ATP13A2-mediated endo-lysosomal polyamine export counters mitochondrial oxidative stress

Recessive loss-of-function mutations in ATP13A2 (PARK9) are associated with a spectrum of neurodegenerative disorders, including Parkinson’s disease (PD). We recently revealed that the late endo-lysosomal transporter ATP13A2 pumps polyamines like spermine into the cytosol, whereas ATP13A2 dysfunction causes lysosomal polyamine accumulation and rupture. Here, we investigate how ATP13A2 provides protection against mitochondrial toxins such as rotenone, an environmental PD risk factor. Rotenone promoted mitochondrial-generated superoxide (MitoROS), which was exacerbated by ATP13A2 deficiency in SH-SY5Y cells and patient-derived fibroblasts, disturbing mitochondrial functionality and inducing toxicity and cell death. Moreover, ATP13A2 knockdown induced an ATF4-CHOP-dependent stress response following rotenone exposure. MitoROS and ATF4-CHOP were blocked by MitoTEMPO, a mitochondrial antioxidant, suggesting that the impact of ATP13A2 on MitoROS may relate to the antioxidant properties of spermine. Pharmacological inhibition of intracellular polyamine synthesis with α-difluoromethylornithine (DFMO) also increased MitoROS and ATF4 when ATP13A2 was deficient. The polyamine transport activity of ATP13A2 was required for lowering rotenone/DFMO-induced MitoROS, whereas exogenous spermine quenched rotenone-induced MitoROS via ATP13A2. Interestingly, fluorescently labeled spermine uptake in the mitochondria dropped as a consequence of ATP13A2 transport deficiency. Our cellular observations were recapitulated in vivo, in a Caenorhabditis elegans strain deficient in the ATP13A2 ortholog catp-6. These animals exhibited a basal elevated MitoROS level, mitochondrial dysfunction, and enhanced stress response regulated by atfs-1, the C. elegans ortholog of ATF4, causing hypersensitivity to rotenone, which was reversible with MitoTEMPO. Together, our study reveals a conserved cell protective pathway that counters mitochondrial oxidative stress via ATP13A2-mediated lysosomal spermine export.

Loss-of-function mutations in ATP13A2 (PARK9) are causative for a spectrum of neurodegenerative disorders, including Kufor-Rakeb syndrome (KRS, a juvenile onset parkinsonism with dementia) (1), early-onset Parkinson’s disease (PD) (2, 3), hereditary spastic paraplegia (HSP) (4), neuronal ceroid lipofuscinosis (5), and amyotrophic lateral sclerosis (6), which are commonly hallmarked by lysosomal and mitochondrial dysfunction (4, 6, 7). Also, ATP13A2 deficiency causes lysosomal and mitochondrial impairment in various models, as evidenced by decreased lysosomal functionality (8, 9), reduced mitochondrial clearance capacity (8–10), mitochondrial fragmentation, mitochondrial DNA damage, and increased oxygen consumption (11, 12).We recently discovered that ATP13A2 transports the polyamines spermidine and spermine from the late endo/lysosome to the cytosol (9). Polyamines are ubiquitous polycationic aliphatic amines that stabilize nucleic acids, influence protein folding, regulate ion channels, and modulate cell proliferation and differentiation (13–15). We found that the late endo-lysosomal transporter ATP13A2 strongly contributes to the total cellular polyamine content via a two-step process: Firstly, polyamines enter the cell via endocytosis and subsequently, polyamines are transported by ATP13A2 into the cytosol (9). This process complements polyamine biosynthesis via the ornithine decarboxylase (ODC) pathway (9). Importantly, ATP13A2’s polyamine transport function is crucial for its neuroprotective effect, since it prevents lysosomal polyamine accumulation and subsequent lysosomal rupture, while improving lysosomal health and functionality (9). Moreover, when activated by its two regulatory lipids—phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2] and phosphatidic acid (PA)—ATP13A2 exerts a cell protective effect against the mitochondrial neurotoxin rotenone (16), an environmental risk factor for PD (17). Rotenone is a mitochondrial complex I inhibitor, which leads to high levels of reactive oxygen species (ROS), promoting protein aggregation and damaging organelles. However, how ATP13A2’s polyamine transport function exerts a cell protective effect against rotenone, or other mitochondrial neurotoxins, is not yet clear.Interestingly, the transported substrates spermine and spermidine reduce oxidative stress (14, 15). Spermine is a potent free radical scavenger (18) and a biologically important antioxidant (19–23). We therefore hypothesize that ATP13A2-mediated polyamine transport may counteract oxidative stress (16, 24) and preserve mitochondrial health (11, 12). Here, we demonstrate in complementary human cell models and Caenorhabditis elegans that lysosomal polyamine export by ATP13A2 effectively lowers ROS levels and promotes mitochondrial health and functionality, pointing to a lysosomal-dependent cell protective pathway that may be implicated in ATP13A2-related neurodegenerative disorders.     

