Short-term exposure to HIV Tat induces glial activation and changes in perineuronal nets.

Despite widespread use of combination antiretroviral therapy (cART), there remains a subset of individuals who display cognitive impairment broadly known as HIV-associated neurocognitive disorder (HAND). Interestingly, HIV-infected cells continuously release the HIV-1 protein Tat even in the presence of cART. Persistent exposure to Tat is proposed to increase both neuroinflammation and neurotoxicity. In vitro evidence shows that matrix metalloproteinases (MMPs) are among the neuroinflammatory molecules induced by Tat, which are known to disrupt specialized neuronal extracellular matrix structures called perineuronal nets (PNNs). PNNs predominantly surround parvalbumin interneurons and help to buffer these cells from oxidant stress and to independently increase their excitability. In order to better understand the link between short-term exposure to Tat, neuroinflammation, and PNNs, we explored the direct effects of Tat on glial cells and neurons. Herein, we report that in mixed glial cultures, Tat directly increases the expression of proinflammatory molecules, including MMP-9. Moreover, direct injection of Tat protein into mouse hippocampus increases the expression of astrocyte and microglia markers as well as MMP-9. The number of PNNs is decreased following Tat exposure, followed later by decreased numbers of hippocampal parvalbumin-expressing neurons. In older mice, Tat induced significant increases in the gene expression of proinflammatory molecules including markers of gliosis, MMPs and complement system proteins. Taken together, these data support a direct effect of Tat on glial-derived MMP expression subsequently affecting PNNs and neuronal health, with older mice more susceptible to Tat-induced inflammation.

Jedi-1/MEGF12-mediated phagocytosis controls the pro-neurogenic properties of microglia in the ventricular-subventricular zone.

Microglia are the primary phagocytes in the central nervous system and are responsible for clearing dead cells generated during development or disease. The phagocytic process shapes the phenotype of the microglia, which affects the local environment. A unique population of microglia reside in the ventricular-subventricular zone (V-SVZ) of neonatal mice, but how they influence this neurogenic niche is not well-understood. Here, we demonstrate that phagocytosis creates a pro-neurogenic microglial phenotype in the V-SVZ and that these microglia phagocytose apoptotic cells via the engulfment receptor Jedi-1. Deletion of Jedi-1 decreases apoptotic cell clearance, triggering the development of a neuroinflammatory phenotype, reminiscent of neurodegenerative and-age-associated microglia, that reduces neural precursor proliferation via elevated interleukin (IL)-1ß signaling; inhibition of IL-1 receptor rescues precursor proliferation in vivo. Together, these results reveal a critical role for Jedi-1 in connecting microglial phagocytic activity to a phenotype that promotes neurogenesis in the developing V-SVZ.

Polychlorinated biphenyls induce oxidative stress and metabolic responses in astrocytes.

Exposure to environmental toxicants is prevalent, hazardous and linked to varied detrimental health outcomes and disease. Polychlorinated biphenyls (PCBs), a class of hazardous organic chlorines once widely used for industrial purposes, are associated with neurodegenerative disease and oxidative stress in both in vitro and in vivo models. Here, we investigated the impact of Aroclor 1254, a commercially available PCB mixture, on primary murine astrocytes to determine the response to this once ubiquitously used toxicant on the most numerous cells of the central nervous system (CNS). Astrocytes are a critical component of homeostasis throughout the CNS, including at the blood-brain barrier, where they serve as the primary defense against xenobiotics entering the CNS, and at the synapse, where they are closely coupled to neurons through several metabolic pathways. We hypothesized that PCBs cause astrocytic oxidative stress and related dysfunction including altered metabolism. We exposed primary murine cortical astrocytes to PCBs and report an increased expression of antioxidant genes (Prdx1, Gsta2, Gfap, Amigo2) in response to oxidative stress. Our data show increased ATP production and spare respiratory capacity in astrocytes exposed to 10 µM (∼ 3 ppm) PCBs. This dose also causes an increase in glucose uptake that is not seen at a higher dose (50 µM) suggesting that, at a lower dose, astrocytes are able to engage compensatory mechanisms to promote survival. Together, these data suggest that exposure to PCBs impact astrocytic metabolism, which is important to consider both in the context of human health and disease and in in vitro and in vivo disease models.

