Hexokinase activity is required for recruitment of parkin to depolarized mitochondria.

Autosomal recessive parkinsonism genes contribute to maintenance of mitochondrial function. Two of these, PINK1 and parkin, act in a pathway promoting autophagic removal of depolarized mitochondria. Although recruitment of parkin to mitochondria is PINK1-dependent, additional components necessary for signaling are unclear. We performed a screen for endogenous modifiers of parkin recruitment to depolarized mitochondria and identified hexokinase 2 (HK2) as a novel modifier of depolarization-induced parkin recruitment. Hexose kinase activity was required for parkin relocalization, suggesting the effects are shared among hexokinases including the brain-expressed hexokinase 1 (HK1). Knockdown of both HK1 and HK2 led to a stronger block in parkin relocalization than either isoform alone, and expression of HK2 in primary neurons promoted YFP-parkin recruitment to depolarized mitochondria. Mitochondrial parkin recruitment was attenuated with AKT inhibition, which is known to modulate HK2 activity and mitochondrial localization. We, therefore, propose that Akt-dependent recruitment of hexokinases is a required step in the recruitment of parkin prior to mitophagy.

DJ-1 acts in parallel to the PINK1/parkin pathway to control mitochondrial function and autophagy.

Mutations in DJ-1, PINK1 (PTEN-induced putative kinase 1) and parkin all cause recessive parkinsonism in humans, but the relationships between these genes are not clearly defined. One event associated with loss of any of these genes is altered mitochondrial function. Recent evidence suggests that turnover of damaged mitochondria by autophagy might be central to the process of recessive parkinsonism. Here, we show that loss of DJ-1 leads to loss of mitochondrial polarization, fragmentation of mitochondria and accumulation of markers of autophagy (LC3 punctae and lipidation) around mitochondria in human dopaminergic cells. These effects are due to endogenous oxidative stress, as antioxidants will reverse all of them. Similar to PINK1 and parkin, DJ-1 also limits mitochondrial fragmentation in response to the mitochondrial toxin rotenone. Furthermore, overexpressed parkin will protect against loss of DJ-1 and, although DJ-1 does not alter PINK1 mitochondrial phenotypes, DJ-1 is still active against rotenone-induced damage in the absence of PINK1. None of the three proteins complex together using size exclusion chromatography. These data suggest that DJ-1 works in parallel to the PINK1/parkin pathway to maintain mitochondrial function in the presence of an oxidative environment.

GSK3 inhibition reduces ECM production and prevents age-related macular degeneration-like pathology.

Malattia Leventinese/Doyne Honeycomb Retinal Dystrophy (ML/DHRD) is an age-related macular degeneration (AMD)-like retinal dystrophy caused by an autosomal dominant R345W mutation in the secreted glycoprotein, fibulin-3 (F3). To identify new small molecules that reduce F3 production from retinal pigmented epithelium (RPE) cells, we knocked-in a luminescent peptide tag (HiBiT) into the endogenous F3 locus which enabled simple, sensitive, and high throughput detection of the protein. The GSK3 inhibitor, CHIR99021 (CHIR), significantly reduced F3 burden (expression, secretion, and intracellular levels) in immortalized RPE and non-RPE cells. Low-level, long-term CHIR treatment promoted remodeling of the RPE extracellular matrix (ECM), reducing sub-RPE deposit-associated proteins (e.g., amelotin, complement component 3, collagen IV, and fibronectin), while increasing RPE differentiation factors (e.g., tyrosinase, and pigment epithelium derived factor). In vivo, treatment of 8 mo R345W+/+ knockin mice with CHIR (25 mg/kg i.p., 1 mo) was well tolerated and significantly reduced R345W F3-associated AMD-like basal laminar deposit number and size, thereby preventing the main pathological feature in these mice. This is the first demonstration of small molecule-based prevention of AMD-like pathology in ML/DHRD mice and may herald a rejuvenation of interest in GSK3 inhibition for the treatment of neurodegenerative diseases, including, potentially AMD itself.

