Inflammasome Activation by Methamphetamine Potentiates Lipopolysaccharide Stimulation of IL-1Β Production in Microglia

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Theses & Dissertations Graduate Studies

Summer 8-18-2017

Inflammasome Activation by Methamphetamine Potentiates Lipopolysaccharide Stimulation of IL-1β Production in Microglia

Enquan Xu University of Nebraska Medical Center

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Inflammasome Activation by Methamphetamine Potentiates Lipopolysaccharide

Stimulation of IL-1β Production in Microglia

Enquan Xu

A DISSERTATION

Presented to the Faculty of The University of Nebraska Graduate College In Partial Fulfillment of Requirements for the Degree of Doctor of Philosophy

Pharmacology & Experimental Neuroscience

Under the Supervision of Professor Huangui Xiong University of Nebraska Medical Center Omaha, Nebraska May, 2017

Supervisory Committee:

Jianuo Liu, Ph.D. Jyothi Arikkath, Ph.D. R. Lee Mosley, Ph.D. Woo-Yang Kim, Ph.D. Santhi Gorantal, Ph.D. ii

Acknowledgements

Firstly, I would like to express my sincere gratitude to my advisor Prof. Huangui

Xiong, who expertly guided me through my graduate education. Without his continuous support, patience, motivation, and immense knowledge, I could not complete my Ph.D. study. His professional guidance kept me constantly engaged with my research, and his personal generosity made my overseas-study at UNMC enjoyable.

A very special gratitude goes out to Prof. Jianuo Liu, who help me with every detail of my experimental design and encourage me when I was down.

Besides my advisor, I would like to thank the rest of my thesis committee: Prof.

Jyothi Arikkath, Prof. R. Lee Mosley, Prof. Woo-Yang Kim, and Prof. Santhi Gorantal, for their insightful comments, encouragement, and instructive questions.

My appreciation also extends to Prof. Tammy L. Kielian, Prof. Matthew C.

Zimmerman, and Prof. Adam J. Case for their generous help on my project. I also would like to thank Prof. Myron Toews for training my scientific writing and presentation skills.

Finally, my sincere thanks go to my life-coach, my father, for I owe it all to you. My dearest Mom, thanks for your unselfish love. My deepest lover, Xiaobei Wang, who gave me the most precious gift: a new life to my family. I will never forget your innumerable sacrifices made by you, many thanks!

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Inflammasome Activation by Methamphetamine Potentiates Lipopolysaccharide

Stimulation of IL-1β Production in Microglia

Enquan Xu, Ph.D.

University of Nebraska Medical Center, 2017

Supervisor: Huangui Xiong, M.D., Ph.D.

Methamphetamine (Meth) is a psychostimulant drug that is widely abused all around the world. The administration of Meth causes a strong instant euphoria effect, and long- term of abuse is correlative of drug-dependence and neurotoxicity. The neuroimaging studies demonstrated that the long-term abuse of Meth is associated with the reduction of the dopamine transporter (DAT) and vesicular monoamine transporter (VMAT2) in the striatum. Neuroinflammation is well-accepted as an important mechanism underlying the

Meth-induced neurotoxicity. The over-activated microglia were found both in Meth human abusers and animal models.

NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome is the most predominant Nod-like receptor (NLR) expressed in microglia and is involved in the pathogenesis of many neurodegenerative diseases. In recent years, multiple lines of evidence suggest that the activation of NLRP3 inflammasome is associated with drug abuse induced innate immune system activation both in central nervous system (CNS) and peripheral nervous system (PNS). We investigated the role of NLRP3 inflammasome in Meth-induced microglial activation. Meth induced the production of mitochondrial ROS and disruption of lysosomal membrane integrity, which served as the second activation signal of NLRP3 inflammasome. The activation of NLRP3 inflammasome led to the cleavage of pro-IL-1β and subsequent release of biologically

active IL-1β. By blocking the inflammasome activation, we successfully attenuated the neuronal apoptosis induced by supernatants of Meth-treated microglia.

It is well-known that Meth abuse exacerbates HIV-1-associated neurocognitive disorders (HAND). However, the mechanism of how Meth potentiates HAND is not fully understood. Ample evidence indicates that both Meth and HIV-1 cause microglial activation and resultant secretion of proinflammatory molecules leading to neuronal injury and ultimately the development of HAND. Inflammasome is the key signaling platform involved in HIV-1-associated microglia activation and the production of proinflammatory molecules. We studied the synergistic effects of HIV-1 glycoprotein protein 120 (gp120) and Meth in microglial NLRP3 inflammasome activation. Gp120 upregulated the pro-IL-1β and thus, primed the microglia as the first signal. The subsequent stimulation of Meth as the second signal further activates the inflammasome that promotes the processing and release of IL-1β. The overactivated ROS system is potentially relative to gp120- and Meth-induced inflammasome activation.

Table of contents

Contents:

Acknowledgments…………………………………………………………………………… ii

Table of Contents……………………………………………………………………………. v

List of figures and tables……………………………………………………………………. ix

List of abbreviations…………………………………………………………………………. xi

Chapter 1……………………………………………………………………………………... 1

Review of the role of microglia in methamphetamine (Meth)-induced neurotoxicity and inflammasome in drug abuse-associated neurotoxicity

Abstract……………………………………………………………………………………...... 2

1. Introduction……………………………………………………………………………….. 3

2. The acute and chronic consequences of Meth abuse……………………………….. 5

3. Microgliosis: a neurotoxic marker of long-term Meth addiction……………………... 6

4. Microglia: a potential intersecting point for Meth and HIV…………………………... 11

5. Meth and neuronal immunosuppressive signals…………………………………...... 13

6. Prevention and therapy of Meth-induced neural injury…………………………...... 15

7. Inflammasome activation in Drug Abuse…………………………………………...... 21

1.1. Overview of inflammasome in CNS…………………………………………...... 21

1.2. Inflammasome in alcohol abuse………………………………………………...... 24

1.3. Inflammasome in cocaine abuse………………………………………………..... 28

1.4. Inflammasome in morphine…………………………………………………...... 30

8. Summary and Discussion…………………………………………………...... 34

Chapter 2……………………………………………………………………………………… 36

Inflammasome activation by Meth potentiates lipopolysaccharide (LPS) stimulation of IL-

1β production in microglia

Abstract……………………………………………………………………………………...... 37

1. Introduction………………………………...…………………………………………...... 38

2. Materials and Methods……………………………………………...……………...... 42

2.1. Materials………………………………...…………………………………………... 42

2.2. Animals………………………………...………………………………..…………... 42

2.3. Isolation and culture of microglial and Neuronal cells………………………….. 42

2.4. Brain slice culture……………………...……………………………….…………... 44

2.5. Quantitative PCR analysis…………...……………………………….……….…... 47

2.6. Total protein extraction and concentration determination………………….…... 47

2.7. Western blotting……………………...…………………………………………...... 48

2.8. Immunofluorescences……………...……………………………….…………...... 48

2.9. MTT assay……………...………………………………….…...…….…………...... 49

2.10. TUNEL staining……………...……………………………….…………..……...... 49

2.11. Cytokine analysis by ELISA……………...….…………………….…………...... 49

2.12. Migration assay……………...……………………………….…………...... 50

2.13. Colorimetric assay for caspase-1 activity……………...……………………..... 51

2.14. Data analyses……………...……………………………………….…………...... 51

3. Results……………...………………………………………..…………….…………...... 53

3.1. Microglial morphologic, migration, and proliferation are not affected by Meth

treatment alone……………...……………………………….…………………...... 53

3.2. Analyses of Meth-induced change of proinflammatory cytokines…………...... 59

3.3. Meth potentiate LPS-induced microglia activation and neurotoxicity…..…...... 62

3.4. Enhancement of processing and release of IL-1β by Meth treatment….…...... 68

3.5. Activation of inflammasome after Meth treatment in LPS-primed microglia..... 76

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4. Discussion……………...……………………………….……………………………...... 80