Environmental toxicants in the brain: A review of astrocytic metabolic dysfunction.

Exposure to environmental toxicants is linked to long-term adverse outcomes in the brain and involves the dysfunction of glial and neuronal cells. Astrocytes, the most numerous cell type, are increasingly implicated in the pathogenesis of many diseases of the central nervous system, including neurodegenerative diseases. Astrocytes are critical for proper brain function in part due to their robust antioxidant and unique metabolic capabilities. Additionally, astrocytes are positioned both at the blood-brain barrier, where they are the primary responders to xenobiotic penetrance of the CNS, and at synapses where they are in close contact with neurons and synaptic machinery. While exposure to several classes of environmental toxicants, including chlorinated and fluorinated compounds, and trace metals, have been implicated in neurodegenerative diseases, their impact on astrocytes represents an important and growing field of research. Here, we review existing literature focused on the impact of a range of synthetic compounds on astrocytic function. We focus specifically on perturbed metabolic processes in response to these compounds and consider how perturbation of these pathways impacts disease pathogenesis.

HIV-1 Tat promotes astrocytic release of CCL2 through MMP/PAR-1 signaling.

The HIV-1 protein Tat is continually released by HIV-infected cells despite effective combination antiretroviral therapies (cART). Tat promotes neurotoxicity through enhanced expression of proinflammatory molecules from resident and infiltrating immune cells. These molecules include matrix metalloproteinases (MMPs), which are pathologically elevated in HIV, and are known to drive central nervous system (CNS) injury in varied disease settings. A subset of MMPs can activate G-protein coupled protease-activated receptor 1 (PAR-1), a receptor that is highly expressed on astrocytes. Although PAR-1 expression is increased in HIV-associated neurocognitive disorder (HAND), its role in HAND pathogenesis remains understudied. Herein, we explored Tat's ability to induce expression of the PAR-1 agonists MMP-3 and MMP-13. We also investigated MMP/PAR-1-mediated release of CCL2, a chemokine that drives CNS entry of HIV infected monocytes and remains a significant correlate of cognitive dysfunction in the era of cART. Tat exposure significantly increased the expression of MMP-3 and MMP-13. These PAR-1 agonists both stimulated the release of astrocytic CCL2, and both genetic knock-out and pharmacological inhibition of PAR-1 reduced CCL2 release. Moreover, in HIV-infected post-mortem brain tissue, within-sample analyses revealed a correlation between levels of PAR-1-activating MMPs, PAR-1, and CCL2. Collectively, these findings identify MMP/PAR-1 signaling to be involved in the release of CCL2, which may underlie Tat-induced neuroinflammation.

Toll-Like Receptor 2 Signaling and Current Approaches for Therapeutic Modulation in Synucleinopathies.

The innate immune response in the central nervous system (CNS) is implicated as both beneficial and detrimental to health. Integral to this process are microglia, the resident immune cells of the CNS. Microglia express a wide variety of pattern-recognition receptors, such as Toll-like receptors, that detect changes in the neural environment. The activation of microglia and the subsequent proinflammatory response has become increasingly relevant to synucleinopathies, including Parkinson's disease the second most prevalent neurodegenerative disease. Within these diseases there is evidence of the accumulation of endogenous α-synuclein that stimulates an inflammatory response from microglia via the Toll-like receptors. There have been recent developments in both new and old pharmacological agents designed to target microglia and curtail the inflammatory environment. This review will aim to delineate the process of microglia-mediated inflammation and new therapeutic avenues to manage the response.

Inflammation alters AMPA-stimulated calcium responses in dorsal striatal D2 but not D1 spiny projection neurons.

