ATM kinase inhibitor AZD0156 in combination with irinotecan and 5-fluorouracil in preclinical models of colorectal cancer.

BACKGROUND: AZD0156 is an oral inhibitor of ATM, a serine threonine kinase that plays a key role in DNA damage response (DDR) associated with double-strand breaks. Topoisomerase-I inhibitor irinotecan is used clinically to treat colorectal cancer (CRC), often in combination with 5-fluorouracil (5FU). AZD0156 in combination with irinotecan and 5FU was evaluated in preclinical models of CRC to determine whether low doses of AZD0156 enhance the cytotoxicity of irinotecan in chemotherapy regimens used in the clinic. METHODS: Anti-proliferative effects of single-agent AZD0156, the active metabolite of irinotecan (SN38), and combination therapy were evaluated in 12 CRC cell lines. Additional assessment with clonogenic assay, cell cycle analysis, and immunoblotting were performed in 4 selected cell lines. Four colorectal cancer patient derived xenograft (PDX) models were treated with AZD0156, irinotecan, or 5FU alone and in combination for assessment of tumor growth inhibition (TGI). Immunofluorescence was performed on tumor tissues. The DDR mutation profile was compared across in vitro and in vivo models. RESULTS: Enhanced effects on cellular proliferation and regrowth were observed with the combination of AZD0156 and SN38 in select models. In cell cycle analysis of these models, increased G2/M arrest was observed with combination treatment over either single agent. Immunoblotting results suggest an increase in DDR associated with irinotecan therapy, with a reduced effect noted when combined with AZD0156, which is more pronounced in some models. Increased TGI was observed with the combination of AZD0156 and irinotecan as compared to single-agent therapy in some PDX models. The DDR mutation profile was variable across models. CONCLUSIONS: AZD0156 and irinotecan provide a rational and active combination in preclinical colorectal cancer models. Variability across in vivo and in vitro results may be related to the variable DDR mutation profiles of the models evaluated. Further understanding of the implications of individual DDR mutation profiles may help better identify patients more likely to benefit from treatment with the combination of AZD0156 and irinotecan in the clinical setting.

The lysosomal enzyme alpha-Galactosidase A is deficient in Parkinson's disease brain in association with the pathologic accumulation of alpha-synuclein.

The aberrant accumulation of alpha-synuclein (α-syn) is believed to contribute to the onset and pathogenesis of Parkinson's disease (PD). The autophagy-lysosome pathway (ALP) is responsible for the high capacity clearance of α-syn. ALP dysfunction is documented in PD and pre-clinical evidence suggests that inhibiting the ALP promotes the pathological accumulation of α-syn. We previously identified the pathological accumulation of α-syn in the brains of mice deficient for the soluble lysosomal enzyme alpha-Galactosidase A (α-Gal A), a member of the glycosphingolipid metabolism pathway. In the present study, we quantified α-Gal A activity and levels of its glycosphingolipid metabolites in postmortem temporal cortex specimens from control individuals and in PD individuals staged with respect to α-syn containing Lewy body pathology. In late-state PD temporal cortex we observed significant decreases in α-Gal A activity and the 46kDa "active" species of α-Gal A as determined respectively by fluorometric activity assay and western blot analysis. These decreases in α-Gal A activity/levels correlated significantly with increased α-syn phosphorylated at serine 129 (p129S-α-syn) that was maximal in late-stage PD temporal cortex. Mass spectrometric analysis of 29 different isoforms of globotriaosylceramide (Gb3), a substrate of α-Gal A indicated no significant differences with respect to different stages of PD temporal cortex. However, significant correlations were observed between increased levels of several Gb3 isoforms and with decreased α-Gal A activity and/or increased p129S-α-syn. Deacylated Gb3 (globotriaosylsphingosine or lyso-Gb3) was also analyzed in PD brain tissue but was below the limit of detection of 20pmol/g. Analysis of other lysosomal enzymes revealed a significant decrease in activity for the lysosomal aspartic acid protease cathepsin D but not for glucocerebrosidase (GCase) or cathepsin B in late-stage PD temporal cortex. However, a significant correlation was observed between decreasing GCase activity and increasing p129S-α-syn. Together our findings indicate α-Gal A deficiency in late-stage PD brain that correlates significantly with the pathological accumulation of α-syn, and further suggest the potential for α-Gal A and its glycosphingolipid substrates as putative biomarkers for PD.