Chapter 3……………...……………………………………………………….…………...... 85

Mitochondrial ROS production and lysosomal membrane permeabilization are molecular mechanisms of Meth-induced inflammasome activation

Abstract……………...……………………………….……………………………….…….... 86

1. Introduction……………...……………………………….…………………………...... 87

2. Materials and methods……………...……………………………….……..………...... 89

2.1. Immunocytochemistry……………...……………………………….…………...... 89

2.2. ELISA……………...……………………………….…………...... 89

2.3. Fluorescent dye loading and imaging…………….……………….…………...... 89

2.4. Measurement of nitric oxide (NO) production……………...………………….... 90

2.5. Tunel staining……………...……………………………………..….…………...... 90

2.6. Statistical analyses……………...……………………………….…….………...... 90

3. Results……………...……………………………………………………..…………...... 92

3.1. Identification of danger signaling-associated molecular pathways leading to

Meth-induced inflammasome activation……………...……………...………...... 92

3.2. Production of mitochondrial ROS after Meth treatmen………….…………...... 96

3.3. Permeabilization of lysosomal membrane in Meth-treated microglia……...... 101

3.4. Inhibition of inflammasome activation block the Meth-potentiated neurotoxicity

……...……………………………….……………………………….……...... 106

4. Discussion……...……………………………….………………………………………. 108

Chapter 4……...……………………………….……………………………….……...... 113

Meth potentiated HIV-1 gp120-induced inflammasome activation

Abstract……...……………………………….……………………………….……...... 114

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1. Introduction……...……………………………….…………………………….……..... 115

2. Materials and methods……...……………………………….……………….……..... 117

2.1. Western blotting……...……………………………….……………………….…….... 117

2.2. ELISA……...……………………………….……………………………….……...... 117

2.3. Immunofluorescent staining……...……………………………….………….…….... 117

2.4. Fluorescent dye loading and imaging……...……………………………….……..... 118

3. Results……...……………………………….……………………………….……...... 119

3.1. HIV-1 gp120 works as the priming signal for Meth-induced inflammasome

activation……...……………………………….…………………...………….……..... 119

3.2. Activation of ROS contribute to the Meth-induced inflammasome activation in

gp120-primed microglia……………………………………………………….…….... 124

4. Discussion……...……………………………….…………………………………….... 126

Chapter 5……...……………………………….……………………………….……...... 128

General discussion……...……………………………….…………………………..…...... 129

References……...……………………………….…….……………………….……...... 131

List of figures and tables

Figure 1. Mechanisms of microglia-induced neurotoxicity in Meth abuse

Figure 2. Illustration of brain slice cutting and culturing technique

Figure 3. Meth did not increase the activated morphological change in cultured microglia

Figure 4. Meth did not promote the migration of microglia

Figure 5. Meth did not induce the proliferation of microglia

Figure 6. Meth did not stimulate the production of proinflammatory cytokines in transcriptional level

Figure 7. Potentiation of the LPS-induced morphological change by Meth

Figure 8. Potentiation of the LPS-induced neurotoxicity by Meth

Figure 9. Meth did not potentiate the expression of proinflammatory cytokines induced by LPS

Figure 10. Enhancement of IL-1β release in cultured microglia and brain slice after Meth treatment

Figure 11. Meth promoted the processing of IL-1β in cultured microglia

Figure 12. Meth activated the caspase-1 in culture microglia

Figure 13. Blockade of caspase-1 activation attenuated the Meth-mediated processing of

IL-1β

Figure 14. Aggregation of ASC protein in Meth-treated microglia

Figure 15. Co-localization of NLRP3 and caspase-1 in Meth-treated microglia

Figure 16. Meth did not regulate the expression of NLRP3

Figure 17. Involvement of Mitochondrial ROS and lysosomal cathepsin B in Meth- induced IL-1β release

Figure 18. Blockade of mitochondrial ROS and lysosomal cathepsin B prevented the

Meth-induced ASC protein aggregation

Figure 19. Meth increased the production of total ROS and mitochondrial ROS

Figure 20. Meth did not increase the NO in microglial supernatant

Figure 21. Time-dependent activation of mitochondrial ROS after Meth treatment

Figure 22. Increased permeabilization of lysosomal membrane in Meth-treated microglia

Figure 23. Release of cathepsin B in Meth-treated microglia

Figure 24. Time-dependent disruption of lysosomal membrane and cathepsin B release after Meth treatment

Figure 25. Inhibition of mitochondrial ROS and lysosomal cathepsin B attenuated the

Meth-potentiated neurotoxicity

Figure 26. Meth promoted the HIV-1 gp120-induced IL-1β processing

Figure 27. Meth potentiated the HIV-1 gp120-induced IL-1β release

Figure 28. Meth induced the co-localization of NLRP3 and caspase-1 in gp120-primed microglia.

Figure 29. Meth activated the total ROS synergistically with HIV-1 gp120

Table 1. Release of inflammatory-related cytokines in microglia after Meth treatment

Table 2. Meth did not alter the LPS-induced release of TNF-α, IL-6, IL-4, and IL-10

List of abbreviations

ASC: Apoptosis-associated speck-like protein containing a CARD

BBB: Blood Brain Barrier

BMVEC: Brain microvascular endothelial cells

Casepase-1: Cysteine-aspartic proteases 1

CA-074 Me: N-(L-3-trans-Propylcarbonyl-oxirane-2-carbonyl)-L-isoleucyl-L-proline methyl ester cART: combined antiretroviral therapy

CD: Cluster of differentiation

CNS: Central nervous system

COX-2: Cyclooxygenase 2

DA: Dopamine

DAT: Dopamine transporter

DAMPs: Dangerous-associated molecular patterns

DTG: Ditolylguanidine

FBS: Fetal bovine serum

Gp120: glycoprotein 120

HAART: Highly active antiretroviral therapy

HAD: HIV-1-associated dementia

HAND: HIV-1-associated neurocognitive disorder

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HBSS: Hank’s buffered salt solution

HIV-1: Human immunodeficiency virus 1

HIVE: HIV encephalitis iNOS: Inducible nitric oxide synthase

IL: Interleukin

IFNβ: Interferon beta

IP3Rs: Inositol 1,4,5-triphosphate receptors

Kv: Voltage-gated potassium channels

LPS: Lipopolysaccharide

LTP: Long-term potentiation

MAM: Mitochondria-associated ER membrane

METH: Methamphetamine miRNA: microRNA

MMPs: Matrix metalloproteinases

MRI: Magnetic resonance imaging

MTT: 3-(4,5-dimethlthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

NLRP3: NACHT, LRR and PYD domains-containing protein 3

NLRs: NOD-like receptors

NMDA: N-methyl-D-aspartate

NO: Nitric oxide

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RNS: Reactive nitrogen species

ROS: Reactive oxidative species

PAMPs: Pathogen-associated molecular patterns

PBS: Phosphate-buffered saline

PET: Positron emission tomography

PLA2: Phospholipase A2

PNS: Peripheral nervous system

Sig-R: Sigma receptor

Tat: trans-activator of transcription

TBS: Tris buffer solution

TEER: Transendothelial electrical resistance

TGF-β: Transforming growth factor beta

TLR: Toll-like receptor

TNF-α: Tumor necrosis factor alpha

TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling

VMAT2: Vesicular monoamine transporter 2

Chapter 1 Review of the microglial activation in methamphetamine (Meth)-induced neurotoxicity

and the role of inflammasome in drug abuse-

associated neurotoxicity

Abstract

Methamphetamine (Meth) is an addictive psychostimulant widely abused around the world. The chronic use of Meth produces the neurotoxicity featured by dopaminergic terminal damage and microgliosis, resulting in serious neurological and behavioral consequences. Ample evidence indicates that Meth causes microglial activation and resultant secretion of pro-inflammatory molecules leading to neural injury. However, the mechanisms underlying Meth-induced microglial activation remain to be determined. In this review, we attempt to address the effects of Meth on human immunodeficiency virus (HIV)-associated microglia activation both in vivo and in-vitro. As Meth abuse not only increases HIV transmission but also exacerbates progression of HIV-associated neurocognitive disorders (HAND) through activation of microglia. In addition, the therapeutic potential of anti-inflammatory drugs on ameliorating Meth-induced microglia activation and resultant neuronal injury is discussed.

