects in ribosome-associated quality control and its associated factors have been implicated in the accumulation of aberrant proteins and neurodegeneration. C9orf72 repeat-associated non-AUG translation has been suggested to involve inefficient translation elongation, lead to ribosomal pausing and activation of ribosome-associated quality control pathways. However, the role of the ribosome-associated quality control complex in the processing of proteins generated through this non-canonical translation is not well understood. Here we use reporter constructs containing the C9orf72-associated hexanucleotide repeat, ribosome-associated quality control complex deficient cell models and stain for ribosome-associated quality control markers in C9orf72-expansion carrier human tissue to understand its role in dipeptide-repeat protein pathology. Our studies show that canonical ribosome-associated quality control substrates products are efficiently cleared by the ribosome-associated quality control complex in mammalian cells. Furthermore, using stalling reporter constructs, we show that repeats associated with the C9orf72-expansion induce ribosomal stalling when arginine (R)-rich dipeptide-repeat proteins are synthesized in a length-dependent manner. However, despite triggering this pathway, these arginine-rich dipeptide-repeat proteins are not efficiently processed by the core components of the ribosome-associated quality control complex (listerin, nuclear-export mediator factor and valosin containing protein) partly due to lack of lysine residues, which precludes ubiquitination. Deficient processing by this complex may be implicated in C9orf72-expansion associated disease as dipeptide-repeat protein inclusions were observed to be predominantly devoid of ubiquitin and co-localize with nuclear-export mediator factor in mutation carriers' frontal cortex and cerebellum tissue. These findings suggest that impaired processing of these arginine-rich dipeptide-repeat proteins derived from repeat-associated non-AUG translation by the ribosome-associated quality control complex may contribute to protein homeostasis dysregulation observed in C9orf72-expansion amyotrophic lateral sclerosis and frontotemporal degeneration neuropathogenesis.

High density SNP array and reanalysis of genome sequencing uncovers CNVs associated with neurodevelopmental disorders in KOLF2.1J iPSCs.

The KOLF2.1J iPSC line was recently proposed as a reference iPSC to promote the standardization of research studies in the stem cell field. Due to overall good performance differentiating to neural cell lineages, high gene editing efficiency, and absence of genetic variants associated to neurological disorders KOLF2.1J iPSC line was particularly recommended for neurodegenerative disease modeling. However, our work uncovers that KOLF2.1J hPSCs carry heterozygous small copy number variants (CNVs) that cause DTNBP1, JARID2 and ASTN2 haploinsufficiencies, all of which are associated with neurological disorders. We further determine that these CNVs arose in vitro over the course of KOLF2.1J iPSC generation from a healthy donor-derived KOLF2 iPSC line and affect the expression of DNTBP1, JARID2 and ASTN2 proteins in KOLF2.1J iPSCs and neural progenitors. Therefore, our study suggests that KOLF2.1J iPSCs carry genetic variants that may be deleterious for neural cell lineages. This data is essential for a careful interpretation of neural cell studies derived from KOLF2.1J iPSCs and highlights the need for a catalogue of iPSC lines that includes a comprehensive genome characterization analysis.

Increased LRRK2 kinase activity alters neuronal autophagy by disrupting the axonal transport of autophagosomes.

Parkinson's disease-causing mutations in the leucine-rich repeat kinase 2 (LRRK2) gene hyperactivate LRRK2 kinase activity and cause increased phosphorylation of Rab GTPases, important regulators of intracellular trafficking. We found that the most common LRRK2 mutation, LRRK2-G2019S, dramatically reduces the processivity of autophagosome transport in neurons in a kinase-dependent manner. This effect was consistent across an overexpression model, neurons from a G2019S knockin mouse, and human induced pluripotent stem cell (iPSC)-derived neurons gene edited to express the G2019S mutation, and the effect was reversed by genetic or pharmacological inhibition of LRRK2. Furthermore, LRRK2 hyperactivation induced by overexpression of Rab29, a known activator of LRRK2 kinase, disrupted autophagosome transport to a similar extent. Mechanistically, we found that hyperactive LRRK2 recruits the motor adaptor JNK-interacting protein 4 (JIP4) to the autophagosomal membrane, inducing abnormal activation of kinesin that we propose leads to an unproductive tug of war between anterograde and retrograde motors. Disruption of autophagosome transport correlated with a significant defect in autophagosome acidification, suggesting that the observed transport deficit impairs effective degradation of autophagosomal cargo in neurons. Our results robustly link increased LRRK2 kinase activity to defects in autophagosome transport and maturation, further implicating defective autophagy in the pathogenesis of Parkinson's disease.