Catecholamines up integrates dopamine synthesis and synaptic trafficking.

The highly reactive nature of dopamine renders dopaminergic neurons vulnerable to oxidative damage. We recently demonstrated that loss-of-function mutations in the Drosophila gene Catecholamines up (Catsup) elevate dopamine pools but, paradoxically, also confer resistance to paraquat, an herbicide that induces oxidative stress-mediated toxicity in dopaminergic neurons. We now report a novel association of the membrane protein, Catsup, with GTP cyclohydrolase rate-limiting enzyme for tetrahydrobiopterin (BH(4)) biosynthesis and tyrosine hydroxylase, rate-limiting enzyme for dopamine biosynthesis, which requires BH(4) as a cofactor. Loss-of-function Catsup mutations cause dominant hyperactivation of both enzymes. Elevated dopamine levels in Catsup mutants coincide with several distinct characteristics, including hypermobility, minimal basal levels of 3,4-dihydroxy-phenylacetic acid, an oxidative metabolite of dopamine, and resistance to the vesicular monoamine transporter inhibitor, reserpine, suggesting that excess dopamine is synaptically active and that Catsup functions in the regulation of synaptic vesicle loading and release of dopamine. We conclude that Catsup regulates and links the dopamine synthesis and transport networks.

Molecular profile of osteoprogenitor cells seeded on allograft bone.

In order to optimize and modulate bone formation it is essential to understand the expression patterns of key bone-specific growth factors, as osteoprogenitor cells undergo the processes of proliferation, differentiation and maturation. This study reports the sequential expression of bone-related growth and transcription factors when bone marrow-derived osteoprogenitor cells from C57BL mice were cultured on allograft bone discs. Mineralization and osteocalcin protein levels were used to track osteogenic differentiation and maturation. Bone-related growth factors, such as Bmp-2, Bmp-7, Ctnnb-1, Fgf-2, Igf-1, Vegf-a and Tgf-ß1, and transcription factors, such as Runx-2 and osteocalcin, were examined by enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR). Total density of mineralized bone was significantly increased 7.6 ± 0.7% in allografts cultured with cells, compared with a 0.5 ± 2.0% increase in the controls without cells (p < 0.01). Osteocalcin protein levels peaked at day 4. Protein expression showed peaks of BMP-2 and TGF-ß1 on day 2, with VEGF peaking on day 8, and IGF-1 decreasing on day 2. mRNA for Pdgf-a peaked on day 2; Bmp-2 on days 4 and 16; Ctnnb-1 on days 8 and 20; Vegf-a, Fgf-2, Runx-2 and Igf-1 on day 12; Tgf-ß1 on day 16; and Pdgf-b on day 20. Osteogenic growth factors correlated with Runx-2 and Ctnnb-1, whereas a predominant vascular growth factor, Vegf-a, did not follow this pattern. Specific bone-related genes and proteins were expressed in a time-dependent manner when osteoprogenitor cells were cultured on cortico-cancellous bone discs in vitro.

Role of the Toll-like receptor pathway in the recognition of orthopedic implant wear-debris particles.

The inflammatory response to prosthetic implant-derived wear particles is the primary cause of bone loss and aseptic loosening of implants, but the mechanisms by which macrophages recognize and respond to particles remain unknown. Studies of innate immunity demonstrate that Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPS). All TLRs signal through myeloid differentiation factor 88 (MyD88), except TLR3 which signals through TIR domain containing adapter inducing interferon-beta (TRIF), and TLR4 which signals through both MyD88 and TRIF. We hypothesized that wear-debris particles may act as PAMPs/DAMPs and activate macrophages via TLRs. To test this hypothesis, we first demonstrated that inhibition of MyD88 decreases polymethylmethacrylate (PMMA) particle-induced production of TNF-α in RAW 264.7 macrophages. Next we compared particle-induced production of TNF-α among MyD88 knockout (MyD88(-/-)), TRIF knockout (TRIF(-/-)), and wild type (WT) murine macrophages. Relative to WT, disruption of MyD88 signaling diminished, and disruption of TRIF amplified the particle-induced production of TNF-α. Gene expression data indicated that this latter increase in TNF-α was due to a compensatory increase in expression of MyD88 associated components of the TLR pathway. Finally, using an in vivo model, MyD88(-/-) mice developed less particle-induced osteolysis than WT mice. These results indicate that the response to PMMA particles is partly dependent on MyD88, presumably as part of TLR signaling; MyD88 may represent a therapeutic target for prevention of wear debris-induced periprosthetic osteolysis.