1. Introduction

Methamphetamine (Meth) is a highly-abused psychostimulant and the second most widely used illicit drug worldwide (after cannabis) (Degenhardt et al. 2010). Chronic abuse eventually leads to the neurotoxic regimen, which induces the psychological and behavioral abnormalities, such as increased aggressive behavior and craving for the drug (Goldstein and Volkow 2011; Sekine et al. 2006; Volkow et al. 2001b; Volkow and

Li 2004). The long-term neurotoxic effects of Meth are well established by neuroimaging studies and psychological tests and confirmed in both rodents and non-human primates

(Melega et al. 1998; Panenka et al. 2013; Villemagne et al. 1998). Although the primary target of Meth is dopaminergic terminal, the neuropathological changes are not only limited in the striatum. A broader scope of the neural injury has been observed in human subjects with a decreased volume of the hippocampus and the hypertrophy in white matter (Thompson et al. 2004). Repeated administration of Meth also impaired cognitive function, which could be partially explained by current dopamine-based neurotoxic mechanisms (Kalechstein et al. 2003; Marshall et al. 2007; O'Dell et al. 2011;

Schroder et al. 2003), suggesting other mechanisms may be involved in Meth- associated neuropathology. Increasing evidence indicate that neuroinflammation featured by microglial activation plays an important role in Meth-induced neurotoxicity.

The notion is supported by recent studies that anti-inflammatory drug ibudilast attenuated Meth dependence and Meth-induced neural injury (Beardsley et al. 2010;

Charntikov et al. 2015; Snider et al. 2012; Worley et al. 2016).

Studies have shown that microgliosis is an early response to Meth abuse and such a response lasts for a long time even after abstinence (LaVoie et al. 2004; Sekine et al.

2008). Over-activated microglia are found in multiple brain regions in individuals with

Meth abuse, but not in those with cocaine use (Narendran et al. 2014; Sekine et al.

2008). The time-course, dose-response, and pharmacological profiles of Meth-induced microglial activation indicate that over-activated microglia are not merely a response subsequent to nerve terminal damage, but a specific pharmacological marker of Meth- induced neurotoxicity (LaVoie et al. 2004; Thomas et al. 2004b). As microglia are regulated by a variety of inhibitory signals such as CX3CL1, CD200, CD22, or CD172

(Biber et al. 2007; Hellwig et al. 2013; Prinz and Mildner 2011), the impact of repeated

Meth administration on inhibitory signaling molecules in CNS were investigated as the potential therapeutic targets (Thomas et al. 2008a; Yue et al. 2012). On the other hand, since the release of neurotoxins including pro-inflammatory cytokines and super oxidative factors are the primary neuronal toxic mediators of microglia (Block et al.

2007), the production of these pro-inflammatory mediators after Meth treatment was analyzed (Loftis et al. 2011). However, the results on the immune modulatory effects of

Meth are inconsistent. While its suppressive role was reported in most studies in the peripheral immune system (Harms et al. 2012; In et al. 2004; Mata et al. 2015; Nair et al.

2006), Meth was primarily considered as a pro-inflammatory mediator in the CNS

(Downey and Loftis 2014; McConnell et al. 2015; Najera et al. 2016). A recent study on cultured microglia showed that Meth had limited impacts on microglia production of proinflammatory cytokines indicating the biological and molecular intricacy of this drug

(Frank et al. 2016). Further investigations on potential roles of the other CNS-specific factors and neuronal danger-associated molecular patterns (DAMPs) are needed. Meth dependence is one of the most common co-morbid conditions among the HIV-infected population (Colfax and Shoptaw 2005). In comparison with the independent effect of

Meth abuse or HIV infection, the combined action of HIV infection and Meth dependence resulted in more severe impairment on neurocognition (Jernigan et al.

2005; Kesby et al. 2015a; Kesby et al. 2015b). Studies on the interactions between

Meth and HIV proteins in animal models have demonstrated their synergistic effects on

cognitive deficits (Hoefer et al. 2015; Kesby et al. 2015b) and altered behaviors (Henry et al. 2013; Roberts et al. 2010). However, the precise mechanisms underlying Meth exacerbation of HAND remain unclear. It has been shown that Meth-taking patients with

HIV encephalitis (HIVE) exhibited a significant microgliosis, but not astrogliosis

(Langford et al. 2003), suggesting that enhanced microglial activation underlies the cross-talk of HIV-1 infection and Meth dependence. Moreover, Meth abuse enhanced opportunistic microbial brain infection (Eugenin et al. 2013; Patel et al. 2013) and increase LPS-mediated human macrophage production of proinflammatory cytokines

(Liu et al. 2012b). LPS was found to potentiate Meth-associated neurotoxicity (Jung et al. 2010). Based on these results, whether chronic Meth abuse could enhance the pre- existing neuroinflammation and promote the progression of another comorbid disease are emerging topics that are worthy of further investigation.

2. Acute and chronic consequences of Meth abuse

Meth is a synthetic lipophilic drug that is blood brain barrier (BBB) permeable

(Cruickshank and Dyer 2009). It derived from amphetamine, but with higher binding affinity to monoamine transporters (Kish 2008). Meth administration induces a remarkable outflow of monoamines from synaptic vesicles to cytosol on presynaptic neuron (Brown et al. 2000; Fleckenstein et al. 1997; Sulzer et al. 2005). By reverse transportation and freely diffusion, Meth induces potent efflux of dopamine to postsynaptic causing strong and long-lasting euphoric effect. A comparable dopamine dynamic study suggested that single injection of Meth on the concentration of 2.5 mg/kg induced around 5 times of dopamine release compared to cocaine on 40 mg/kg

(Kuczenski et al. 1991). Because it is easy to synthesize and is much more potent on psychostimulant effect than other stimulants, Meth has already widely abused all around the world.

The instant effects of Meth include increased attention, activity, and wakefulness; decreased fatigue and appetite; euphoria and rush experience. Hyperthermia and irregular heartbeat are two most prominent toxic effects of hyperthermia and irregular heartbeat overdose (Cruickshank and Dyer 2009). On the other hand, chronic use of

Meth induces a series of negative consequences including addiction, anxiety, confusion, insomnia, mood disturbances, and violent behaviors (Darke et al. 2008). In addition to these psychotic symptoms, chronic abusers are often associated with cognitive deficits ranging from impaired pulse control, working memory and decision-making (Johanson et al. 2006; Simon et al. 2002; Volkow 2001). These symptoms are accompanied by the

Meth-induced neuropathological changes including the damage to dopamine and serotonin axons, loss of gray matter, hypertrophy of the white matter, and microgliosis.

Neuroimaging studies with specific probes implicated a monoamine transporter reduction and dopaminergic terminal degeneration (Frey et al. 1997; Sekine et al. 2001).

Moreover, magnetic resonance imaging (MRI) studies indicated that Meth-associated neurodegeneration was not restricted to the striatum (Bae et al. 2006; Thompson et al.