The impact of hypoglycaemia awareness status on regional brain responses to acute hypoglycaemia in men with type 1 diabetes

Aims/hypothesis

Impaired awareness of hypoglycaemia (IAH) in type 1 diabetes increases the risk of severe hypoglycaemia sixfold and can be resistant to intervention. We explored the impact of IAH on central responses to hypoglycaemia to investigate the mechanisms underlying barriers to therapeutic intervention.

Methods

We conducted [¹⁵O]water positron emission tomography studies of regional brain perfusion during euglycaemia (target 5 mmol/l), hypoglycaemia (achieved level, 2.4 mmol/l) and recovery (target 5 mmol/l) in 17 men with type 1 diabetes: eight with IAH, and nine with intact hypoglycaemia awareness (HA).

Results

Hypoglycaemia with HA was associated with increased activation in brain regions including the thalamus, insula, globus pallidus (GP), anterior cingulate cortex (ACC), orbital cortex, dorsolateral frontal (DLF) cortex, angular gyrus and amygdala; deactivation occurred in the temporal and parahippocampal regions. IAH was associated with reduced catecholamine and symptom responses to hypoglycaemia vs HA (incremental AUC: autonomic scores, 26.2?±?35.5 vs 422.7?±?237.1; neuroglycopenic scores, 34.8?±?88.8 vs 478.9?±?311.1; both p?<?0.002). There were subtle differences (p?<?0.005, k?≥?50 voxels) in brain activation at hypoglycaemia, including early differences in the right central operculum, bilateral medial orbital (MO) cortex, and left posterior DLF cortex, with additional differences in the ACC, right GP and post- and pre-central gyri in established hypoglycaemia, and lack of deactivation in temporal regions in established hypoglycaemia.

Conclusions/interpretation

Differences in activation in the post- and pre-central gyri may be expected in people with reduced subjective responses to hypoglycaemia. Alterations in the activity of regions involved in the drive to eat (operculum), emotional salience (MO cortex), aversion (GP) and recall (temporal) suggest differences in the perceived importance and urgency of responses to hypoglycaemia in IAH compared with HA, which may be key to the persistence of the condition.

     

Meropenem-RPX7009 Concentrations in Plasma,Epithelial Lining Fluid,and Alveolar Macrophages of Healthy Adult Subjects