ATP6V0C knockdown in neuroblastoma cells alters autophagy-lysosome pathway function and metabolism of proteins that accumulate in neurodegenerative disease.

ATP6V0C is the bafilomycin A1-binding subunit of vacuolar ATPase, an enzyme complex that critically regulates vesicular acidification. We and others have shown previously that bafilomycin A1 regulates cell viability, autophagic flux and metabolism of proteins that accumulate in neurodegenerative disease. To determine the importance of ATP6V0C for autophagy-lysosome pathway function, SH-SY5Y human neuroblastoma cells differentiated to a neuronal phenotype were nucleofected with non-target or ATP6V0C siRNA and following recovery were treated with either vehicle or bafilomycin A1 (0.3-100 nM) for 48 h. ATP6V0C knockdown was validated by quantitative RT-PCR and by a significant decrease in Lysostracker Red staining. ATP6V0C knockdown significantly increased basal levels of microtubule-associated protein light chain 3-II (LC3-II), α-synuclein high molecular weight species and APP C-terminal fragments, and inhibited autophagic flux. Enhanced LC3 and LAMP-1 co-localization following knockdown suggests that autophagic flux was inhibited in part due to lysosomal degradation and not by a block in vesicular fusion. Knockdown of ATP6V0C also sensitized cells to the accumulation of autophagy substrates and a reduction in neurite length following treatment with 1 nM bafilomycin A1, a concentration that did not produce such alterations in non-target control cells. Reduced neurite length and the percentage of propidium iodide-positive dead cells were also significantly greater following treatment with 3 nM bafilomycin A1. Together these results indicate a role for ATP6V0C in maintaining constitutive and stress-induced ALP function, in particular the metabolism of substrates that accumulate in age-related neurodegenerative disease and may contribute to disease pathogenesis.

Autophagy-lysosome pathway associated neuropathology and axonal degeneration in the brains of alpha-galactosidase A-deficient mice.

BACKGROUND: Mutations in the gene for alpha-galactosidase A result in Fabry disease, a rare, X-linked lysosomal storage disorder characterized by a loss of alpha-galactosidase A enzymatic activity. The resultant accumulation of glycosphingolipids throughout the body leads to widespread vasculopathy with particular detriment to the kidneys, heart and nervous system. Disruption in the autophagy-lysosome pathway has been documented previously in Fabry disease but its relative contribution to nervous system pathology in Fabry disease is unknown. Using an experimental mouse model of Fabry disease, alpha-galactosidase A deficiency, we examined brain pathology in 20-24 month old mice with particular emphasis on the autophagy-lysosome pathway. RESULTS: Alpha-galactosidase A-deficient mouse brains exhibited enhanced punctate perinuclear immunoreactivity for the autophagy marker microtubule-associated protein light-chain 3 (LC3) in the parenchyma of several brain regions, as well as enhanced parenchymal and vascular immunoreactivity for lysosome-associated membrane protein-1 (LAMP-1). Ultrastructural analysis revealed endothelial cell inclusions with electron densities and a pronounced accumulation of electron-dense lipopigment. The pons of alpha-galactosidase A-deficient mice in particular exhibited a striking neuropathological phenotype, including the presence of large, swollen axonal spheroids indicating axonal degeneration, in addition to large interstitial aggregates positive for phosphorylated alpha-synuclein that co-localized with the axonal spheroids. Double-label immunofluorescence revealed co-localization of phosphorylated alpha-synuclein aggregates with ubiquitin and LC3. CONCLUSION: Together these findings indicate widespread neuropathology and focused axonal neurodegeneration in alpha-galactosidase A-deficient mouse brain in association with disruption of the autophagy-lysosome pathway, and provide the basis for future mechanistic assessment of the contribution of the autophagy-lysosome pathway to this histologic phenotype.

LRRK2 inhibition attenuates microglial inflammatory responses.