2004). Considerable shrinkage of hippocampi, gray matter, cingulate cortex, limbic cortex and paralimbic cortex was observed in recreational abusers (Thompson et al.

2004). Multiple neurotoxic events are associated with Meth abuse including oxidative stress, excitotoxicity, hyperthermia, mitochondrial dysfunction, endoplasmic reticulum stress, and neuroinflammatory responses. Here, we primarily focus on neuroinflammation associated with microglial activation.

3. Microgliosis: a neurotoxic marker of Meth addiction

Microglial activation is characterized by proliferation, morphologically change, migration and inflammatory secretion profiles (Kettenmann et al. 2011). It is well-established that the activation of microglia is relative to neurodegenerative diseases, brain injury and

toxicant-induced damage to the CNS (Lull and Block 2010). In animal model injected with Meth, microgliosis was found in multiple brain areas (Escubedo et al. 1998). A human study validated the remarkable microglial activation in all brain regions using positron emission tomography (PET) with a specific radiotracer (Sekine et al. 2008).

The dose- and time-response of Meth-induced microglial activation performed on Meth- administrated rat indicated that microglial response preceded both terminal neuronal degeneration and astrocyte activation (LaVoie et al. 2004). As it is difficult to differentiate infiltrated monocyte/macrophage with activated microglia by immunohistochemistry staining, the possibility that Meth increases the trafficking of peripheral immune cells into the brain needs to be further proved. The irradiated mice were rescued with bone marrow transplantation from “green mice”, a transgenic mouse line with an enhanced GFP (eGFP) expression in all tissues except for erythrocytes and hair. To determine if Meth increases the peripheral monocyte infiltration, the peripheral monocytes in the brain with eGFP were detected after Meth administration. Two days after four injections of neurotoxic regimen of Meth (5 mg/kg) and/or physiological saline in with a 2-h interval, the brains were harvested, fixed and the sectioned. The stratum sections were examined under fluorescent microscope. While the resident microglia were significantly activated in the striatum, no infiltrated eGFP-expressing cells

(migrated hematopoietic cells) were detected (Thomas et al. 2008a). Although this study suggests that there is no evidence of transmigration of peripheral hematopoietic cells cross the BBB in response to Meth administration, the role of BBB impairment in

Meth-induced neuroinflammation is still in debate. The alteration of BBB permeability was found shortly after Meth administration and lasted long in multiple brain areas including stratum, amygdala, and hippocampus (Bowyer and Ali 2006; Quinton and

Yamamoto 2006). Moreover, Meth application was found to induce the oxidative stress in cultured primary human brain microvascular endothelial cells (BMVEC) (Ramirez et

al. 2009). The impairment of barrier function of BBB was determined both in vivo using fluorescent tracer and in vitro via transendothelial electrical resistance (TEER) test. The application of Meth diminished the tightness of BMVEC monolayers by decreasing the expression of cell membrane-associated tight junction proteins and thus, enhanced the monocyte transendothelial migration (Ramirez et al. 2009). The increased monocytes passage through the endothelial cells monolayer can be blocked by a specific inhibitor of Arp 2/3 complex, indicating that actin cytoskeletal dynamics play an important role in

Meth-induced transendothelial monocyte migration (Park et al. 2013). Taken together, although the Meth-induced microglial activation is validated in both animal models and human abusers, it is still controversial that whether Meth-associated inflammatory response is mediated by the resident microglia other than the peripheral immune cells infiltration.

To determine the mechanism of microglial activation, various experimental groupings were designed to mimic selected pharmacological elements of Meth action (Thomas et al. 2004b). As another psychostimulant with high binding affinity with plasma dopamine transporter, cocaine failed to trigger microglial activation in Meth administrated rat

(Thomas et al. 2004b). Also, the administration of cocaine was found less relative to long-term neurotoxicity (Bennett et al. 1993; Narendran et al. 2014). The involvement of dopamine receptors in Meth-induced microglial activation was also evaluated. However, neither D1 nor D2 receptor agonist replicated Meth-induced microglial activation, which suggested that microglial activation is independent of Meth-induced release of dopamine (Thomas et al. 2004b). Furthermore, the levorotary enantiomer of Meth (L-

Meth), a molecular sharing the most receptor targets with Meth but with much less binding affinities, did not activate microglia. Because L-Meth is much less neurotoxic compared to dextrorotary enantiomer (O'Callaghan and Miller 1994), its failure in

inducing microglial activation further proved that Meth-induced neuroinflammation is an essential event in the Meth neurotoxic cascade. On the other hand, the factors mitigate neurotoxicity of Meth such as lower ambient temperature and NMDA receptor antagonists also reduce microgliosis along with their neuroprotective effects (LaVoie et al. 2004; Thomas and Kuhn 2005c). Moreover, the tolerance of Meth toxicity is also associated with attenuated microglial activation. After a neurotoxic challenge with Meth, tolerance was developed to the subsequent neurotoxic effects of Meth (Gygi et al. 1996;

Riddle et al. 2002; Stephans and Yamamoto 1996). Although the second Meth stimulation induced the same extent of hyperthermia and astrocyte activities, microglial activation was blunted (Friend and Keefe 2013; Thomas and Kuhn 2005a). Thus, attenuated microglial activation was considered as an important mechanism underlying the reduced Meth-induced neurotoxicity. In contrast, classical immunogen lipopolysaccharide administration significantly increased the microglial activation and potentiated the Meth-induced neurotoxicity (Buchanan et al. 2010; Jung et al. 2010).

While the astrocytes remain reactive even 30 days after Meth administration, microglial activation subsides within 7 days, which is more reflecting the acute Meth-induced neurotoxicity (Friend and Keefe 2013). Based aforementioned evidence, microglial activation could be an important indicator of Meth-induced toxicity.

Although in vivo experiments proved a solid link between neurotoxicity and microglia- mediated neuroinflammation (Clark et al. 2013), there is a rare study of Meth effects on cultured microglia. In the case that Meth receptors on microglia were undetermined, the mechanism of whether Meth-induced microglial activation is a receptor-mediated effect or not is inconclusive. Therefore, testing enantiomers on isolated microglia may address this question because of the different binding affinities of the dextrorotatory/levorotary forms to certain receptors. Although the specific receptors to Meth on microglia are

unknown, emerging studies examined the cellular effects of Meth-associated microglia- induced neurotoxicity. Because activated microglial primarily contribute to neuronal death by releasing various neurotoxic factors, the current studies narrowed their research interest on the activation of inflammatory signaling pathway and subsequent production of neurotoxins after Meth exposure. The latest study evaluated mRNA level of inflammatory-associated genes on cultured microglia stimulated by various concentrations of Meth (Frank et al. 2016). Proinflammatory cytokines upregulated in vivo were found unchanged in Meth-treated microglial cultures (Frank et al. 2016). The inflammatory responses on both whole brain microglia and isolated striatum microglia are consistent, which means there is no regional specificity of microglia activation.

Moreover, the IL-1β level and cell viability were found decreased after administration of

1 mM Meth (Frank et al. 2016). This is consistent with previous experiments on cultured microglia, which suggested the apoptotic effect of Meth in 500 μM, and it could be reversed by TNFα and IL-6 through IL-6 receptor and JAK-STAT3 pathway (Coelho-

Santos et al. 2012). While most of the studies indicated that there is less possibility for

Meth to stimulate the pro-inflammatory response directly, a study performed on CHME-

5 cell line (Human fetal microglial immortalized with large T antigen of simian virus 40) suggested that Meth could reactivate HIV transcription in an NF-κB- dependent manner.