Small Molecule-mediated Insulin Hypersecretion Induces Transient ER Stress Response and Loss of Beta Cell Function.

Pancreatic islet beta cells require a fine-tuned endoplasmic reticulum (ER) stress response for normal function; abnormal ER stress contributes to diabetes pathogenesis. Here, we identified a small molecule, SW016789, with time-dependent effects on beta cell ER stress and function. Acute treatment with SW016789 potentiated nutrient-induced calcium influx and insulin secretion, while chronic exposure to SW016789 transiently induced ER stress and shut down secretory function in a reversible manner. Distinct from the effects of thapsigargin, SW016789 did not affect beta cell viability or apoptosis, potentially due to a rapid induction of adaptive genes, weak signaling through the eIF2α kinase PERK, and lack of oxidative stress gene Txnip induction. We determined that SW016789 acted upstream of voltage-dependent calcium channels (VDCCs) and potentiated nutrient- but not KCl-stimulated calcium influx. Measurements of metabolomics, oxygen consumption rate, and G protein-coupled receptor signaling did not explain the potentiating effects of SW016789. In chemical cotreatment experiments, we discovered synergy between SW016789 and activators of protein kinase C and VDCCs, suggesting involvement of these pathways in the mechanism of action. Finally, chronically elevated calcium influx was required for the inhibitory impact of SW016789, as blockade of VDCCs protected human islets and MIN6 beta cells from hypersecretion-induced dysfunction. We conclude that beta cells undergoing this type of pharmacological hypersecretion have the capacity to suppress their function to mitigate ER stress and avoid apoptosis. These results have the potential to uncover beta cell ER stress mitigation factors and add support to beta cell rest strategies to preserve function.

Regulator of G-protein signaling 10 promotes dopaminergic neuron survival via regulation of the microglial inflammatory response.

Epidemiological studies suggest that chronic use of nonsteroidal anti-inflammatory drugs lowers the incidence of Parkinson's disease (PD) in humans and implicate neuroinflammatory processes in the death of dopamine (DA) neurons. Here, we demonstrate that regulator of G-protein signaling 10 (RGS10), a microglia-enriched GAP (GTPase accelerating protein) for Galpha subunits, is an important regulator of microglia activation. Flow-cytometric and immunohistochemical analyses indicated that RGS10-deficient mice displayed increased microglial burden in the CNS, and exposure to chronic systemic inflammation induced nigral DA neuron loss measured by unbiased stereology. Primary microglia isolated from brains of RGS10-deficient mice displayed dysregulated inflammation-related gene expression profiles under basal and stimulated conditions in vitro compared with that of primary microglia isolated from wild-type littermates. Similarly, knockdown of RGS10 in the BV2 microglia cell line resulted in dysregulated inflammation-related gene expression, overproduction of tumor necrosis factor (TNF), and enhanced neurotoxic effects of BV2 microglia on the MN9D dopaminergic cell line that could be blocked by addition of the TNF decoy receptor etanercept. Importantly, ablation of RGS10 in MN9D dopaminergic cells further enhanced their vulnerability to microglial-derived death-inducing inflammatory mediators, suggesting a role for RGS10 in modulating the sensitivity of dopaminergic neurons against inflammation-mediated cell death. Together, our findings indicate that RGS10 limits microglial-derived TNF secretion and regulates the functional outcome of inflammatory stimuli in the ventral midbrain. RGS10 emerges as a novel drug target for prevention of nigrostriatal pathway degeneration, the neuropathological hallmark of PD.

Intranigral lentiviral delivery of dominant-negative TNF attenuates neurodegeneration and behavioral deficits in hemiparkinsonian rats.

