Marc P.M. Soutar

Inhibition of LRRK2 kinase activity stimulates macroautophagy.

Leucine Rich Repeat Kinase 2 (LRRK2) is one of the most important genetic contributors to Parkinsons disease. LRRK2 has been implicated in a number of cellular processes, including macroautophagy. To test whether LRRK2 has a role in regulating autophagy, a specific inhibitor of the kinase activity of LRRK2 was applied to human neuroglioma cells and downstream readouts of autophagy examined. The resulting data demonstrate that inhibition of LRRK2 kinase activity stimulates macroautophagy in the absence of any alteration in the translational targets of mTORC1, suggesting that LRRK2 regulates autophagic vesicle formation independent of canonical mTORC1 signaling. This study represents the first pharmacological dissection of the role LRRK2 plays in the autophagy/lysosomal pathway, emphasizing the importance of this pathway as a marker for LRRK2 physiological function. Moreover it highlights the need to dissect autophagy and lysosomal activities in the context of LRRK2 related pathologies with the final aim of understanding their aetiology and identifying specific targets for disease modifying therapies in patients.

Evidence that glycogen synthase kinase-3 isoforms have distinct substrate preference in the brain

J. Neurochem. (2010) 115, 974–983.

Increased CRMP2 phosphorylation is observed in Alzheimer's disease; does this tell us anything about disease development?

Collapsin response mediator protein-2 (CRMP2) was recently identified as a physiological substrate for GSK3 and Cdk5, two protein kinases suggested to exhibit greater activity in Alzheimers disease (AD). Indeed, phosphorylation of CRMP2, at the residues targeted by GSK3 and Cdk5, is relatively high in cortex isolated from human AD brain, as well as in the brains of animal models of AD, while phospho-CRMP2 is found in neurofibrillary tangles. In mouse models of AD, increased phosphorylation occurs prior to pathology. Although CRMP2 has no known enzymatic activity, a great deal of information is appearing on its importance in neuronal development and polarity, as well as in axon growth and guidance. In this mini-review, we examine what is known about CRMP2 function, how that is controlled by phosphorylation, what alterations in molecular mechanisms could lead to the abnormally high CRMP2 phosphorylation in AD, and whether this is likely to be specific to AD or occur in other forms of neurodegeneration. This will include discussion of the evidence for increased GSK3 or Cdk5 activity, for decreased phosphatase activity, or the upregulation of other CRMP2 protein kinases in AD. Importantly, we will compare the processes that may contribute to increased CRMP2 phosphorylation with those known to increase tau hyperphosphorylation in AD, and whether these are likely to be part of disease development or a useful early marker for AD.

Relative Resistance of Cdk5-phosphorylated CRMP2 to Dephosphorylation

Collapsin response mediator protein 2 (CRMP2) binds to microtubules and regulates axon outgrowth in neurons. This action is regulated by sequential phosphorylation by the kinases cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3 (GSK3) at sites that are hyperphosphorylated in Alzheimer disease. The increased phosphorylation in Alzheimer disease could be due to increases in Cdk5 and/or GSK3 activity or, alternatively, through decreased activity of a CRMP phosphatase. Here we establish that dephosphorylation of CRMP2 at the residues targeted by GSK3 (Ser-518/Thr-514/Thr-509) is carried out by a protein phosphatase 1 family member in vitro, in neuroblastoma cells, and primary cortical neurons. Inhibition of GSK3 activity using insulin-like growth factor-1 or the highly selective inhibitor CT99021 causes rapid dephosphorylation of CRMP2 at these sites. In contrast, pharmacological inhibition of Cdk5 using purvalanol results in only a gradual and incomplete dephosphorylation of CRMP2 at the site targeted by Cdk5 (Ser-522), suggesting a distinct phosphatase targets this residue. A direct comparison of dephosphorylation at the Cdk5 versus GSK3 sites in vitro shows that the Cdk5 site is comparatively resistant to phosphatase treatment. The presence of the peptidyl-prolyl isomerase enzyme, Pin1, does not affect dephosphorylation of Ser-522 in vitro, in cells, or in Pin1 transgenic mice. Instead, the relatively high resistance of this site to phosphatase treatment is at least in part due to the presence of basic residues located nearby. Similar sequences in Tau are also highly resistant to phosphatase treatment. We propose that relative resistance to phosphatases might be a common feature of Cdk5 substrates and could contribute to the hyperphosphorylation of CRMP2 and Tau observed in Alzheimer disease.