The NF-κB reporter assay (Luciferase System) and the p65 ELISA of nuclear extracts were used to examine the activity of this cellular transcription factors. The activation of

NF-κB was observed in CHME-5 cell treated with 500 µM of Meth. The activation effect of Meth started as early as 0.5 h and lasted for 24 h. Moreover, the IκB dominant- negative construct, which lacked the phosphorylation site and could not dissociated from NF-κB, blocked the Meth-induced activation (Wires et al. 2012). It is worth to note that the concentration of Meth used in those experiments was more than 500 µM, which is too high to represent the condition of long-term recreational users. On the other hand,

the activation of microglia might also be a consequence of the neuronal release of

DAMPs that stimulated by application of Meth. High mobility group box-1 (HMGB1) was found upregulated after Meth administration and mediated the neuroinflammatory response in multiple brain areas (Frank et al. 2016). Another candidate neuronal DAMP that might mediate Meth-induced microglial activation is DA-quinones (DAQ). The excess outflow of dopamine induced by Meth could be self-oxidized to DAQ that might play an obligatory role in various Meth-induced neurotoxic effects (Kuhn et al. 2006;

Miyazaki et al. 2006). Researchers found DAQ causes a time-dependent activation of cultured microglia. Gene expression study analyzed 101 genes alteration, in which inflammation cytokines, chemokines, and prostaglandins were upregulated, whereas protective neuronal genes were downregulated (Kuhn et al. 2006). The critical roles of dopamine and its oxidative form in Meth-induced microglial activation were further demonstrated by the disruption of DA release from the newly synthesized pool in vivo, which abrogated the microgliosis (Thomas et al. 2008b). Thus, the excessive release of dopamine from vesicles and outflow outside neuronal terminals might function as neuronal DAMPs that could be sensed by regional microglia and initiates the neurotoxic signal cascades (Thomas et al. 2008b). The patterns and mechanisms of microglia activation in the brain need to be further investigated.

The microglia neurotoxicity was initially demonstrated in vitro with primary cultured microglia. After challenge with proinflammatory stimuli such as LPS, IFNβ, or TNFα, the supernatants were transferred to cultured neurons, and the neuronal apoptosis was observed (Chao et al. 1992). Ever since these experiments, it has been well established that the toxic microglial secretory products are the major mediators of microglia-induced neurotoxicity (Gao et al. 2011; Lehnardt et al. 2008; Levesque et al. 2010; Pais et al.

2008). For this reason, it is critical to determine whether the proinflammatory factors,

released by microglia in response to Meth, are toxic to neurons. It was shown that microglia-associated factors, for example, IL-1α, IL-6, CCL2, and TNFα, were upregulated in mice with a single low-dose regimen of Meth (Sriram et al. 2006). The microglial-mediated proinflammatory responses were attenuated by minocycline, a selective inhibitor of microglial activation (Sriram et al. 2006). However, the experiment using mice with genetic deficiency in IL-1α, IL-6, and CCL2 did not show neuroprotection against Meth (Sriram et al. 2006). Only the mice lack of the TNF1/2 receptors showed attenuated neurotoxicity, indicating that TNF-α is a critical factor in

Meth-induced neurotoxicity. Thereby, it is hypothesized that the failure of minocycline in neuroprotection against Meth-associated neuronal damages could be attributed to its incomplete inhibition of TNF-α signaling pathway (Sriram et al. 2006). Currently, neuroprotective effects targeting microglial activation on Meth-related neurotoxicity in an animal model is controversial (Hashimoto et al. 2007; Kelly et al. 2012; Sriram et al.

2006; Zhang et al. 2007). The disparity between these mixed results probably attribute to the differences in the dosing regimen or the time of anti-inflammatory drug administration. However, lack of specific selectivity against downstream pathway(s) might also contribute to these ambiguous results. Further studies on identification and inhibition of more specific molecular targets mediating microglia-mediated neuroinflammation are imperative.

4. Microglia: a potential intersecting point for Meth and HIV

Despite of successes with highly active antiretroviral therapy (HAART), 50% of the HIV- infected population still suffers from HIV-associated neurological disorder (HAND)

(Chen et al. 2014). The persistent neuroinflammation was found in HAND patients, even in the HAART era (Eden et al. 2007; Everall et al. 2009; Harezlak et al. 2011;

Kumar et al. 2007). A neuroimaging study revealed that the degree of

neuroinflammation marked by the activated microglia inversely correlated with the executive function even in the patient receiving HAART (Garvey et al. 2014). Meth abuse is prevalent among HIV-infected patients, and the interaction between Meth abuse and HIV pathogenesis is an interesting research topic for many neurovirologists

(Colfax and Shoptaw 2005; Halkitis et al. 2001; Letendre et al. 2005). In comparison to their independent effects, Meth exposure combined with HIV infection produces severer impairment on neurocognition (Jernigan et al. 2005; Kesby et al. 2015a). Studies on the interactions between HIV proteins and Meth in animal models have further demonstrated their synergistic effects on cognitive dysfunctions (Hoefer et al. 2015;

Kesby et al. 2015b) and altered behaviors (Henry et al. 2013; Roberts et al. 2010).

However, the precise mechanisms for Meth exacerbation of HAND are still not fully understood. More significant microgliosis was observed in Meth-taking patients with HIV encephalitis (HIVE), while astrogliosis was not changed (Langford et al. 2003). Thus, enhanced microglial activation could be the intersecting point for the cross-talk of HIV infection and Meth dependence. Because neurons are refractory to the HIV infection, the activated microglia are most likely the contributors to neuronal damages and cognitive dysfunctions due to the release of neurotoxic substances (Gonzalez-Scarano and Martin-Garcia 2005). On the one hand, Meth is associated with increased entry of virus into CNS system, which could be attributed to several potential mechanisms including increased peripheral viral load (Ellis et al. 2003), reduced adherence to antiretroviral therapy (Carrico et al. 2010), and disruption of the blood-brain barrier

(BBB) (Bowyer and Ali 2006; Turowski and Kenny 2015). Furthermore, Meth can regulate the HIV replication by targeting HIV-associated cellular factors (Liang et al.

2008; Nair et al. 2009). The potential of dopamine to promote HIV replication is corroborated in rat and monkey models with administration of L-DOPA (Czub et al.

2004; Czub et al. 2001). However, the dopamine concentrations used in the above

studies were very high; it is questionable whether such high dopamine concentrations could be found in Meth abusers (Purohit et al. 2011).

Recently, common molecular targets for Meth and HIV intersecting effects gain more attention. It has been suggested that voltage-gated potassium channels (Kv) are involved with neuronal damage and microglial function (Stebbing et al. 2015). HIV protein Tat and gp120 were reported to increase the outward potassium current and proinflammatory cytokines release, resulting in neurotoxic activity (Liu et al. 2013; Xu et al. 2011). The potassium channels on microglia were also associated with Meth neurotoxicity (Wang et al. 2014). Thus, it is worth to investigate further whether Meth application could potentiate the Tat- or gp120-induced microglial inflammatory response through specific potassium channels. In addition, some microglia-associated proinflammatory cytokines that are crucial for the progression of Meth- and HIV-induced neuropathological changes are also needed to be further investigated (Brabers and

Nottet 2006; Goncalves et al. 2008; Lane et al. 2001; Nuovo and Alfieri 1996). The enhanced TNFα secreted by microglia after Meth stimulation might be responsible for the increased HIV replication (Munoz-Fernandez et al. 1997), NMDA receptor neurotoxicity (Fine et al. 1996), and BBB disruption (Mediouni et al. 2015). Likewise, upregulated IL-1β and IL-8 expression may also contribute to the Meth and HIV- associated neurotoxic activities (Brabers and Nottet 2006; Lane et al. 2001) as demonstrated by their compromising effects on Long-term potentiation (LTP) and cognitive function (Bellinger et al. 1993; Xiong et al. 2003).