Neuroinflammation precedes neuronal loss in striatal neurodegenerative diseases and can be exacerbated by the release of proinflammatory molecules by microglia. These molecules can affect trafficking of AMPARs. The preferential trafficking of calcium-permeable versus impermeable AMPARs can result in disruptions of [Ca2+ ]i and alter cellular functions. In striatal neurodegenerative diseases, changes in [Ca2+ ]i and L-type voltage-gated calcium channels (VGCCs) have been reported. Therefore, this study sought to determine whether a proinflammatory environment alters AMPA-stimulated [Ca2+ ]i through calcium-permeable AMPARs and/or L-type VGCCs in dopamine-2- and dopamine-1-expressing striatal spiny projection neurons (D2 and D1 SPNs) in the dorsal striatum. Mice expressing the calcium indicator protein, GCaMP in D2 or D1 SPNs, were utilized for calcium imaging. Microglial activation was assessed by morphology analyses. To induce inflammation, acute mouse striatal slices were incubated with lipopolysaccharide (LPS). Here we report that LPS treatment potentiated AMPA responses only in D2 SPNs. When a nonspecific VGCC blocker was included, we observed a decrease of AMPA-stimulated calcium fluorescence in D2 but not D1 SPNs. The remaining agonist-induced [Ca2+ ]i was mediated by calcium-permeable AMPARs because the responses were completely blocked by a selective calcium-permeable AMPAR antagonist. We used isradipine, the highly selective L-type VGCC antagonist to determine the role of L-type VGCCs in SPNs treated with LPS. Isradipine decreased AMPA-stimulated responses selectively in D2 SPNs after LPS treatment. Our findings suggest that dorsal striatal D2 SPNs are specifically targeted in proinflammatory conditions and that L-type VGCCs and calcium-permeable AMPARs are important mediators of this effect.

Protease induced plasticity: matrix metalloproteinase-1 promotes neurostructural changes through activation of protease activated receptor 1.

Matrix metalloproteinases (MMPs) are a family of secreted endopeptidases expressed by neurons and glia. Regulated MMP activity contributes to physiological synaptic plasticity, while dysregulated activity can stimulate injury. Disentangling the role individual MMPs play in synaptic plasticity is difficult due to overlapping structure and function as well as cell-type specific expression. Here, we develop a novel system to investigate the selective overexpression of a single MMP driven by GFAP expressing cells in vivo. We show that MMP-1 induces cellular and behavioral phenotypes consistent with enhanced signaling through the G-protein coupled protease activated receptor 1 (PAR1). Application of exogenous MMP-1, in vitro, stimulates PAR1 dependent increases in intracellular Ca2+ concentration and dendritic arborization. Overexpression of MMP-1, in vivo, increases dendritic complexity and induces biochemical and behavioral endpoints consistent with increased GPCR signaling. These data are exciting because we demonstrate that an astrocyte-derived protease can influence neuronal plasticity through an extracellular matrix independent mechanism.

Activation of MyD88-dependent TLR1/2 signaling by misfolded α-synuclein, a protein linked to neurodegenerative disorders.

Synucleinopathies, such as Parkinson's disease and diffuse Lewy body disease, are progressive neurodegenerative disorders characterized by selective neuronal death, abnormal accumulation of misfolded α-synuclein, and sustained microglial activation. In addition to inducing neuronal toxicity, higher-ordered oligomeric α-synuclein causes proinflammatory responses in the brain parenchyma by triggering microglial activation, which may exacerbate pathogenic processes by establishing a chronic neuroinflammatory milieu. We found that higher-ordered oligomeric α-synuclein induced a proinflammatory microglial phenotype by directly engaging the heterodimer TLR1/2 (Toll-like receptor 1 and 2) at the cell membrane, leading to the nuclear translocation of NF-κB (nuclear factor κB) and the increased production of the proinflammatory cytokines TNF-α (tumor necrosis factor-α) and IL-1ß (interleukin-1ß) in a MyD88-dependent manner. Blocking signaling through the TLR1/2 heterodimer with the small-molecule inhibitor CU-CPT22 reduced the nuclear translocation of NF-κB and secretion of TNF-α from cultured primary mouse microglia. Candesartan cilexetil, a drug approved for treating hypertension and that inhibits the expression of TLR2, reversed the activated proinflammatory phenotype of primary microglia exposed to oligomeric α-synuclein, supporting the possibility of repurposing this drug for synucleinopathies.