Continuous infusion of UHMWPE particles induces increased bone macrophages and osteolysis.

BACKGROUND: Aseptic loosening and periprosthetic osteolysis resulting from wear debris are major complications of total joint arthroplasty. Monocyte/macrophages are the key cells related to osteolysis at the bone-implant interface of joint arthroplasties. Whether the monocyte/macrophages found at the implant interface in the presence of polyethylene particles are locally or systemically derived is unknown. QUESTIONS/PURPOSES: We therefore asked (1) whether macrophages associated with polyethylene particle-induced chronic inflammation are recruited locally or systemically and (2) whether the recruited macrophages are associated with enhanced osteolysis locally. METHODS: Noninvasive in vivo imaging techniques (bioluminescence and microCT) were used to investigate initial macrophage migration systemically from a remote injection site to polyethylene wear particles continuously infused into the femoral canal. We used histologic and immunohistologic staining to confirm localization of migrated macrophages to the polyethylene particle-treated femoral canals and monitor cellular markers of bone remodeling. RESULTS: The values for bioluminescence were increased for animals receiving UHMWPE particles compared with the group in which the carrier saline was infused. At Day 8, the ratio of bioluminescence (operated femur divided by nonoperated contralateral femur of each animal) for the UHMWPE group was 13.95 ± 5.65, whereas the ratio for the saline group was 2.60 ± 1.14. Immunohistologic analysis demonstrated the presence of reporter macrophages in the UHMWPE particle-implanted femora only. MicroCT scans showed the bone mineral density for the group with both UHMWPE particles and macrophage was lower than the control groups. CONCLUSIONS: Infusion of clinically relevant polyethylene particles, similar to the human scenario, stimulated systemic migration of remotely injected macrophages and local net bone resorption.

Effects of orthopedic polymer particles on chemotaxis of macrophages and mesenchymal stem cells.

Wear particles generated from total joint arthroplasty (TJA) stimulate macrophages to release chemokines. The role of chemokines released from wear particle-stimulated macrophages on the migration of macrophages and osteoprogenitor cells in vitro has not been elucidated. In this study, we challenged murine macrophages (RAW 264.7) with clinically relevant polymethyl methacrylate (PMMA, 1-10 microm) and ultra high molecular weight polyethylene (UHMWPE, 2-3 microm) particles. The chemotactic effects of the conditioned media (CM) were tested in vitro using human macrophages (THP-1) and human mesenchymal stem cells (MSCs) as the migrating cells. CM collected from both particle types had a chemotactic effect on human macrophages, which could be eliminated by monocyte chemotactic protein-1 (MCP-1) neutralizing antibody. Blocking the CCR1 receptor eliminated the chemotactic effect, while CCR2 antibody only partially decreased THP-1 cell migration. CM from PMMA but not UHMWPE-exposed macrophages led to chemotaxis of MSCs; this effect could be eliminated by macrophage inflammatory protein-1 alpha (MIP-1alpha) neutralizing antibody. Neither CCR1 nor CCR2 blocking antibodies showed an effect on the migration of MSCs. Chemokines released by macrophages stimulated by wear particles can have an effect on the migration of macrophages and MSCs. This effect seems to be dependent on the particle type, and may be modulated by MCP-1 and MIP-1alpha, however, more than one chemokine may be necessary for chemotaxis.

Modulating osteogenesis of mesenchymal stem cells by modifying growth factor availability.

Growth factors control the proliferation and differentiation of osteoprogenitor cells. This study explores the effects of modulating growth factors (VEGF, IGF-1, FGF-2 and BMP-2) on osteogenesis of mesenchymal stem cells (MSCs) in vitro. Constant and profiled delivery protocols, in accordance with protein expression in vitro, were applied to deliver or neutralize growth factors. Cell number, alkaline phosphatase (ALP-2) and osteocalcin (OC) expression, and mineralization were measured as outcome variables. Profiled addition of VEGF increased MSC proliferation. Constant and profiled application of FGF-2 and neutralization of IGF-1 and BMP-2 decreased ALP-2 levels. Profiled addition of BMP-2 vastly increased OC release from MSCs, but constant addition of IGF-1, constant and profiled neutralization of IGF-1 and FGF-2 reduced OC levels. Constant addition of IGF-1 and FGF-2, as well as profiled loading of FGF-2 decreased mineralization of MSCs. This study indicated that endogenous IGF-1 and FGF-2 are essential to osteogenesis; excess IGF-1 and FGF-2 were inhibitory to bone formation. Selective, temporally specific addition of growth factors, such as BMP-2 and VEGF appears to be an important strategy to enhance osteogenesis.