5. Meth and neuronal immunosuppressive signals

The neuronal cells are vulnerable to the potential detrimental immune reactions. Except for a few limited brain areas, most neurons are incompetents in regenerating themselves (Gage and Temple 2013). Thus, in addition to the physical isolation by

blood brain barrier, neurons express multiple immune suppressive signals to provide a restricted and immunosuppressed microenvironment, which rapidly turns down uncontrolled microglial activation to prevent secondary neuronal damage (Biber et al.

2007). Those signals may be secreted by neurons including TGF-β, CX3CL1, and

CD22 or expressed on the membrane such as CD200 and CD47. Among these suppressive signals exchanging between the neuron and glial cells, CX3CL1-CX3CR1 and CD200-CD200R are two most well-studied axes in Meth-associated neuronal dysfunction (Thomas et al. 2008a; Yue et al. 2012). Ample evidence shows CX3CL1 is the most prominent neuron-derived signal that restricts microglial activation after harmful environmental stimulization (Pabon et al. 2011). Mice homozygous with

CX3CR1-deficient have been widely studied in various neurodegenerative models. Mice with deficient CX3CL1-CXCR1 signaling showed an enhanced microglial activation, resulting in greater neurotoxicity (Cardona et al. 2006). In Meth-induced neurotoxicity, while the genes of many inflammatory molecules were found upregulated, the expression levels of CX3CR1 and CX3CL1 did not change (Thomas et al. 2004a).

Unlike the hypersensitivity of the neurons to MPTP-induced neurotoxicity in a CX3CR1 knockout mouse model, the level of Meth-induced hyperthermia and dopaminergic terminal damage were not enhanced in CX3CR1 deficient mice (Thomas et al. 2008a).

It is still unclear, however, if the overexpressed exogenous CX3CL1 could downregulate Meth-induced microglial activation. Thus, although current evidence supports that microglial activation and neuronal damage induced by Meth are not mediated by CX3CR1 signaling, the therapeutic potential of this signaling axis needs further investigation.

Another well-studied neuronal immunosuppressive signal in Meth-induced neurotoxicity is CD200. The neuroprotective roles of CD200 against microglia-induced neurotoxicity

were reported in multiple neurodegenerative disease models (Chitnis et al. 2007; Zhang et al. 2011b). Consistently, CD200 exerts protective effects on Meth neurotoxicity via decreasing microglial activation (Yue et al. 2012). The Fc region of CD200 (CD200-Fc) was tested on neuron-microglia co-culture system and on rats before Meth treatment

(Yue et al. 2012). CD200-Fc has been proved in vitro that it suppressed the microglial activation and secretion of inflammatory cytokines (IL-1β and TNFα) (Yue et al. 2012).

Meanwhile, the Meth-induced striatal neurotoxicity was also attenuated by the CD200-

Fc application (Yue et al. 2012). However, the Meth-associated microglial activation and neuronal damage were not completely blocked by CD200-Fc, indicating the insufficiency of the solo neuronal suppressive signal. Also, because Meth-induced neuronal damages are consisted of multifactorial and complicated processes, the inhibition of microglial activation might not be enough to reverse the toxicity of Meth completely. Lastly, it is still challenging to deliver the antibody drugs into the specific brain areas. Thus, improved delivery systems must facilitate developing the immunosuppressive neuronal signals administration as a therapeutic strategy (Chacko et al. 2013).

6. Therapeutic studies of Meth-induced neural injury

Currently, the emerging cures for Meth addiction are cognitive behavioral therapy and inpatient treatment. However, the therapeutic effect is barely satisfied. Only 33% of

Meth users finished 16 weeks of outpatient counseling and merely 45% patients achieved 3 weeks of Meth abstinence (Hillhouse et al. 2007). Even inpatient treatment can reach only 30% long-term abstinent (Brecht and Herbeck 2014). The reason for this high relapse rate is the Meth-induced neuropsychiatric impairments (Hoffman et al.

2006; London et al. 2004). Long-term abuse of Meth causes structural damages to multiple brain areas followed by, the impairment of the cognitive and psychiatric

functions (Berman et al. 2008; Hoffman et al. 2008; Schwartz et al. 2010). The Meth abstainers with brain dysfunctions are more likely to relapse (Volkow 2001; Volkow and

Li 2004). This feedback loop is the major obstacle to the current therapy for Meth dependence. However, with limited successfulness of drug development based on neurotransmitter systems, alternative therapeutic strategies are needed. Because the neuroinflammation plays a critical role in Meth-induced neurotoxicity, overactivated microglia seems to be a promising target.

In recent years, exciting progresses have been made on developing an anti- inflammation strategy against microglia-mediated neuronal damages (Beardsley and

Hauser 2014; Kim and Suh 2009). Minocycline is the most lipophilic tetracycline antibiotic that is proved for an anti-inflammatory effect through inhibition of key inflammatory enzymes, like inducible nitric oxide synthase (iNOS) (Amin et al. 1997),

Matrix metalloproteinases (MMPs) (Golub et al. 1991), cyclooxygenase-2 (COX2)

(Yrjanheikki et al. 1999) and Phospholipase A2 (PLA2) (Pruzanski et al. 1992).

Minocycline blocked the microglial activation and attenuated the Meth-induced neurotoxicity (Hashimoto et al. 2007; Tanibuchi et al. 2010; Zhang et al. 2006). After pretreatment of minocycline (40 mg/kg), the behavioral sensitization induced by repeated administration of Meth (3 mg/kg/day) was significantly attenuated (Zhang et al.

2006). Furthermore, the reduction of DA and DAT after Meth are also rescued by application of minocycline (Zhang et al. 2006). A study performed on monkeys further confirmed the neuroprotective effect of minocycline against Meth (Hashimoto et al.

2007). A reduction of DAT after repeated administration of Meth (2 mg/kg) was observed by PET, with or without minocycline (200 mg). The treatment of minocycline, either pre- or post-Meth administration, significantly blocked the DAT reduction.

(Hashimoto et al. 2007). Administration of minocycline was also effective for Meth-

related psychotic disorders as revealed in a clinical case report from Japan (Tanibuchi et al. 2010). However, only one patient was involved in that clinical study, which was insufficient to perform the statistical analysis. In the latest years, minocycline was found to block the rewarding effect of Meth and reduce the self-administrated amount of Meth on both experimental animal models and human studies (Fujita et al. 2012; Snider et al.

2013). Consecutive administration of minocycline (20-40 mg/kg) ameliorated the Meth- induced impairment of long-term memory (Mizoguchi et al. 2008). Most importantly, minocycline attenuated the maintenance and reinstatement of Meth-seeking behaviors, which indicated that minocycline treatment might be able to reduce the craving and relapse for Meth addiction (Attarzadeh-Yazdi et al. 2014). Taken together, minocycline might break the aforementioned feedback loop to help Meth-dependent individuals quit from relapse. However, we also noticed that there are some divergent views in this regard. In contrast to the researches mentioned above, there are some other studies suggesting that minocycline is unable to protect neurons from Meth challenge (Boger et al. 2009; Sriram et al. 2006). The discrepancies may, at least in part, be attributed by different doses of Meth employed in these studies (10 mg/kg and 20 mg/kg). The total amount of Meth administrated with multiple times was much higher in those other studies.