From student to steward: the Interdisciplinary Program in Neuroscience at Georgetown University as a case study in professional development during doctoral training.

A key facet of professional development is the formation of professional identity. At its most basic level, professional identity for a scientist centers on mastery of a discipline and the development of research skills during doctoral training. To develop a broader understanding of professional identity in the context of doctoral training, the Carnegie Initiative on the Doctorate (CID) ran a multi-institutional study from 2001 to 2005. A key outcome of the CID was the development of the concept of 'stewards of the discipline'. The Interdisciplinary Program in Neuroscience (IPN) at Georgetown University participated in CID from 2003 to 2005. Here, we describe the IPN and highlight the programmatic developments resulting from participation in the CID. In particular, we emphasize programmatic activities that are designed to promote professional skills in parallel with scientific development. We describe activities in the domains of leadership, communication, teaching, public outreach, ethics, collaboration, and mentorship. Finally, we provide data that demonstrate that traditional metrics of academic success are not adversely affected by the inclusion of professional development activities in the curricula. By incorporating these seven 'professional development' activities into the required coursework and dissertation research experience, the IPN motivates students to become stewards of the discipline.

The beneficial effect of a prolyl oligopeptidase inhibitor, KYP-2047, on alpha-synuclein clearance and autophagy in A30P transgenic mouse.

The misfolding and aggregation of α-synuclein (aSyn) eventually lead to an accumulation of toxic forms that disturb normal neuronal function and result in cell death. aSyn rich inclusions are seen in Parkinson's disease, dementia with Lewy bodies and other synucleinopathies. Prolyl oligopeptidase (PREP) can accelerate the aggregation process of aSyn and the inhibition of PREP leads to a decreased amount of aggregated aSyn in cell models and in aSyn transgenic mice. In this study, we investigated the effect of 5- and 28-day PREP inhibitor (KYP-2047) treatments on a mouse strain carrying a point mutation in the aSyn coding gene. Following PREP inhibition, we found a decrease in high molecular-weight oligomeric aSyn and a concomitant increase in the amount of the autophagosome marker, LC3BII, suggesting enhanced macroautophagy (autophagy) and aSyn clearance by KYP-2047. Moreover, 28-day treatment with KYP-2047 caused significant increases in striatal dopamine levels. In cell culture, overexpression of PREP reduced the autophagy. Furthermore, the inhibition of PREP normalized the changes on autophagy markers (LC3BII and p62) caused by an autophagy inhibition or aSyn overexpression, and induced the expression of beclin 1, a positive regulator of autophagy. Taken together, our results suggest that PREP inhibition accelerates the clearance of protein aggregates via increased autophagy and thus normalizes the cell functions in vivo and in vitro. Therefore, PREP inhibition may have future potential in the treatment of synucleinopathies.

Isolation of cortical microglia with preserved immunophenotype and functionality from murine neonates.

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-κB (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-α (TNF-α), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

Development of inducible leucine-rich repeat kinase 2 (LRRK2) cell lines for therapeutics development in Parkinson's disease.

The pathogenic mechanism(s) contributing to loss of dopamine neurons in Parkinson's disease (PD) remain obscure. Leucine-rich repeat kinase 2 (LRRK2) mutations are linked, as a causative gene, to PD. LRRK2 mutations are estimated to account for 10% of familial and between 1 % and 3 % of sporadic PD. LRRK2 proximate single nucleotide polymorphisms have also been significantly associated with idiopathic/sporadic PD by genome-wide association studies. LRRK2 is a multidomain-containing protein and belongs to the protein kinase super-family. We constructed two inducible dopaminergic cell lines expressing either human-LRRK2-wild-type or human-LRRK2-mutant (G2019S). Phenotypes of these LRRK2 cell lines were examined with respect to cell viability, morphology, and protein function with or without induction of LRRK2 gene expression. The overexpression of G2019S gene promoted (1) low cellular metabolic activity without affecting cell viability, (2) blunted neurite extension, and (3) increased phosphorylation at S910 and S935. Our observations are consistent with reported general phenotypes in LRRK2 cell lines by other investigators. We used these cell lines to interrogate the biological function of LRRK2, to evaluate their potential as a drug-screening tool, and to investigate screening for small hairpin RNA-mediated LRRK2 G2019S gene knockdown as a potential therapeutic strategy. A proposed LRRK2 kinase inhibitor (i.e., IN-1) decreased LRRK2 S910 and S935 phosphorylation in our MN9DLRRK2 cell lines in a dose-dependent manner. Lentivirus-mediated transfer of LRRK2 G2019S allele-specific small hairpin RNA reversed the blunting of neurite extension caused by LRRK2 G2019S overexpression. Taken together, these inducible LRRK2 cell lines are suitable reagents for LRRK2 functional studies, and the screening of potential LRRK2 therapeutics.

