Missense mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset Parkinson disease, and common genetic variation in LRRK2 modifies susceptibility to Crohn 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 pro-inflammatory responses and morphology. In a murine model of neuroinflammation, we observe robust induction of LRRK2 in microglia. Experiments with 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 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 iNOS induction, suggesting 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.

cAMP, the classical second messenger, regulates many diverse cellular functions. The primary effector of cAMP signals, protein kinase A, differentially phosphorylates hundreds of cellular targets. Little is known, however, about the spatial and temporal nature of cAMP signals and their information content. Thus, it is largely unclear how cAMP, in response to different stimuli, orchestrates such a wide variety of cellular responses. Previously, we presented evidence that cAMP is produced in subcellular compartments near the plasma membrane, and that diffusion of cAMP from these compartments to the bulk cytosol is hindered. Here we report that a uniform extracellular stimulus initiates distinct cAMP signals within different cellular compartments. By using cyclic nucleotide-gated ion channels engineered as cAMP biosensors, we found that prostaglandin E 1 stimulation of human embryonic kidney cells caused a transient increase in cAMP concentration near the membrane. Interestingly, in the same time frame, the total cellular cAMP rose to a steady level. The decline in cAMP levels near the membrane was prevented by pretreatment with phosphodiesterase inhibitors. These data demonstrate that spatially and temporally distinct cAMP signals can coexist within simple cells.

Phosphodiesterases (PDEs) catalyze the hydrolysis of the second messengers cAMP and cGMP. However, little is known about how PDE activity regulates cyclic nucleotide signals in vivo because, outside of specialized cells, there are few methods with the appropriate spatial and temporal resolution to measure cyclic nucleotide concentrations. We have previously demonstrated that adenovirus-expressed, olfactory cyclic nucleotide–gated channels provide real-time sensors for cAMP produced in subcellular compartments of restricted diffusion near the plasma membrane (Rich, T.C., K.A. Fagan, H. Nakata, J. Schaack, D.M.F. Cooper, and J.W. Karpen. 2000. J. Gen. Physiol. 116:147–161). To increase the utility of this method, we have modified the channel, increasing both its cAMP sensitivity and specificity, as well as removing regulation by Ca2+-calmodulin. We verified the increased sensitivity of these constructs in excised membrane patches, and in vivo by monitoring cAMP-induced Ca2+ influx through the channels in cell populations. The improved cAMP sensors were used to monitor changes in local cAMP concentration induced by adenylyl cyclase activators in the presence and absence of PDE inhibitors. This approach allowed us to identify localized PDE types in both nonexcitable HEK-293 and excitable GH4C1 cells. We have also developed a quantitative framework for estimating the KI of PDE inhibitors in vivo. The results indicate that PDE type IV regulates local cAMP levels in HEK-293 cells. In GH4C1 cells, inhibitors specific to PDE types I and IV increased local cAMP levels. The results suggest that in these cells PDE type IV has a high K m for cAMP, whereas PDE type I has a low K m for cAMP. Furthermore, in GH4C1 cells, basal adenylyl cyclase activity was readily observable after application of PDE type I inhibitors, indicating that there is a constant synthesis and hydrolysis of cAMP in subcellular compartments near the plasma membrane. Modulation of constitutively active adenylyl cyclase and PDE would allow for rapid control of cAMP-regulated processes such as cellular excitability.

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 mocktreated 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 ␤-cell function in transplants. Diabetes 55:2429 -2436, 2006 T he Edmonton protocol for islet transplantation depends on the infusion of ϳ10,000 IE (islet equivalents)/kg body wt, requiring multiple cadaver pancreata per diabetic recipient (1-4). Despite the implantation of such a large quantity of islets, Ͻ30% of transplanted islets can survive the procedure and gain stable engraftment, and yet the mechanism underlying the loss of a vast majority of islet mass in the early posttransplantation phase remains elusive (4,5). Unlike whole-organ transplantation, by which grafts are implanted as vascularized tissue, islets are transplanted as single islets or islet clusters that are considered avascular following collagenase digestion and isolation. Although residual endothelial cells in isolated islets may contribute to islet revascularization (6,7), adequate intraislet blood flow requires the formation of a functional microvascular network that links engrafted islets to surrounding tissues. These data suggest that microvascular perfusion to newly transplanted islets does not resume immediately after transplantation and can take up to 2 weeks before the reestablishment of a functional microvasculature in islet grafts (8,9). This delay in islet revascularization can potentially deprive islets of oxygen and nutrients, resulting in islet cell death, particularly within the core of engrafted islets. There is mounting evidence that impaired islet revascularization is an independent factor that limits the success rate...

.-High-fructose consumption is associated with insulin resistance and diabetic dyslipidemia, but the underlying mechanism is unclear. We show in hamsters that highfructose 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-␥ coactivator-1␤ 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-␣ (PPAR␣). PPAR␣ 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 PPAR␣ constitutes an important mechanism by which fibrates act to curb apoC-III overproduction and ameliorate hypertriglyceridemia.hypertriglyceridemia; forkhead box O1; peroxisome proliferator-activated receptor-␣; apolipoprotein C-III; very-low-density lipoproteintriglyceride; microsomal triglyceride transfer protein HYPERTRIGLYCERIDEMIA IS CHARACTERIZED by increased production of very-low-density lipoprotein (VLDL) and/or decreased clearance of triglyceride (TG)-rich particles (4). Due to its proatherogenic profile, hypertriglyceridemia is a major contributing factor for the pathogenesis of atherosclerosis and coronary artery disease in obesity and type 2 diabetes. While the pathophysiology of hypertriglyceridemia is poorly understood, its close association with visceral adiposity and type 2 diabetes implicates insulin resistance as a causative factor in the development of hypertriglyceridemia (5,12,22,29,42). However, the molecular events that link insulin resistance to the pathogenesis of hypertriglyceridemia remain elusive.An important player in plasma VLDL-TG metabolism is apolipoprotein C-III (apoC-III). It functions as an inhibitor of lipoprotein lipase and hepatic lipase, playing a pivotal role in the hydrolysis and clearance of TG-rich particles such as VLDL-TG and chylomicrons (27,36,51,53). Elevated plasma apoC-III levels are associated with impaired clearance of TG-rich particles, leading to the accumulation of TG-rich lipoprotein remnants in plasma and the development of hypertriglyceridemia.Our recent data indicate that forkhead box O1 (FoxO1), a nuclear transcription factor known to be a mediator of insulin signaling, plays an important role in hepatic regulation of apoC-III. FoxO1 stimulates hepatic apoC-III expression, which is counteracted by insulin (2). Elevated FoxO1 production in liver augments hepatic apoC-III expression, r...

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.

BackgroundMutations 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.ResultsAlpha-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.ConclusionTogether 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.Electronic supplementary materialThe online version of this article (doi:10.1186/2051-5960-2-20) contains supplementary material, which is available to authorized users.

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 (Gb), 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 Gb isoforms and with decreased α-Gal A activity and/or increased p129S-α-syn. Deacylated Gb (globotriaosylsphingosine or lyso-Gb) 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.