The interaction of the stress and Meth abuse has been well-studied. The unpredictable stress was not only considered as a potentiating factor but also a relapse inducer

(Beardsley et al. 2010; Tata et al. 2007). Ketoprofen, an FDA-proved medication previously used to treat arthritis has recently been found to have a potential therapeutic effect on stress-induced inflammatory response in Meth-administrated rats (Asanuma et al. 2003; Northrop and Yamamoto 2012). Moreover, persistent stress was also linked with increased microglial activation (Nair and Bonneau 2006; Perry 2007). To

investigate the neuroinflammatory effects on stress-induced potentiation of Meth toxicity, the researchers examined the activity of cyclooxygenase (COX), a well-known neuroinflammatory mediator, in rats exposed to both stress and Meth. Their results showed that the COX inhibitor, ketoprofen, attenuated the enhanced monoaminergic toxicity induced by stress as well as Meth administration (Northrop and Yamamoto

2013). Further experiments demonstrated that an increased permeability of BBB induced by neuroinflammation might underlie the synergistic mechanism of stress and

Meth. Ketoprofen, applied either during or post the treatment, significantly reduced the impairment of BBB (Northrop and Yamamoto 2012). Despite its therapeutic potential on synergistic effects of Meth and stress, ketoprofen, however, did not have a protective effect on Meth alone. This is consistent with a previous report regarding to the development of COX as a potential intervention target on Meth toxicity (Thomas and

Kuhn 2005b). The current results also suggest that neuroinflammation may exacerbate the existing monoaminergic damage, and thus, promote the disease progression

(Thrash et al. 2009).

Sigma receptor (Sig-R), an endoplasmic reticulum protein, has two subtypes expressed in the brain (Sig-1R and Sig-2R) (Maurice and Su 2009). Meth binds to Sig-R with preferential binding affinity on Sig-1R (2-4 μM), ten times higher than Sig-2R (16-47 μM)

(Yasui and Su 2016). Sig-1R has been implicated in the addiction and toxicity induced by Meth (Itzhak 1993; Matsumoto et al. 2008; Nguyen et al. 2005; Takahashi et al. 2000;

Ujike et al. 1992). The adaptive upregulation of Sig-1R in the mid-brain was observed after 5 weeks of self-administrated with Meth (Stefanski et al. 2004). Antagonists of Sig-

1R block the Meth-induced neurotoxic effects (Matsumoto et al. 2008) and prevent the development of behavioral sensitization to Meth (Takahashi et al. 2000). In the CNS,

Sig-1R is expressed in microglia (Gekker et al. 2006). A sigma receptor antagonist

(SN79) was found to suppress microglial activation and proinflammatory cytokine release in vivo, which blocks the subsequent Meth-induced neurotoxicity. In vitro, inhibition of Sig-1R blocked the Meth-induced microglial apoptosis (Shen et al. 2016) and with pretreatment of Ditolylguanidine (DTG) [rlm1]or afobazole, sigma receptor agonists, the ATP-induced Ca2+ in microglia decreased and proinflammatory cytokine expression reduced (Cuevas et al. 2011). Although these results suggest sigma receptor as a viable target, it is still too early to conclude that blocking Sig-1R reverse the Meth-induced toxicity by modulation of microglial activity. First, localization studies indicated that Sig-1R was not exclusively expressed in microglia (Matsumoto et al.

2007). Second, SN79 is also reported with attenuation of Meth-induced astrogliosis

(Robson et al. 2014). In cultured astrocyte, Sig-1R is involved in Meth-induced astrocyte activation in a positive feedback manner (Zhang et al. 2015). More studies focused on the exact biochemical relationship between Sig-1R and Meth in microglia are needed. The interaction of Sig-1R and inositol 1,4,5-triphosphate receptors (IP3Rs) on mitochondria-associated ER membrane (MAM) is worth to be investigated on cultured microglia treated with Meth. The potentiation of Ca2+ transmission between ER and mitochondria might play a role in Meth-induced microglial activation (van Vliet et al.

2014).

The most striking progress having been made recent years based on neuroinflammation is the use of ibudilast to treat Meth dependence. Ibudilast exerts its anti-inflammation effect by inhibition of phsphodiesterase-4 (Huang et al. 2006;

Suzumura et al. 1999). Because its ability to cross the BBB and suppress the microglial activation, ibudilast has been thoroughly studied for potential efficacy on Meth addiction.

To date, Ibudilast has been demonstrated to be effective in reducing the self- administration of Meth and rate of stress-induced Meth relapse (Beardsley et al. 2010;

Snider et al. 2013). Moreover, it is also showed to modulate Meth-induced behavioral change (Snider et al. 2012). The neuroprotective role of this drug has been attributed to its inhibition of inflammatory response (Mizuno et al. 2004). Given the fact that Meth is prevalent in HIV-infected patients and microglia are one the most important common targets that mediate the rapid progression of HAND in HIV patients with Meth abuse, it is very meaningful that ibudilast also inhibits Tat-induced proinflammatory cytokines release from microglia (Kiebala and Maggirwar 2011). Altogether, this pharmacotherapy is quite promising for Meth addiction and may block the potentiation effect of Meth on the pre-existing neurodegenerative disorders, such as HAND. Currently, using ibudilast for Meth dependence has completed the phase I clinical trial for drug safety, and now is undertaking the phase II clinical trial (NCT01860807).

Figure 1. Mechanisms of microglia-induced neurotoxicity in Meth abuse. The simplified schematic of neuroimmune mechanisms of Microglia-mediated neurotoxicity in

Methamphetamine abusers.

7. Inflammasome activation in Drug Abuse

7.1. Overview of inflammasome in CNS system

Due to the physical isolation of blood-brain barrier (BBB), the peripheral immune cell infiltration and free passage of blood molecules into the healthy central nervous system (CNS) are highly restricted (Broadwell and Sofroniew 1993; Bush et al. 1999;

Medawar 1948; Muldoon et al. 2013). For this reason, the CNS resident innate immune system primarily response to the invading pathogens and/or tissue damages

(Hanamsagar et al. 2012). In recent years, increasing attention has been focused on

Nod-like receptors (NLRs), a cytoplasmic pattern recognition receptor (PRRs) that is responsible for the processing and release of IL-1β and IL-18 (Strowig et al. 2012). Up to now, there are 22 members of NLRs have been found in human and 34 in mice, which can be divided into four subfamilies based on their different N-terminal regions

(NLRA, NLRB, NLRC, and NLRP) (Kong et al. 2016; Motta et al. 2015). As critical cytosolic sensors, NLRs are responsible for the reorganization of pathogen-associated molecular patterns (PAMPs) and DAMPs (Martinon et al. 2009).

In a recently published review, the author analyzed an RNA-sequencing transcriptome and splicing database of neuronal cells including the expression level of

NLRs in the brain (Kong et al. 2016). In brief, all cell types in the brain express NLRs, and NLRP3 is mainly expressed by the microglia. Current researches suggested that drug abuse-induced inflammasome activation are primarily medicated by NLRP3.

Therefore, this review focuses on microglial NLRP3 inflammasome activation in the context of drug abuse. The roles of NLRs in the neuronal pathogenesis have been well- studied in recent years. The NLRPs (1, 3, 10, 12) are found to be involved in the development of neurodegenerative diseases including multiple sclerosis (MS),

Alzheimer’s disease (AD) and Parkinson’s disease (PD) (Gharagozloo et al. 2015;

Gustot et al. 2015; Heneka et al. 2013; Kaushal et al. 2015; Murphy et al. 2014). Among

all these NLRs in the CNS, NLRP3 is the most-studied. The NLRP3 inflammasome is a multimeric protein complexe composed of cytosolic sensor NLRP3, bridge protein apoptosis-associated speck-like protein containing a CARD (ASC), and cysteine protease caspase-1 (Guo et al. 2015a). In response to stimuli, NLRP3 recruits the ASC protein and serves as a caspase-1-activating scaffold. The inactive pro-caspase-1 oligomerizes and is auto-proteolyticly cleaved into the active form (Guo et al. 2015a).