Surveillance of systemic trafficking of macrophages induced by UHMWPE particles in nude mice by noninvasive imaging.

Macrophages constitute a major part of the cell response to wear particles produced at articulating and nonarticulating interfaces of joint replacements. This foreign body reaction can result in periprosthetic osteolysis and implant loosening. We demonstrate that ultra-high molecular weight polyethylene (UHMWPE) particles induce systemic trafficking of macrophages by noninvasive in vivo imaging and immunohistochemistry. The distal femora of nude mice were injected with 60 mg/mL UHMWPE suspension or saline alone. Reporter RAW264.7 macrophages that stably expressed the bioluminescent reporter gene and the fluorescence reporter gene were injected intravenously. Bioluminescence imaging was performed using an in vivo imaging system immediately after macrophage injection and at 2-day intervals. Compared with the nonoperated contralateral femora, at day 4, 6, and 8, the bioluminescent signal of femora containing UHMWPE suspension increased 1.30 +/- 0.09-, 2.36 +/- 0.92-, and 10.32 +/- 7.61-fold, respectively. The values at same time points for saline-injected control group were 1.08 +/- 0.07-, 1.14 +/- 0.27-, and 1.14 +/- 0.35-fold, respectively. The relative bioluminescence of the UHMWPE group was higher at all postinjection days and significantly greater than the saline group at day 8 (p < 0.05). Histological analysis confirmed the presence of reporter macrophages within the medullary canal of mice with implanted UHMWPE particles. The presence of UHMWPE particles induced enhanced bone remodeling activity. Clinically relevant UHMWPE particles stimulated the systemic recruitment of macrophages during an early time course using the murine femoral implant model. Interference with systemic macrophage trafficking may potentially mitigate UHMWPE particle-induced periprosthetic osteolysis.

Polymethylmethacrylate particle exposure causes changes in p38 MAPK and TGF-beta signaling in differentiating MC3T3-E1 cells.

Periprosthetic osteolysis of joint replacements caused by wear debris is a significant complication of joint replacements. Polymethylmethacrylate (PMMA) particles have been shown to inhibit osteogenic differentiation, but the molecular mechanism has not been previously determined. In this study, we exposed differentiating MC3T3-E1 preostoblast cells to PMMA particles and determined the changes that occurred with respect to p38 mitogen-activated protein kinase (MAPK) activity and the transforming growth factor (TGF)-beta1 and bone morphogenetic protein (BMP) signaling pathways. In the absence of particles, MC3T3-E1 cells demonstrate activation of p38 MAPK on day 8 of differentiation; however, when treated with PMMA particles, differentiating MC3T3-E1 cells demonstrate the suppression of p38 activity on day 8 and show activation of p38 on days 1 and 4. On day 4 of particle exposure, the differentiating MC3T3-E1 cells show significant downregulation of TGF-beta1 expression, which is involved in osteoblast differentiation, and a significant upregulation of the expression of BMP3 and Sclerostin (SOST), which are negative regulators of osteoblast differentiation. By day 8 of particle exposure, the changes in TGF-beta1, BMP3, and SOST expression are opposite of those seen on day 4. This study has demonstrated the distinct changes in the molecular profile of MC3T3-E1 cells during particle-induced inhibition of osteoblast differentiation. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

In vivo murine model of continuous intramedullary infusion of particles--a preliminary study.

Continued production of wear debris affects both initial osseointegration and subsequent bone remodeling of total joint replacements (TJRs). However, continuous delivery of clinically relevant particles using a viable, cost effective, quantitative animal model to simulate the scenario in humans has been a challenge for orthopedic researchers. In this study, we successfully infused blue-dyed polystyrene particles, similar in size to wear debris in humans, to the intramedullary space of the mouse femur for 4 weeks using an osmotic pump. Approximately 40% of the original particle load (85 microL) was delivered into the intramedullary space, an estimate of 3 x 10(9) particles. The visible blue dye carried by the particles confirmed the delivery. This model demonstrated that continuous infusion of particles to the murine bone-implant interface is possible. In vivo biological processes associated using wear debris particles can be studied using this new animal model.

Effect of nanofiber-coated surfaces on the proliferation and differentiation of osteoprogenitors in vitro.