Microglial activation and antioxidant responses induced by the Parkinson's disease protein α-synuclein.

Parkinson's disease (PD) is the second most common age-related neurodegenerative disorder typified by tremor, rigidity, akinesia and postural instability due in part to the loss of dopamine within the nigrostriatal system. The pathologic features of this disorder include the loss of substantia nigra dopamine neurons and attendant striatal terminals, the presence of large protein-rich neuronal inclusions containing fibrillar α-synuclein and increased numbers of activated microglia. Evidence suggests that both misfolded α-synuclein and oxidative stress play an important role in the pathogenesis of sporadic PD. Here we review evidence that α-synuclein activates glia inducing inflammation and that Nrf2-directed phase-II antioxidant enzymes play an important role in PD. We also provide new evidence that the expression of antioxidant enzymes regulated in part by Nrf2 is increased in a mouse model of α-synuclein overexpression. We show that misfolded α-synuclein directly activates microglia inducing the production and release of the proinflammatory cytokine, TNF-α, and increasing antioxidant enzyme expression. Importantly, we demonstrate that the precise structure of α-synuclein is important for induction of this proinflammatory pathway. This complex α-synuclein-directed glial response highlights the importance of protein misfolding, oxidative stress and inflammation in PD and represents a potential locus for the development of novel therapeutics focused on induction of the Nrf2-directed antioxidant pathway and inhibition of protein misfolding.

Parkinson's disease.

Parkinson's disease (PD) is the most common age-related motoric neurodegenerative disease initially described in the 1800's by James Parkinson as the 'Shaking Palsy'. Loss of the neurotransmitter dopamine was recognized as underlying the pathophysiology of the motor dysfunction; subsequently discovery of dopamine replacement therapies brought substantial symptomatic benefit to PD patients. However, these therapies do not fully treat the clinical syndrome nor do they alter the natural history of this disorder motivating clinicians and researchers to further investigate the clinical phenotype, pathophysiology/pathobiology and etiology of this devastating disease. Although the exact cause of sporadic PD remains enigmatic studies of familial and rare toxicant forms of this disorder have laid the foundation for genome wide explorations and environmental studies. The combination of methodical clinical evaluation, systematic pathological studies and detailed genetic analyses have revealed that PD is a multifaceted disorder with a wide-range of clinical symptoms and pathology that include regions outside the dopamine system. One common thread in PD is the presence of intracytoplasmic inclusions that contain the protein, α-synuclein. The presence of toxic aggregated forms of α-synuclein (e.g., amyloid structures) are purported to be a harbinger of subsequent pathology. In fact, PD is both a cerebral amyloid disease and the most common synucleinopathy, that is, diseases that display accumulations of α-synuclein. Here we present our current understanding of PD etiology, pathology, clinical symptoms and therapeutic approaches with an emphasis on misfolded α-synuclein.

Matrix metalloproteinase dependent cleavage of cell adhesion molecules in the pathogenesis of CNS dysfunction with HIV and methamphetamine.

Physiologically appropriate levels of matrix metalloproteinases (MMPs) are likely important to varied aspects of CNS function. In particular, these enzymes may contribute to neuronal activity dependent synaptic plasticity and to cell mobility in processes including stem cell migration and immune surveillance. Levels of MMPs may, however, be substantially increased in the setting of HIV infection with methamphetamine abuse. Elevated MMP levels might in turn influence integrity of the blood brain barrier, as has been demonstrated in published work. Herein we suggest that elevated levels of MMPs can also contribute to microglial activation as well as neuronal and synaptic injury through a mechanism that involves cleavage of specific cell and synaptic adhesion molecules.

