Mutations in PTEN-induced kinase 1 (PINK1) cause early onset autosomal recessive Parkinson's disease (PD). PINK1 is a 63 kDa protein kinase, which exerts a neuroprotective function and is known to localize to mitochondria. Upon entry into the organelle, PINK1 is cleaved to produce a ∼53 kDa protein (ΔN-PINK1). In this paper, we show that PINK1 is cleaved between amino acids Ala-103 and Phe-104 to generate ΔN-PINK1. We demonstrate that a reduced ability to cleave PINK1, and the consequent accumulation of full-length protein, results in mitochondrial abnormalities reminiscent of those observed in PINK1 knockout cells, including disruption of the mitochondrial network and a reduction in mitochondrial mass. Notably, we assessed three N-terminal PD-associated PINK1 mutations located close to the cleavage site and, while these do not prevent PINK1 cleavage, they alter the ratio of full-length to ΔN-PINK1 protein in cells, resulting in an altered mitochondrial phenotype. Finally, we show that PINK1 interacts with the mitochondrial protease presenilin-associated rhomboid-like protein (PARL) and that loss of PARL results in aberrant PINK1 cleavage in mammalian cells. These combined results suggest that PINK1 cleavage is important for basal mitochondrial health and that PARL cleaves PINK1 to produce the ΔN-PINK1 fragment.

SummaryMotor neurons (MNs) and astrocytes (ACs) are implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), but their interaction and the sequence of molecular events leading to MN death remain unresolved. Here, we optimized directed differentiation of induced pluripotent stem cells (iPSCs) into highly enriched (> 85%) functional populations of spinal cord MNs and ACs. We identify significantly increased cytoplasmic TDP-43 and ER stress as primary pathogenic events in patient-specific valosin-containing protein (VCP)-mutant MNs, with secondary mitochondrial dysfunction and oxidative stress. Cumulatively, these cellular stresses result in synaptic pathology and cell death in VCP-mutant MNs. We additionally identify a cell-autonomous VCP-mutant AC survival phenotype, which is not attributable to the same molecular pathology occurring in VCP-mutant MNs. Finally, through iterative co-culture experiments, we uncover non-cell-autonomous effects of VCP-mutant ACs on both control and mutant MNs. This work elucidates molecular events and cellular interplay that could guide future therapeutic strategies in ALS.

Mutations in PINK1 cause early-onset Parkinson's disease (PD). Studies in Drosophila melanogaster have highlighted mitochondrial dysfunction on loss of Pink1 as a central mechanism of PD pathogenesis. Here we show that global analysis of transcriptional changes in Drosophila pink1 mutants reveals an upregulation of genes involved in nucleotide metabolism, critical for neuronal mitochondrial DNA synthesis. These key transcriptional changes were also detected in brains of PD patients harbouring PINK1 mutations. We demonstrate that genetic enhancement of the nucleotide salvage pathway in neurons of pink1 mutant flies rescues mitochondrial impairment. In addition, pharmacological approaches enhancing nucleotide pools reduce mitochondrial dysfunction caused by Pink1 deficiency. We conclude that loss of Pink1 evokes the activation of a previously unidentified metabolic reprogramming pathway to increase nucleotide pools and promote mitochondrial biogenesis. We propose that targeting strategies enhancing nucleotide synthesis pathways may reverse mitochondrial dysfunction and rescue neurodegeneration in PD and, potentially, other diseases linked to mitochondrial impairment.The role of mitochondrial impairment in PD has long been debated. Recently, the identification of causative mutations in PINK1, a gene encoding a mitochondrial kinase in PD patients has renewed interest in the role of mitochondrial damage in PD (ref. 1).

Disruption of synaptic integrity, loss of connectivity and axodendritic degeneration are early and essential components of neurodegeneration. Although neuronal cell death mechanisms have been thoroughly investigated, less is known about the signals involved in axodendritic damage and the processes involved in regeneration. Here we conducted a genome-wide RNA interference-based forward genetic screen, using small interfering RNA targeting all human kinases, and identified clusters of kinases families essential for growth cone collapse, neurite retraction and neurite outgrowth. Of 59 kinases identified as positive regulators of neurite outgrowth, almost 50% were in the tyrosine kinase/tyrosine kinase-like (TK/TKL) receptor subgroups, underlining the importance of extracellular ligands in this process. Neurite outgrowth was inhibited by 66 other kinases, none of which were TK/TKL members, whereas 79 kinases inhibited lysophosphatidic acid-induced neurite retraction. Twenty kinases were involved in both inhibitory processes suggesting shared mechanisms. Within this group of 20 kinases, some (ULK1, PDK1, MAP4K4) have been implicated previously in axonal events, but others (MAST2, FASTK, CKM and DGUOK) have not. For a subset of kinases, the effect on neurite outgrowth was validated in rat primary cerebellar cultures. The ability to affect regeneration was further tested in a model of axodendritic lesion using primary rat midbrain cultures. Finally, we demonstrated that haploinsufficiency of two members of the AGC kinase subgroup, ROCK1 and PKN1, was able to suppress retinal degeneration in Drosophila model of class III Autosomal Dominant Retinitis Pigmentosa.