Mouse hypothalamic GT1-7 cells demonstrate AMPK-dependent intrinsic glucose-sensing behaviour

Aims/hypothesisHypothalamic glucose-excited (GE) neurons contribute to whole-body glucose homeostasis and participate in the detection of hypoglycaemia. This system appears defective in type 1 diabetes, in which hypoglycaemia commonly occurs. Unfortunately, it is at present unclear which molecular components required for glucose sensing are produced in individual neurons and how these are functionally linked. We used the GT1-7 mouse hypothalamic cell line to address these issues.MethodsElectrophysiological recordings, coupled with measurements of gene expression and protein levels and activity, were made from unmodified GT1-7 cells and cells in which AMP-activated protein kinase (AMPK) catalytic subunit gene expression and activity were reduced.ResultsHypothalamic GT1-7 neurons express the genes encoding glucokinase and ATP-sensitive K+ channel (KATP) subunits Kir6.2 and Sur1 and exhibit GE-type glucose-sensing behaviour. Lowered extracellular glucose concentration hyperpolarised the cells in a concentration-dependent manner, an outcome that was reversed by tolbutamide. Inhibition of glucose uptake or metabolism hyperpolarised cells, showing that energy metabolism is required to maintain their resting membrane potential. Short hairpin (sh)RNA directed to Ampkα2 (also known as Prkaa2) reduced GT1-7 cell AMPKα2, but not AMPKα1, activity and lowered the threshold for hypoglycaemia-induced hyperpolarisation. shAmpkα1 (also known as Prkaa1) had no effect on glucose-sensing or AMPKα2 activity. Decreased uncoupling protein 2 (Ucp2) mRNA was detected in AMPKα2-reduced cells, suggesting that AMPKα2 regulates UCP2 levels.Conclusions/interpretationWe have demonstrated that GT1-7 cells closely mimic GE neuron glucose-sensing behaviour, and reducing AMPKα2 blunts their responsiveness to hypoglycaemic challenge, possibly by altering UCP2 activity. These results show that suppression of AMPKα2 activity inhibits normal glucose-sensing behaviour and may contribute to defective detection of hypoglycaemia.

mTOR independent regulation of macroautophagy by Leucine Rich Repeat Kinase 2 via Beclin-1.

Leucine rich repeat kinase 2 is a complex enzyme with both kinase and GTPase activities, closely linked to the pathogenesis of several human disorders including Parkinson’s disease, Crohn’s disease, leprosy and cancer. LRRK2 has been implicated in numerous cellular processes; however its physiological function remains unclear. Recent reports suggest that LRRK2 can act to regulate the cellular catabolic process of macroautophagy, although the precise mechanism whereby this occurs has not been identified. To investigate the signalling events through which LRRK2 acts to influence macroautophagy, the mammalian target of rapamycin (mTOR)/Unc-51-like kinase 1 (ULK1) and Beclin-1/phosphatidylinositol 3-kinase (PI3K) pathways were evaluated in astrocytic cell models in the presence and absence of LRRK2 kinase inhibitors. Chemical inhibition of LRRK2 kinase activity resulted in the stimulation of macroautophagy in a non-canonical fashion, independent of mTOR and ULK1, but dependent upon the activation of Beclin 1-containing class III PI3-kinase.

Intracellular pH Modulates Autophagy and Mitophagy

The specific autophagic elimination of mitochondria (mitophagy) plays the role of quality control for this organelle. Deregulation of mitophagy leads to an increased number of damaged mitochondria and triggers cell death. The deterioration of mitophagy has been hypothesized to underlie the pathogenesis of several neurodegenerative diseases, most notably Parkinson disease. Although some of the biochemical and molecular mechanisms of mitochondrial quality control are described in detail, physiological or pathological triggers of mitophagy are still not fully characterized. Here we show that the induction of mitophagy by the mitochondrial uncoupler FCCP is independent of the effect of mitochondrial membrane potential but dependent on acidification of the cytosol by FCCP. The ionophore nigericin also reduces cytosolic pH and induces PINK1/PARKIN-dependent and -independent mitophagy. The increase of intracellular pH with monensin suppresses the effects of FCCP and nigericin on mitochondrial degradation. Thus, a change in intracellular pH is a regulator of mitochondrial quality control.

