Blocking dPerk in the intestine suppresses neurodegeneration in a Drosophila model of Parkinson's disease.

Parkinson's disease (PD) is characterised by selective death of dopaminergic (DA) neurons in the midbrain and motor function impairment. Gastrointestinal issues often precede motor deficits in PD, indicating that the gut-brain axis is involved in the pathogenesis of this disease. The features of PD include both mitochondrial dysfunction and activation of the unfolded protein response (UPR) in the endoplasmic reticulum (ER). PINK1 is a mitochondrial kinase involved in the recycling of defective mitochondria, and PINK1 mutations cause early-onset PD. Like PD patients, pink1 mutant Drosophila show degeneration of DA neurons and intestinal dysfunction. These mutant flies also lack vital proteins due to sustained activation of the kinase R-like endoplasmic reticulum kinase (dPerk), a kinase that induces the UPR. Here, we investigated the role of dPerk in intestinal dysfunction. We showed that intestinal expression of dPerk impairs mitochondrial function, induces cell death, and decreases lifespan. We found that suppressing dPerk in the intestine of pink1-mutant flies rescues intestinal cell death and is neuroprotective. We conclude that in a fly model of PD, blocking gut-brain transmission of UPR-mediated toxicity, is neuroprotective.

Profiling tumour heterogeneity through circulating tumour DNA in patients with pancreatic cancer.

The majority of pancreatic ductal adenocarcinomas (PDAC) are diagnosed late so that surgery is rarely curative. Earlier detection could significantly increase the likelihood of successful treatment and improve survival. The aim of the study was to provide proof of principle that point mutations in key cancer genes can be identified by sequencing circulating free DNA (cfDNA) and that this could be used to detect early PDACs and potentially, premalignant lesions, to help target early effective treatment. Targeted next generation sequencing (tNGS) analysis of mutation hotspots in 50 cancer genes was conducted in 26 patients with PDAC, 14 patients with chronic pancreatitis (CP) and 12 healthy controls with KRAS status validated by digital droplet PCR. A higher median level of total cfDNA was observed in patients with PDAC (585 ng/ml) compared to either patients with CP (300 ng/ml) or healthy controls (175 ng/ml). PDAC tissue showed wide mutational heterogeneity, whereas KRAS was the most commonly mutated gene in cfDNA of patients with PDAC and was significantly associated with a poor disease specific survival (p=0.018). This study demonstrates that tNGS of cfDNA is feasible to characterise the circulating genomic profile in PDAC and that driver mutations in KRAS have prognostic value but cannot currently be used to detect early emergence of disease. Importantly, monitoring total cfDNA levels may have utility in individuals "at risk" and warrants further investigation.

dATF4 regulation of mitochondrial folate-mediated one-carbon metabolism is neuroprotective.

Neurons rely on mitochondria as their preferred source of energy. Mutations in PINK1 and PARKIN cause neuronal death in early-onset Parkinson's disease (PD), thought to be due to mitochondrial dysfunction. In Drosophila pink1 and parkin mutants, mitochondrial defects lead to the compensatory upregulation of the mitochondrial one-carbon cycle metabolism genes by an unknown mechanism. Here we uncover that this branch is triggered by the activating transcription factor 4 (ATF4). We show that ATF4 regulates the expression of one-carbon metabolism genes SHMT2 and NMDMC as a protective response to mitochondrial toxicity. Suppressing Shmt2 or Nmdmc caused motor impairment and mitochondrial defects in flies. Epistatic analyses showed that suppressing the upregulation of Shmt2 or Nmdmc deteriorates the phenotype of pink1 or parkin mutants. Conversely, the genetic enhancement of these one-carbon metabolism genes in pink1 or parkin mutants was neuroprotective. We conclude that mitochondrial dysfunction caused by mutations in the Pink1/Parkin pathway engages ATF4-dependent activation of one-carbon metabolism as a protective response. Our findings show a central contribution of ATF4 signalling to PD that may represent a new therapeutic strategy. A video abstract for this article is available at https://youtu.be/cFJJm2YZKKM.

LRRK2, but not pathogenic mutants, protects against H2O2 stress depending on mitochondrial function and endocytosis in a yeast model.

BACKGROUND: Mutations in LRRK2 are the most common genetic cause of Parkinson's disease (PD). Studies in the yeast Saccharomyces cerevisiae have provided valuable insights into the mechanisms of cellular dysfunction associated with the expression of faulty PD genes. METHODS: We developed a yeast model for full-length LRRK2 studies. We expressed wild-type (wt) LRRK2 and mutations and evaluated their role during oxidative stress conditions. The involvement of mitochondria was assessed by using rho-zero mutants and by evaluating reactive oxygen species (ROS) production and mitochondrial membrane potential by flow cytometry. The involvement of endocytosis was also studied by testing several endocytic mutants and by following the vacuolar delivery of the probe FM4-64. RESULTS: Expression of LRRK2 in yeast was associated to increased hydrogen peroxide resistance. This phenotype, which was dependent on mitochondrial function, was not observed for PD-mutants G2019S and R1441C or in the absence of the kinase activity and the WD40 repeat domain. Expression of the pathogenic mutants stimulated ROS production and increased mitochondrial membrane potential. For the PD-mutants, but not for wild-type LRRK2, endocytic defects were also observed. Additionally, several endocytic proteins were required for LRRK2-mediated protection against hydrogen peroxide. CONCLUSIONS: Our results indicate that LRRK2 confers cellular protection during oxidative stress depending on mitochondrial function and endocytosis. GENERAL SIGNIFICANCE: Both the loss of capacity of LRRK2 pathogenic mutants to protect against oxidative stress and their enhancement of dysfunction may be important for the development of PD during the aging process.

HtrA2 deficiency causes mitochondrial uncoupling through the F1F0-ATP synthase and consequent ATP depletion.

Loss of the mitochondrial protease HtrA2 (Omi) in mice leads to mitochondrial dysfunction, neurodegeneration and premature death, but the mechanism underlying this pathology remains unclear. Using primary cultures from wild-type and HtrA2-knockout mice, we find that HtrA2 deficiency significantly reduces mitochondrial membrane potential in a range of cell types. This depolarisation was found to result from mitochondrial uncoupling, as mitochondrial respiration was increased in HtrA2-deficient cells and respiratory control ratio was dramatically reduced. HtrA2-knockout cells exhibit increased proton translocation through the ATP synthase, in combination with decreased ATP production and truncation of the F1 α-subunit, suggesting the ATP synthase as the source of the proton leak. Uncoupling in the HtrA2-deficient mice is accompanied by altered breathing pattern and, on a cellular level, ATP depletion and vulnerability to chemical ischaemia. We propose that this vulnerability may ultimately cause the neurodegeneration observed in these mice.