Neuroinflammatory processes have been implicated in the progressive loss of ventral midbrain dopaminergic (DA) neurons that give rise to Parkinson's disease (PD), a late-onset movement disorder that affects 2% of the population over the age of 70 years. We have shown earlier, in two rat models of PD, that inhibition of the proinflammatory cytokine tumor necrosis factor (TNF) through nigral infusion of dominant-negative (DN-TNF) protein (XENP345) attenuates DA neuron loss. The objectives of this study were to develop a constitutive lentiviral vector encoding dominate-negative TNF, and to determine whether a gene therapy approach to deliver DN-TNF directly into the rodent substantia nigra could prevent or attenuate neurotoxin-induced DA neuron loss and associated behavioral deficits. Here we demonstrate that a single injection of lentivirus-expressing DN-TNF into rat substantia nigra, administered concomitant with a striatal 6-hydroxydopamine lesion, results in sufficiently high expression of inhibitor in vivo to attenuate both DA neuron loss and behavioral deficits resulting from striatal dopamine depletion. Our findings demonstrate the feasibility and efficacy of dominant-negative TNF gene transfer as a novel neuroprotective strategy to prevent or delay nigrostriatal pathway degeneration. This strategy holds the potential for therapeutic application in the treatment of PD.

TNF signaling inhibition in the CNS: implications for normal brain function and neurodegenerative disease.

The role of tumor necrosis factor (TNF) as an immune mediator has long been appreciated but its function in the brain is still unclear. TNF receptor 1 (TNFR1) is expressed in most cell types, and can be activated by binding of either soluble TNF (solTNF) or transmembrane TNF (tmTNF), with a preference for solTNF; whereas TNFR2 is expressed primarily by microglia and endothelial cells and is preferentially activated by tmTNF. Elevation of solTNF is a hallmark of acute and chronic neuroinflammation as well as a number of neurodegenerative conditions including ischemic stroke, Alzheimer's (AD), Parkinson's (PD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). The presence of this potent inflammatory factor at sites of injury implicates it as a mediator of neuronal damage and disease pathogenesis, making TNF an attractive target for therapeutic development to treat acute and chronic neurodegenerative conditions. However, new and old observations from animal models and clinical trials reviewed here suggest solTNF and tmTNF exert different functions under normal and pathological conditions in the CNS. A potential role for TNF in synaptic scaling and hippocampal neurogenesis demonstrated by recent studies suggest additional in-depth mechanistic studies are warranted to delineate the distinct functions of the two TNF ligands in different parts of the brain prior to large-scale development of anti-TNF therapies in the CNS. If inactivation of TNF-dependent inflammation in the brain is warranted by additional pre-clinical studies, selective targeting of TNFR1-mediated signaling while sparing TNFR2 activation may lessen adverse effects of anti-TNF therapies in the CNS.

The synthetic triterpenoid CDDO-methyl ester modulates microglial activities, inhibits TNF production, and provides dopaminergic neuroprotection.