The osteoconductive property of titanium (Ti) surfaces is important in orthopedic and dental implant devices. Surface modifications of Ti have been proposed to further improve osseointegration. In this study, three different materials, silicon (Si), silicon oxide (SiO(2)), and titanium oxide (TiO(2)), were used to construct nanofibers for surface coating of Ti alloy Ti-6Al-4 V (Ti alloy). MC3T3-E1 osteoprogenitor cells were seeded on nanofiber-coated discs and cultured for 42 days. DNA, alkaline phosphatase, osteocalcin, and mineralization nodules were measured using PicoGreen, enzyme-linked immunosorbent assay, and calcein blue staining to detect the attachment, proliferation, differentiation, and mineralization of MC3T3-E1 cells, respectively. The results demonstrated that the initial cell attachments on nanofiber-coated discs were significantly lower, although cell proliferation on Si and SiO(2) nanofiber-coated discs was better than on Ti alloy surfaces. TiO(2) nanofibers facilitated a higher cellular differentiation capacity than Ti alloy and tissue culture-treated polystyrene surfaces. Thus, surface modification using nanofibers of various materials can alter the attachment, proliferation, and differentiation of osteoprogenitor cells in vitro.

Direct binding of GTP cyclohydrolase and tyrosine hydroxylase: regulatory interactions between key enzymes in dopamine biosynthesis.

The signaling functions of dopamine require a finely tuned regulatory network for rapid induction and suppression of output. A key target of regulation is the enzyme tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, which is activated by phosphorylation and modulated by the availability of its cofactor, tetrahydrobiopterin. The first enzyme in the cofactor synthesis pathway, GTP cyclohydrolase I, is activated by phosphorylation and inhibited by tetrahydrobiopterin. We previously reported that deficits in GTP cyclohydrolase activity in Drosophila heterozygous for mutant alleles of the gene encoding this enzyme led to tightly corresponding diminution of in vivo tyrosine hydroxylase activity that could not be rescued by exogenous cofactor. We also found that the two enzymes could be coimmunoprecipitated from tissue extracts and proposed functional interactions between the enzymes that extended beyond provision of cofactor by one pathway for another. Here, we confirm the physical association of these enzymes, identifying interacting regions in both, and we demonstrate that their association can be regulated by phosphorylation. The functional consequences of the interaction include an increase in GTP cyclohydrolase activity, with concomitant protection from end-product feedback inhibition. In vivo, this effect would in turn provide sufficient cofactor when demand for catecholamine synthesis is greatest. The activity of tyrosine hydroxylase is also increased by this interaction, in excess of the stimulation resulting from phosphorylation alone. Vmax is elevated, with no change in Km. These results demonstrate that these enzymes engage in mutual positive regulation.

New bone formation by murine osteoprogenitor cells cultured on corticocancellous allograft bone.

The gold standard for bone grafting in orthopedics is autograft, however autograft has a limited supply and is associated with significant morbidity at the harvest site. One alternative, allograft bone, provides an osteoconductive scaffold, is in less limited supply, and it does not require a harvest from the patient. However, allograft lacks both osteogenic cells and osteoinductive proteins that make autograft bone so advantageous. This study provides a model to investigate strategies for augmentation of corticocancellous allograft bone discs with bone marrow-derived osteoprogenitor cells (OPCs) plus exogenous growth factors in vitro. In this model, allograft bone discs were created by cutting 1-mm thick slices from the distal femur and proximal tibia of euthanized mice. The allografts were sterilized and scanned by micro-computed tomography (microCT) to provide the pre-culture graft volume and trabecular characteristics. The discs were then seeded with OPCs harvested from murine bone marrow. The seeded grafts were placed in organ culture until harvest, after which they were re-scanned by microCT and the data compared to the corresponding pre-culture data. In addition, bone morphogenetic protein-7 (BMP-7, also know as osteogenic protein-1 or OP-1), basic fibroblast growth factor (bFGF), and OP-1 combined with bFGF were added on a daily basis to the cultures. After final microCT scanning, all grafts were sectioned and evaluated histologically after hematoxylin and eosin (H&E) staining. microCT scans of cultured allografts with cells at 3, 5, and 9 weeks showed a time-dependent, statistically significant increase in bone volume. The trabecular thickness (Tb.Th.) of grafts, from both groups that were augmented with OP-1, showed a statistically significant increase in trabecular thickness of allografts with OPCs. These data suggest that bone marrow-derived OPCs adhere to, and produce, new bone on corticocancellous allograft in vitro. When exogenous OP-1 is added to this model, an increase in the production of bone onto the corticocancellous allograft bone disc is seen. This model allows for the investigation of the effects of multiple growth factors, and other interventions, on OPCs seeded onto allograft bone in vitro.