The steady-state concentrations of meropenem and the β-lactamase inhibitor RPX7009 in plasma, epithelial lining fluid (ELF), and alveolar macrophage (AM) concentrations were obtained in 25 healthy, nonsmoking adult subjects. Subjects received a fixed combination of meropenem (2 g) and RPX7009 (2 g) administered every 8 h, as a 3-h intravenous infusion, for a total of three doses. A bronchoscopy and bronchoalveolar lavage were performed once in each subject at 1.5, 3.25, 4, 6, or 8 h after the start of the last infusion. Meropenem and RPX7009 achieved a similar time course and magnitude of concentrations in plasma and ELF. The mean pharmacokinetic parameters ± the standard deviations of meropenem and RPX7009 determined from serial plasma concentrations were as follows: C_max = 58.2 ± 10.8 and 59.0 ± 8.4 μg/ml, V_ss = 16.3 ± 2.6 and 17.6 ± 2.6 liters; CL = 11.1 ± 2.1 and 10.1 ± 1.9 liters/h, and t_1/2 = 1.03 ± 0.15 and 1.27 ± 0.21 h, respectively. The intrapulmonary penetrations of meropenem and RPX7009 were ca. 63 and 53%, respectively, based on the area under the concentration-time curve from 0 to 8 h (AUC_0–8) values of ELF and total plasma concentrations. When unbound plasma concentrations were considered, ELF penetrations were 65 and 79% for meropenem and RPX7009, respectively. Meropenem concentrations in AMs were below the quantitative limit of detection, whereas median concentrations of RPX7009 in AMs ranged from 2.35 to 6.94 μg/ml. The results from the present study lend support to exploring a fixed combination of meropenem (2 g) and RPX7009 (2 g) for the treatment of lower respiratory tract infections caused by meropenem-resistant Gram-negative pathogens susceptible to the combination of meropenem-RPX7009.     

Toxicity of Amphotericin B Deoxycholate-Based Induction Therapy in Patients with HIV-Associated Cryptococcal Meningitis

Amphotericin B deoxycholate (AmBd) is the recommended induction treatment for HIV-associated cryptococcal meningitis (CM). Its use is hampered by toxicities that include electrolyte abnormalities, nephrotoxicity, and anemia. Protocols to minimize toxicity are applied inconsistently. In a clinical trial cohort of AmBd-based CM induction treatment, a standardized protocol of preemptive hydration and electrolyte supplementation was applied. Changes in blood counts, electrolyte levels, and creatinine levels over 14 days were analyzed in relation to the AmBd dose, treatment duration (short course of 5 to 7 days or standard course of 14 days), addition of flucytosine (5FC), and outcome. In the 368 patients studied, the hemoglobin levels dropped by a mean of 1.5 g/dl (95% confidence interval [CI], 1.0 to 1.9 g/dl) following 7 days of AmBd and by a mean of 2.3 g/dl (95% CI, 1.1 to 3.6 g/dl) after 14 days. Serum creatinine levels increased by 37 μmol/liter (95% CI, 30 to 45 μmol/liter) by day 7 and by 49 μmol/liter (95% CI, 35 to 64μmol/liter) by day 14 of AmBd treatment. Overall, 33% of patients developed grade III/IV anemia, 5.6% developed grade III hypokalemia, 9.5% had creatinine levels that exceeded 220 μmol, and 6% discontinued AmBd prematurely. The addition of 5FC was associated with a slight increase in anemia but not neutropenia. Laboratory abnormalities stabilized or reversed during the second week in patients on short-course induction. Grade III/IV anemia (adjusted odds ratio [aOR], 2.2; 95% CI, 1.1 to 4.3; P = 0.028) and nephrotoxicity (aOR, 4.5; 95% CI, 1.8 to 11; P = 0.001) were risk factors for 10-week mortality. In summary, routine intravenous saline hydration and preemptive electrolyte replacement during AmBd-based induction regimens for HIV-associated CM minimized the incidence of hypokalemia and nephrotoxicity. Anemia remained a concerning adverse effect. The addition of flucytosine was not associated with increased neutropenia. Shorter AmBd courses were less toxic, with rapid reversibility.     

Inhibition of Aminoglycoside 6′-N-Acetyltransferase Type Ib-Mediated Amikacin Resistance in Klebsiella pneumoniae by Zinc and Copper Pyrithione

The in vitro activity of the aminoglycoside 6′-N-acetyltransferase type Ib [AAC(6′)-Ib] was inhibited by CuCl₂ with a 50% inhibitory concentration (IC₅₀) of 2.8 μM. The growth of an amikacin-resistant Klebsiella pneumoniae strain isolated from a neonate with meningitis was inhibited when amikacin was supplemented by the addition of Zn²⁺ or Cu²⁺ in complex with the ionophore pyrithione. Coordination complexes between cations and ionophores could be developed for their use, in combination with aminoglycosides, to treat resistant infections.