BACKGROUND: Recent animal and human studies implicate chronic activation of microglia in the progressive loss of CNS neurons. The inflammatory mechanisms that have neurotoxic effects and contribute to neurodegeneration need to be elucidated and specifically targeted without interfering with the neuroprotective effects of glial activities. Synthetic triterpenoid analogs of oleanolic acid, such as methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me, RTA 402) have potent anti-proliferative and differentiating effects on tumor cells, and anti-inflammatory activities on activated macrophages. We hypothesized that CDDO-Me may be able to suppress neurotoxic microglial activities while enhancing those that promote neuronal survival. Therefore, the aims of our study were to identify specific microglial activities modulated by CDDO-Me in vitro, and to determine the extent to which this modulation affords neuroprotection against inflammatory stimuli. METHODS: We tested the synthetic triterpenoid methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me, RTA 402) in various in vitro assays using the murine BV2 microglia cell line, mouse primary microglia, or mouse primary peritoneal macrophages to investigate its effects on proliferation, inflammatory gene expression, cytokine secretion, and phagocytosis. The antioxidant and neuroprotective effects of CDDO-Me were also investigated in primary neuron/glia cultures from rat basal forebrain or ventral midbrain. RESULTS: We found that at low nanomolar concentrations, treatment of rat primary mesencephalon neuron/glia cultures with CDDO-Me resulted in attenuated LPS-, TNF- or fibrillar amyloid beta 1-42 (A beta 1-42) peptide-induced increases in reactive microglia and inflammatory gene expression without an overall effect on cell viability. In functional assays CDDO-Me blocked death in the dopaminergic neuron-like cell line MN9D induced by conditioned media (CM) of LPS-stimulated BV2 microglia, but did not block cell death induced by addition of TNF to MN9D cells, suggesting that dopaminergic neuroprotection by CDDO-Me involved inhibition of microglial-derived cytokine production and not direct inhibition of TNF-dependent pro-apoptotic pathways. Multiplexed immunoassays of CM from LPS-stimulated microglia confirmed that CDDO-Me-treated BV2 cells produced decreased levels of specific subsets of cytokines, in particular TNF. Lastly, CDDO-Me enhanced phagocytic activity of BV2 cells in a stimulus-specific manner but inhibited generation of reactive oxygen species (ROS) in mixed neuron/glia basal forebrain cultures and dopaminergic cells. CONCLUSION: The neuroimmune modulatory properties of CDDO-Me indicate that this potent antioxidant and anti-inflammatory compound may have therapeutic potential to modify the course of neurodegenerative diseases characterized by chronic neuroinflammation and amyloid deposition. The extent to which synthetic triterpenoids afford therapeutic benefit in animal models of Parkinson's and Alzheimer's disease deserves further investigation.

Neuroinflammation in Parkinson's disease: is there sufficient evidence for mechanism-based interventional therapy?

The inflammatory response in the brain associated with most chronic neurodegenerative diseases is termed neuroinflammation. Neuropathological and neuroradiological studies indicate that in certain neurodegenerative disorders neuroinflammation may be detectable years before significant loss of neurons occurs. In this review, we discuss the evidence from human studies and experimental models that implicate neuroinflammatory processes in the progressive neurodegeneration of the nigrostriatal pathway, the hallmark of Parkinson's Disease (PD). We discuss the neurotoxic role of microglia-derived inflammatory mediators which are suspected to hasten the death of nigral dopaminergic neurons, in particular the pro-inflammatory cytokine Tumor Necrosis Factor (TNF) and its downstream signaling pathways. We also entertain the possibility that chronic microglia activation links proteinopathies to neurodegeneration. The rationale for current and future use of anti-inflammatory approaches to protect vulnerable neuronal populations in PD is also reviewed.

Blocking soluble tumor necrosis factor signaling with dominant-negative tumor necrosis factor inhibitor attenuates loss of dopaminergic neurons in models of Parkinson's disease.

The mechanisms that trigger or contribute to loss of dopaminergic (DA) neurons in Parkinson's disease (PD) remain unclear and controversial. Elevated levels of tumor necrosis factor (TNF) in CSF and postmortem brains of PD patients and animal models of PD implicate this proinflammatory cytokine in the pathophysiology of the disease; but a role for TNF in mediating loss of DA neurons in PD has not been clearly demonstrated. Here, we report that neutralization of soluble TNF (solTNF) in vivo with the engineered dominant-negative TNF compound XENP345 (a PEGylated version of the TNF variant A145R/I97T) reduced by 50% the retrograde nigral degeneration induced by a striatal injection of the oxidative neurotoxin 6-hydroxydopamine (6-OHDA). XENP345 was neuroprotective only when infused into the nigra, not the striatum. XENP345/6-OHDA rats displayed attenuated amphetamine-induced rotational behavior, indicating preservation of striatal dopamine levels. Similar protective effects were observed with chronic in vivo coinfusion of XENP345 with bacterial lipopolysaccharide (LPS) into the substantia nigra, confirming a role for solTNF-dependent neuroinflammation in nigral degeneration. In embryonic rat midbrain neuron/glia cell cultures exposed to LPS, even delayed administration of XENP345 prevented selective degeneration of DA neurons despite sustained microglia activation and secretion of solTNF. XENP345 also attenuated 6-OHDA-induced DA neuron toxicity in vitro. Collectively, our data demonstrate a role for TNF in vitro and in vivo in two models of PD, and raise the possibility that delaying the progressive degeneration of the nigrostriatal pathway in humans is therapeutically feasible with agents capable of blocking solTNF in early stages of PD.