An in vivo murine model of continuous intramedullary infusion of polyethylene particles.

Wear debris affects both initial osseointegration and subsequent bone remodeling of total joint replacements (TJRs). To study the complex cascade associated with the continuous generation of particles, a robust animal model is essential. To date, an animal model that incorporates continuously delivered particles to an intramedullary orthopaedic implant has not been available. In this study, we successfully infused clinically relevant ultra high molecular weight polyethylene particles, previously isolated from joint simulator tests, to the intramedullary space of the mouse femur for 4 weeks using a subcutaneous osmotic pump. Reduction of bone volume following the 4-week infusion of UHMWPE was detected by microCT. UHMWPE particles also changed the level of Alkaline Phosphatase expression in the infused femurs. Continuous infusion of particles to the murine bone-implant interface simulated the clinical scenario of local polymer wear particle generation and delivery in humans and can be used to further study the biological processes associated with wear debris particles.

Controlled release of growth factors on allograft bone in vitro.

Allografts are important alternatives to autografts for treating defects after major bone loss. Bone growth factors have both local autocrine and paracrine effects and regulate the growth, proliferation, and differentiation of osteoprogenitor cells. To study the effects of prolonged, continuous, local delivery of growth factors on bone growth, we developed a new microelectromechanical system (MEMS) drug delivery device. Bone marrow cells from mice were seeded on mouse allograft discs and cultured in osteogenic media with osteogenic protein 1 (OP-1) and/or basic fibroblast growth factor (FGF-2) delivered from MEMS devices for 6 weeks. We monitored bone formation by changes of bone volume using micro-CT scanning and release of osteocalcin using ELISA. The data suggest the MEMS devices delivered constant concentrations of OP-1 and FGF-2 to the media. Bone marrow cells grew on the allografts and increased bone volume. Addition of OP-1 increased bone formation whereas FGF-2 decreased bone formation. Local delivery of growth factors over a prolonged period modulated the differentiation of osteoprogenitor cells on allograft bone.

Biodegradable micro-osmotic pump for long-term and controlled release of basic fibroblast growth factor.

Microelectromechanical system (MEMS) technology not only provides the possibility of integration of multiple functions but also enables more precise control of dosing of therapeutic agents when the therapeutic window is very limited. Local delivery of basic fibroblast growth factor (bFGF) over a specific dose and time course is critical for mesenchymal tissue regeneration. However, bFGF is degraded quickly in vivo and difficulty of controlling the dose level impedes its effective use in angiogenesis and tissue regeneration. We constructed biodegradable micro-osmotic pumps based on MEMS technology for long-term controlled release of bFGF. The devices were constructed by micro-molding and thermal assembly of 85/15 poly(L-lactide-co-glycolide) sheets. The release of bFGF was regulated at 40 ng/day for four weeks; bioactivity was assessed by monitoring the growth of 3T3 fibroblasts. The proposed devices can be further miniaturized and used for the delivery of multiple therapeutic agents at the individual releasing schedules.

Modulation of allograft incorporation by continuous infusion of growth factors over a prolonged duration in vivo.

Morselized cancellous allograft bone is frequently used in the reconstruction of bone defects in cases of revision total joint replacement, trauma, spine fusion and treated infection. However, the initial lack of viable bone cells in morselized allograft bone significantly slows the process of graft incorporation compared to autograft bone. This study examined the effects of prolonged local infusion of the growth factors bone morphogenic protein-7 (BMP-7 or OP-1) and fibroblast growth factor-2 (FGF-2 or basic FGF) in the process of allograft incorporation using a rabbit tibial chamber model. New bone formation was evaluated by two indices, the activity of alkaline phosphatase and the level of birefringence. The markers of osteoclast-like cells were also measured. Without the infusion of the growth factors, lower levels of new bone formation were observed in the allograft group, compared to the autograft group. Infusion of growth factors FGF-2 and OP-1, singly or in combination, for 4 weeks, diminished this difference. The numbers of osteoclast-like cells were much higher in the allograft group before the growth factors were delivered. The infusion of FGF, singly, diminished this difference. However, the infusion of OP-1 or the combination of FGF and OP-1 did not decrease the number of osteoclast-like cells to a level comparable to autograft only. Local infusion of growth factors appears to be a useful adjunct to promote the incorporation of allograft bone in vivo.