Deficiency of Parkinson's disease-related gene Fbxo7 is associated with impaired mitochondrial metabolism by PARP activation

The Parkinson’s disease (PD)-related protein F-box only protein 7 (Fbxo7) is the substrate-recognition component of the Skp1-Cullin-F-box protein E3 ubiquitin ligase complex. We have recently shown that PD-associated mutations in Fbxo7 disrupt mitochondrial autophagy (mitophagy), suggesting a role for Fbxo7 in modulating mitochondrial homeostasis. Here we report that Fbxo7 deficiency is associated with reduced cellular NAD+ levels, which results in increased mitochondrial NADH redox index and impaired activity of complex I in the electron transport chain. Under these conditions of compromised respiration, mitochondrial membrane potential and ATP contents are reduced, and cytosolic reactive oxygen species (ROS) production is increased. ROS activates poly (ADP-ribose) polymerase (PARP) activity in Fbxo7-deficient cells. PARP inhibitor restores cellular NAD+ content and redox index and ATP pool, suggesting that PARP overactivation is cause of decreased complex I-driven respiration. These findings bring new insight into the mechanism of Fbxo7 deficiency, emphasising the importance of mitochondrial dysfunction in PD.The Parkinsons disease (PD)-related protein F-box only protein 7 (Fbxo7) is the substrate-recognition component of the Skp1-Cullin-F-box protein E3 ubiquitin ligase complex. We have recently shown that PD-associated mutations in Fbxo7 disrupt mitochondrial autophagy (mitophagy), suggesting a role for Fbxo7 in modulating mitochondrial homeostasis. Here we report that Fbxo7 deficiency is associated with reduced cellular NAD+ levels, which results in increased mitochondrial NADH redox index and impaired activity of complex I in the electron transport chain. Under these conditions of compromised respiration, mitochondrial membrane potential and ATP contents are reduced, and cytosolic reactive oxygen species (ROS) production is increased. ROS activates poly (ADP-ribose) polymerase (PARP) activity in Fbxo7-deficient cells. PARP inhibitor restores cellular NAD+ content and redox index and ATP pool, suggesting that PARP overactivation is cause of decreased complex I-driven respiration. These findings bring new insight into the mechanism of Fbxo7 deficiency, emphasising the importance of mitochondrial dysfunction in PD.

Novel Procedure To Investigate the Effect of Phosphorylation on Protein Complex Formation in Vitro and in Cells

The identification of phosphorylation state-dependent interacting proteins provides clues as to the function of the phosphorylation. Techniques such as yeast two hybrid and co-immunoprecipitation do not employ a single species of fully phosphorylated proteins. This is a particular problem for substrates of glycogen synthase kinase-3 (GSK3), where multiple Ser/Thr residues can be targeted, almost always subsequent to a priming phosphorylation by an alternative kinase. We previously identified the brain enriched collapsin response mediator proteins (CRMP2 and CRMP4) as physiological substrates of GSK3. Cdk5 phosphorylates CRMP2 at Ser522, priming for subsequent phosphorylation at three residues by GSK3 in vitro and in vivo. It is clear that phosphorylation of CRMP2 influences axonal growth; however, the molecular processes underlying this action are not fully established. In addition, the role of phosphorylation in other actions of CRMPs has not been elucidated. We developed a novel procedure to isolate CRMP2 and CRMP4 fully phosphorylated at four sites, namely, Ser522 (by CDK5), Ser518, Thr514, and Thr509 (by GSK3). These phosphoproteins were then used to identify binding partners in rat brain lysates in direct comparison with the non-phosphorylated isoforms. We validated the approach by confirming that a previously reported interaction with tubulin-beta is regulated by phosphorylation. We also show that CRMPs (CRMP1, CRMP2, and CRMP4) form heteromers and found that these complexes may also be regulated by phosphorylation. We identified DYRK and Pin1 as novel CRMP4 binding proteins with DYRK interacting preferentially with dephospho-CRMP4 and Pin1 with phospho-CRMP4. Finally, we used this approach to identify the mitochondrial protein ANT as a novel CRMP2 and CRMP4 binding protein. We believe that this approach could be applied generally to the study of phosphorylation-dependent interactions.

Oleate induces KATP channel-dependent hyperpolarization in mouse hypothalamic glucose-excited neurons without altering cellular energy charge

Highlights • Oleate and low glucose hyperpolarize and inhibit GT1-7 and mouse GE neurons by activation of KATP.• Oleate inhibition of GT1-7 neuron activity is not mediated by AMPK or fatty acid oxidation.• Activation of KATP by oleate requires ATP hydrolysis but does not reduce the levels ATP or the ATP:ADP ratio.• GT1-7 hyperpolarization by oleate is not dependent on UCP2.• Oleate and low glucose depolarize a subpopulation of hypothalamic GI neurons.