Insulin promoter-driven Gaussia luciferase-based insulin secretion biosensor assay for discovery of ß-cell glucose-sensing pathways.

High throughput screening of insulin secretion is intractable with current methods. We developed a secreted insulin-luciferase system (Ins-GLuc) in ß cells that is rapid, inexpensive, and amenable to 96- and 384-well formats. We treated stable Ins-GLuc-expressing MIN6 cells overnight with 6298 marine natural product fractions. The cells were then washed to remove media and chemicals, followed by stimulation with glucose in the diazoxide paradigm. These conditions allowed the discovery of many insulin secretion suppressors and potentiators. The mechanisms of action of these natural products must be long-lasting given the continuance of secretory phenotypes in the absence of chemical treatment. We anticipate that these natural products and their target pathways will lead to a greater understanding of glucose-stimulated insulin secretion.

mRNA expression, splicing and editing in the embryonic and adult mouse cerebral cortex.

The complexity of the adult brain is a result of both developmental processes and experience-dependent circuit formation. One way to look at the differences between embryonic and adult brain is to examine gene expression. Previous studies have used microarrays to address this in a global manner. However, the transcriptome is more complex than gene expression levels alone, as alternative splicing and RNA editing generate a diverse set of mature transcripts. Here we report a high-resolution transcriptome data set of mouse cerebral cortex at embryonic and adult stages using RNA sequencing (RNA-Seq). We found many differences in gene expression, splicing and RNA editing between embryonic and adult cerebral cortex. Each data set was validated technically and biologically, and in each case we found our RNA-Seq observations to have predictive validity. We provide this data set and analysis as a resource for understanding gene expression in the embryonic and adult cerebral cortex.

Mitochondrial quality control and dynamics in Parkinson's disease.

SIGNIFICANCE: Studies of sporadic cases, toxin models, and genetic causes of Parkinson's disease suggest that mitochondrial dysfunction may be an early feature of pathogenesis. RECENT ADVANCES: Compelling evidence of a causal relationship between mitochondrial function and disease was found with the identification of several genes for recessive parkinsonism, PINK1, DJ-1, and parkin. There is evidence that each of these regulates responses to cellular stresses, including oxidative stress and depolarization of the mitochondrial membrane. Specifically, PINK1 and parkin modulate mitochondrial dynamics by promoting autophagic removal of depolarized mitochondria. Mutations in all genes linked to Parkinson's disease lead to enhanced sensitivity to mitochondrial toxins and oxidative stress. CRITICAL ISSUES: Both increased mitochondrial damage due to complex 1 inhibition, mishandling of calcium, oxidant stress, or impaired clearance of dysfunctional mitochondria would lead to the accumulation of nonfunctional organelles and could contribute to neuronal dysfunction. However, several unanswered questions remain about the underlying mechanism(s) involved. FUTURE DIRECTIONS: PINK1 and parkin have been demonstrated to regulate mitochondrial dynamics, but the pathways linking PINK1 activity to parkin function are still unclear and warrant further investigation.

DJ-1 regulation of mitochondrial function and autophagy through oxidative stress.

The dysregulation of mitochondrial function has been implicated in the pathogenesis of Parkinson disease. Mutations in the parkin, PINK1 and DJ-1 genes all result in recessive parkinsonism. Although the protein products of these genes have not been fully characterized, it has been established that all three contribute to the maintenance of mitochondrial function. PINK1 and parkin act in a common pathway to regulate the selective autophagic removal of depolarized mitochondria, but the relationship between DJ-1 and PINK1- and/or parkin-mediated effects on mitochondria and autophagy is less clear. We have shown that loss of DJ-1 leads to mitochondrial phenotypes including reduced membrane potential, increased fragmentation and accumulation of autophagic markers. Supplementing DJ-1-deficient cells with glutathione reverses both mitochondrial and autophagic changes suggesting that DJ-1 may act to maintain mitochondrial function during oxidative stress and thereby alter mitochondrial dynamics and autophagy indirectly.