Misfolded α-synuclein and Toll-like receptors: therapeutic targets for Parkinson's disease.

Parkinson's disease (PD) is typified by the loss of midbrain dopamine neurons, the presence of large proteinaceous α-synuclein-positive intracellular inclusions, oxidatively modified molecules and activated microglia. The etiology of sporadic PD is not fully understood but several lines of evidence suggest that genetic vulnerability and environmental toxicants converge to incite pathology--the multiple hit hypothesis. One gene linked to both familial and sporadic PD is SNCA, which encodes for the protein α-synuclein that has a propensity to misfold into toxic moieties. Here we show that α-synuclein directly activates microglia inciting the production of proinflammatory molecules and altering the expression of Toll-like receptors (TLRs). We discuss the role for α-synuclein-directed TLR expression changes in PD and the therapeutic potential of modifying this response.

Dopamine and paraquat enhance α-synuclein-induced alterations in membrane conductance.

We have previously demonstrated that α-synuclein overexpression increases the membrane conductance of dopaminergic-like cells. Although α-synuclein is thought to play a central role in the pathogenesis of several neurodegenerative diseases including Parkinson's disease, multiple system atrophy, and diffuse Lewy body disease, the mechanism of action is not completely understood. In this study, we sought to determine whether multiple factors act together with α-synuclein to engender cell vulnerability through an augmentation of membrane conductance. In this article, we employed a cell model that mimics dopaminergic neurons coupled with α-synuclein overexpression and oxidative stressors. We demonstrate an enhancement of α-synuclein-induced toxicity in the presence of combined treatment with dopamine and paraquat, two molecules known to incite oxidative stress. In addition, we show that combined dopamine and paraquat treatment increases the expression of heme oxygenase-1, an antioxidant response protein. Finally, we demonstrate for the first time that combined treatment of dopaminergic cells with paraquat and dopamine enhances α-synuclein-induced leak channel properties resulting in increased membrane conductance. Importantly, these increases are most robust when both paraquat and dopamine are present suggesting the need for multiple oxidative insults to augment α-synuclein-induced disruption of membrane integrity.

α-Synuclein Alters Toll-Like Receptor Expression.

Parkinson's disease, an age-related neurodegenerative disorder, is characterized by the loss of dopamine neurons in the substantia nigra, the accumulation of α-synuclein in Lewy bodies and neurites, and neuroinflammation. While the exact etiology of sporadic Parkinson's disease remains elusive, a growing body of evidence suggests that misfolded α-synuclein promotes inflammation and oxidative stress resulting in neurodegeneration. α-Synuclein has been directly linked to microglial activation in vitro and increased numbers of activated microglia have been reported in an α-synuclein overexpressing mouse model prior to neuronal loss. However, the mechanism by which α-synuclein incites microglial activation has not been fully described. Microglial activation is governed in part, by pattern recognition receptors that detect foreign material and additionally recognize changes in homeostatic cellular conditions. Upon proinflammatory pathway initiation, activated microglia contribute to oxidative stress through release of cytokines, nitric oxide, and other reactive oxygen species, which may adversely impact adjacent neurons. Here we show that microglia are directly activated by α-synuclein in a classical activation pathway that includes alterations in the expression of toll-like receptors. These data suggest that α-synuclein can act as a danger-associated molecular pattern.

Inhibitors of leucine-rich repeat kinase-2 protect against models of Parkinson's disease.

Leucine-rich repeat kinase-2 (LRRK2) mutations are a common cause of Parkinson's disease. Here we identify inhibitors of LRRK2 kinase that are protective in in vitro and in vivo models of LRRK2-induced neurodegeneration. These results establish that LRRK2-induced degeneration of neurons in vivo is kinase dependent and that LRRK2 kinase inhibition provides a potential new neuroprotective paradigm for the treatment of Parkinson's disease.