The sequential expression profiles of growth factors from osteoprogenitors [correction of osteroprogenitors] to osteoblasts in vitro.

In this study, we delineate the sequential expression of selected growth factors associated with bone formation in vitro. Mineralization, osteocalcin, and alkaline phosphatase (ALP-2) were measured to monitor the differentiation and maturation of osteoprogenitor cells collected from C57BL mice. Bone-related growth factors, including transforming growth factor beta (TGF-beta), fibroblast growth factor 2 (FGF-2), platelet-derived growth factor (PDGF), insulinlike growth factor (IGF)-1, vascular endothelial growth factor (VEGF), bone morphogenetic protein (BMP)-2, and BMP-7, were selected. Enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR) were used to measure growth factors at the protein and messenger ribonucleic acid (mRNA) level, respectively. The results found that ALP-2 expression increased progressively over time, whereas mineralization and osteocalcin did not become evident until culture day 14. VEGF and IGF-1 were upregulated early during proliferation. PDGF and TGF-beta mRNA expression was bimodal. FGF-2 and BMP-2 mRNAs were expressed only later in differentiation. FGF-2 mRNA signal levels were highest at day 14 and remained prominent through day 28 of culture. BMP-2 showed a similar profile as FGF-2. BMP-7 was not detectable using RT-PCR or ELISA. Strong correlations existed for the expression patterns between several early-response growth factors (VEGF, TGF-beta, and IGF-1) and were also evident for several late-response growth factors (BMP-2, PDGF, and FGF-2). Differential expression for grouped sets of growth factors occurs during the temporal acquisition of bone-specific markers as osteoprogenitor cell maturation proceeds in vitro.

A typical N-terminal extensions confer novel regulatory properties on GTP cyclohydrolase isoforms in Drosophila melanogaster.

The cofactor tetrahydrobiopterin plays critical roles in the modulation of the signaling molecules dopamine, serotonin, and nitric oxide. Deficits in cofactor synthesis have been associated with several human hereditary diseases. Responsibility for the regulation of cofactor pools resides with the first enzyme in its biosynthetic pathway, GTP cyclohydrolase I. Because organisms must be able to rapidly respond to environmental and developmental cues to adjust output of these signaling molecules, complex regulatory mechanisms are vital for signal modulation. Mammalian GTP cyclohydrolase is subject to end-product inhibition via an associated regulatory protein and to positive regulation via phosphorylation, although target residues are unknown. GTP cyclohydrolase is composed of a highly conserved homodecameric catalytic core and non-conserved N-terminal domains proposed to be regulatory sites. We demonstrate for the first time in any organism that the N-terminal arms of the protein serve regulatory functions. We identify two different modes of regulation of the enzyme mediated through the N-terminal domains. The first is end-product feedback inhibition, catalytically similar to that of the mammalian enzyme, except that feedback inhibition by the cofactor requires sequences in the N-terminal arms rather than a separate regulatory protein. The second is a novel inhibitory interaction between the N-terminal arms and the active sites, which can be alleviated through the phosphorylation of serine residues within the N termini. Both mechanisms allow for acute and highly responsive regulation of cofactor production as required by downstream signaling pathways.

Mitochondrial pathology and muscle and dopaminergic neuron degeneration caused by inactivation of Drosophila Pink1 is rescued by Parkin.

Mutations in Pink1, a gene encoding a Ser/Thr kinase with a mitochondrial-targeting signal, are associated with Parkinson's disease (PD), the most common movement disorder characterized by selective loss of dopaminergic neurons. The mechanism by which loss of Pink1 leads to neurodegeneration is not understood. Here we show that inhibition of Drosophila Pink1 (dPink1) function results in energy depletion, shortened lifespan, and degeneration of select indirect flight muscles and dopaminergic neurons. The muscle pathology was preceded by mitochondrial enlargement and disintegration. These phenotypes could be rescued by the wild type but not the pathogenic C-terminal deleted form of human Pink1 (hPink1). The muscle and dopaminergic phenotypes associated with dPink1 inactivation show similarity to that seen in parkin mutant flies and could be suppressed by the overexpression of Parkin but not DJ-1. Consistent with the genetic rescue results, we find that, in dPink1 RNA interference (RNAi) animals, the level of Parkin protein is significantly reduced. Together, these results implicate Pink1 and Parkin in a common pathway that regulates mitochondrial physiology and cell survival in Drosophila.