Missense mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset Parkinson's disease (PD), and common genetic variation in LRRK2 modifies susceptibility to Crohn's disease and leprosy. High levels of LRRK2 expression in peripheral monocytes and macrophages suggest a role for LRRK2 in these cells, yet little is known about LRRK2 expression and function in immune cells of the brain. Here, we demonstrate a role for LRRK2 in mediating microglial proinflammatory responses and morphology. In a murine model of neuroinflammation, we observe robust induction of LRRK2 in microglia. Experiments with toll-like receptor 4 (TLR4)-stimulated rat primary microglia show that inflammation increases LRRK2 activity and expression, while inhibition of LRRK2 kinase activity or knockdown of protein attenuates TNFα secretion and nitric oxide synthase (iNOS) induction. LRRK2 inhibition blocks TLR4 stimulated microglial process outgrowth and impairs ADP stimulated microglial chemotaxis. However, actin inhibitors that phenocopy inhibition of process outgrowth and chemotaxis fail to modify TLR4 stimulation of TNFα secretion and inducible iNOS induction, suggesting that LRRK2 acts upstream of cytoskeleton control as a stress-responsive kinase. These data demonstrate LRRK2 in regulating responses in immune cells of the brain and further implicate microglial involvement in late-onset PD.

PPAR{alpha} mediates the hypolipidemic action of fibrates by antagonizing FoxO1.

High-fructose consumption is associated with insulin resistance and diabetic dyslipidemia, but the underlying mechanism is unclear. We show in hamsters that high-fructose feeding stimulated forkhead box O1 (FoxO1) production and promoted its nuclear redistribution in liver, correlating with augmented apolipoprotein C-III (apoC-III) production and impaired triglyceride metabolism. High-fructose feeding upregulated peroxisome proliferator-activated receptor-gamma coactivator-1beta and sterol regulatory element binding protein-1c expression, accounting for increased fat infiltration in liver. High-fructose-fed hamsters developed hypertriglyceridemia, accompanied by hyperinsulinemia and glucose intolerance. These metabolic aberrations were reversible by fenofibrate, a commonly used anti-hypertriglyceridemia agent that is known to bind and activate peroxisome proliferator-activated receptor-alpha (PPARalpha). PPARalpha physically interacted with, but functionally antagonized, FoxO1 in hepatic apoC-III expression. These data underscore the importance of FoxO1 deregulation in the pathogenesis of hypertriglyceridemia in high-fructose-fed hamsters. Counterregulation of hepatic FoxO1 activity by PPARalpha constitutes an important mechanism by which fibrates act to curb apoC-III overproduction and ameliorate hypertriglyceridemia.

Sirolimus is associated with reduced islet engraftment and impaired beta-cell function.

Successful islet transplantation depends on the infusion of sufficiently large quantities of islets, but only a fraction of transplanted islets can survive and become engrafted, and yet the underlying mechanism remains unclear. In this study, we examined the effect of sirolimus, a key component of the immunosuppressive regimen in clinical islet transplantation, on islet engraftment and function. To distinguish the effect of sirolimus on immune rejection from its effect on islet engraftment, we used a syngeneic model. Diabetic mice were transplanted with 250 islets under the renal capsule, followed by treatment with sirolimus or vehicle for 14 days. Thirty days posttransplantation, islet grafts were retrieved for the determination of insulin content and vascular density. Compared with mock-treated controls, diabetic recipient mice receiving sirolimus exhibited impaired blood glucose profiles and reduced glucose-stimulated insulin secretion, correlating with reduced intragraft insulin content and decreased vascular density. Islets exposed to sirolimus for 24 h in culture displayed significantly diminished glucose-stimulated insulin release, coinciding with decreased pancreas duodenum homeobox-1 and GLUT2 expression in cultured islets. Furthermore, sirolimus-treated diabetic recipient mice, as opposed to mock-treated controls, were associated with dyslipidemia. These data suggest that sirolimus, administered in the early posttransplantation phase, is a confounding factor for reduced islet engraftment and impaired beta-cell function in transplants.

Long-term K+ channel-mediated dampening of dopamine neuron excitability by the antipsychotic drug haloperidol.

Antipsychotic drugs require days of treatment to begin to produce therapeutic effects. We report that in vivo treatment with the antipsychotic drug haloperidol acts with a delay of days to slow spontaneous repetitive firing by isolated midbrain dopamine neurons. The decreased excitability is caused by an increased number of functional A-type K+ channels without any change in gating properties. Upregulation of dopamine neuron Kv4.3 mRNA accounts for this effect, demonstrating a role for channel gene expression in this delayed drug action. The resultant long-term dampening of dopamine neuron excitability may serve to tone down the dopamine system.