Autologous transplants of Adipose-Derived Adult Stromal (ADAS) cells afford dopaminergic neuroprotection in a model of Parkinson's disease.

Adult adipose contains stromal progenitor cells with neurogenic potential. However, the stability of neuronal phenotypes adopted by Adipose-Derived Adult Stromal (ADAS) cells and whether terminal neuronal differentiation is required for their consideration as alternatives in cell replacement strategies to treat neurological disorders is largely unknown. We investigated whether in vitro neural induction of ADAS cells determined their ability to neuroprotect or restore function in a lesioned dopaminergic pathway. In vitro-expanded naïve or differentiated ADAS cells were autologously transplanted into substantia nigra 1 week after an intrastriatal 6-hydroxydopamine injection. Neurochemical and behavioral measures demonstrated neuroprotective effects of both ADAS grafts against 6-hydroxydopamine-induced dopaminergic neuron death, suggesting that pre-transplantation differentiation of the cells does not determine their ability to survive or neuroprotect in vivo. Therefore, we investigated whether equivalent protection by naïve and neurally-induced ADAS grafts resulted from robust in situ differentiation of both graft types into dopaminergic fates. Immunohistological analyses revealed that ADAS cells did not adopt dopaminergic cell fates in situ, consistent with the limited ability of these cells to undergo terminal differentiation into electrically active neurons in vitro. Moreover, re-exposure of neurally-differentiated ADAS cells to serum-containing medium in vitro confirmed ADAS cell phenotypic instability (plasticity). Lastly, given that gene expression analyses of in vitro-expanded ADAS cells revealed that both naïve and differentiated ADAS cells express potent dopaminergic survival factors, ADAS transplants may have exerted neuroprotective effects by production of trophic factors at the lesion site. ADAS cells may be ideal for ex vivo gene transfer therapies in Parkinson's disease treatment.

Neuroinflammatory mechanisms in Parkinson's disease: potential environmental triggers, pathways, and targets for early therapeutic intervention.

Most acute and chronic neurodegenerative conditions are accompanied by neuroinflammation; yet the exact nature of the inflammatory processes and whether they modify disease progression is not well understood. In this review, we discuss the key epidemiological, clinical, and experimental evidence implicating inflammatory processes in the progressive degeneration of the dopaminergic (DA) nigrostriatal pathway and their potential contribution to the pathophysiology of Parkinson's disease (PD). Given that interplay between genetics and environment are likely to contribute to risk for development of idiopathic PD, recent data showing interactions between products of genes linked to heritable PD that function to protect DA neurons against oxidative or proteolytic stress and inflammation pathways will be discussed. Cellular mechanisms activated or enhanced by inflammatory processes that may contribute to mitochondrial dysfunction, oxidative stress, or apoptosis of dopaminergic (DA) neurons will be reviewed, with special emphasis on tumor necrosis factor (TNF) and interleukin-1-beta (IL-1beta) signaling pathways. Epigenetic factors which have the potential to trigger neuroinflammation, including environmental exposures and age-associated chronic inflammatory conditions, will be discussed as possible 'second-hit' triggers that may affect disease onset or progression of idiopathic PD. If inflammatory processes have an active role in nigrostriatal pathway degeneration, then evidence should exist to indicate that such processes begin in the early stages of disease and that they contribute to neuronal dysfunction and/or hasten neurodegeneration of the nigrostriatal pathway. Therapeutically, if anti-inflammatory interventions can be shown to rescue nigral DA neurons from degeneration and lower PD risk, then timely use of anti-inflammatory therapies should be investigated further in well-designed clinical trials for their ability to prevent or delay the progressive loss of nigral DA neurons in genetically susceptible populations.