Activation of caspae-1 can directly induce the processing and release of IL-1β and IL-

18.

Compared to other NLRs that require direct binding with their activating ligand, the interaction of ligands with NLRP3 is more complex (Jo et al. 2016). There is an expanding list of NLRP3 ligands, which are mostly structural and functional unrelated

(Vanaja et al. 2015). Currently, the exact mechanisms that how the NLRP3 protein interacts with such a wide variety of ligands are still largely unknown. However, certain common patterns of activating stimulus have been identified. It is widely accepted that the full activation of NLRP3 requires two independent signals. The first priming signal is stimulated by NF-κB pathway to upregulate the major component (NLRP3) and substrate (pro-IL-1β) of inflammasome (Bauernfeind et al. 2009). Emerging evidences indicate that the priming process is more complicated, which involves post-translation regulation of NLRP3 and ASC protein (Hara et al. 2013; Juliana et al. 2012; Lin et al.

2015; Py et al. 2013; Rodgers et al. 2014). As a prototypical first activation signal, LPS is proved priming NLRP3 both in the transcriptional and post-translational manners

(Han et al. 2015). For this reason, LPS is widely applied as the first priming signal in the inflammasome activation, even in the CNS sterile inflammation (Halle et al. 2008;

Shi et al. 2012; Shi et al. 2013). After priming, distinct signals are required to active

NLRP3 and thus promote the assembling of inflammasome complex. After priming, distinct signals are required to active NLRP3 and thus promote the assembling of

inflammasome complex. The potentially involved pathways have been extensively studied, and three activation models were introduced: potassium flux through ion channels, lysosomal membrane destabilization and release of cathepsin, and mitochondrial damage (Guo et al. 2015a).

In the context of traumatic injury, neurodegenerative disease, and long-term drug abuse induced neurotoxicity, the CNS resident immune system primarily deals with self- derived “sterile insults” released by the damaged neurons or other glial cells. The result of the immune cells activation can be either beneficial or detrimental. It is important to determine the timing of engagement and specific downstream inflammatory pathways that directly associates with neuropathology. Otherwise, non-specific immune suppressive therapy will also eliminate the inflammatory cascade that supports healing.

Thus, our ultimate research goal is to promote the resolution of inflammation by dampening the specific signaling pathways tied to neurotoxicity. Abundant evidence indicate that NLRP3 inflammasome is actively involved in the chronic sterile CNS inflammation and leads to detrimental consequences (Codolo et al. 2013; Gris et al.

2010; Hafner-Bratkovic et al. 2012; Halle et al. 2008; Heneka et al. 2013; Inoue et al.

2012; Jha et al. 2010; Meissner et al. 2010; Shi et al. 2012). The critical role of the

NLRP3 inflammasome in promoting neuronal damage was further demonstrated in the traumatic-induced injure (de Rivero Vaccari et al. 2012). In both neurodegenerative disease and actual neuronal injury, the DAMPs are released and quickly initiate a cascade of danger-associated intracellular signaling, which activates the NLRP3 inflammasome that orchestrates the inflammatory signaling in response to neuronal danger signals. One of the most potent effects of NLRP3 inflammasome activation is through processing and release of IL-1β and IL-18, which are critical amplifiers of the innate immune response to CNS damage. The excessive release of pro-inflammatory

cytokine exacerbates excitotoxicity-induced neuronal damage (Hara et al. 1997;

Pearson et al. 1999; Takahashi et al. 2003), and in turn, promotes the DAMPs-induced inflammasome activation. This vicious cycle can be further fueled by neuropathogenic factors such as amyloid-β or α-synuclein. In general, NLRP3 inflammasome signaling is a specific inflammatory pathway tightly associated with the initiation and maintenance of neurotoxicity. Thus, targeting inhibition of NLRP3 inflammasome activation might be a promising therapeutic strategy.

Recently, a novel specific small-molecular inhibitor (MCC950) has been developed and reported to successfully reduce the IL-1β production in vivo (Coll et al. 2015). The specific inhibition of NLRP3 inflammasome not only delayed the onset of experimental autoimmune encephalomyelitis (EAE) but also attenuated the severity of the disease.

Administration of MCC950 to APP/PS1 mice (an animal model of Alzheimer’s disease) significantly reduced the amyloid-β accumulation and thus, significantly improved the cognitive function. The enhanced clearance of amyloid-β was achieved via increased phagocytosis by microglia, which dampens the neuroinflammation-induced neurotoxicity

(Dempsey et al. 2017). In addition to the small-molecular inhibitor, microRNA that negatively regulate NLRP3 expression was also identified (Bauernfeind et al. 2012).

The overexpression of miR-223 reduced the erythrocyte lysis-associated microglia activation and neuronal damage, which suggests a protective role of miR-223 in intracerebral hemorrhage.

Like traumatic injury and neurodegenerative disease, both autonomous and non- autonomous neuronal cell death contribute to the long-term drug abuse-induced neurotoxicity. The microglial NLRP3 is activated in response to DAMPs release, which promotes the proinflammatory-induced secondary neuronal damage. Specific NLRP3 inhibition may cut off the auto-amplification loop between neuronal damage and sterile

inflammation. Hence, the evidence against the roles of NLRP3 activation in neuronal dysfunction induced by long-term abuse of drugs will be further discussed in next part.

7.2. Inflammasome in alcohol abuse

According to the 2015 National Survey on Drug Use and Health (NSDUH), 6.2 percent of people who are at age of 18 and older suffered from the Alcohol Use

Disorder (AUD) in the USA. Around 88000 people died for the alcohol-related reasons

(Alcohol and Public Health: Alcohol-Related Disease Impact. Centers for Disease

Control and Prevention). Ethanol is a small and amphipathic molecule. Therefore it can freely cross the BBB. Long-term abuse of alcohol has been reported associated with several negative impacts on normal neurological function, including habit formation, decision making, stress, and reward (Koob and Volkow 2010). Animal studies indicated that chronic use of alcohol activated microglia and astrocytes accompanied with the increased expression levels of proinflammatory cytokines, which inhibited the neurogenesis and induced long-term behavioral alterations (Fernandez-Lizarbe et al.

2009; He and Crews 2008; Nixon and Crews 2002; Pascual et al. 2007; Qin et al. 2008).

Two important alcohol-related neurotoxic mechanisms-proinflammatory factors release and ROS activation-have been identified (Chastain and Sarkar 2014). Over-activated microglia are considered primarily responsible for the elevated neuroinflammatory signaling and the development of neurological deficit (Lee et al. 2004; Zhao et al. 2013).

In contrast, anti-neuroinflammatory drugs (minocycline and doxycycline) were reported to significantly decrease the alcohol consumption (Agrawal et al. 2011; McIver et al.

2012). These promising therapeutic effects of anti-neuroinflammatory strategy push researchers to further investigate the molecular mechanisms of alcohol-induced neuroinflammation.

IL-1 related signaling pathway has been well-studied in alcohol-associated neurological impairment. In human postmortem brain tissue, the expression levels of IL-

1β and inflammasome proteins (NLRP3 and NLRP1) are significantly upregulated in the hippocampal area (Zou and Crews 2012). The administration of IL-1 receptor antagonist significantly reduced the acute alcohol-induced sedation and promoted the recovery from alcohol-induced motor impairment (Wu et al. 2011). It is well-accepted that IL-1 signaling is regulated both by TLR4-NF-κB pathway and casepase-1-induced maturation process, which is the priming and activation signal of inflammasome (Davis et al. 2011). For this reason, the roles of the inflammasome in alcohol-induced toxicity were extensively studied. As the significant priming signal of the inflammasome, TLR4 was found upregulated in microglia and played a pivotal role in alcohol-induced proinflammatory cytokine production and psychological impairments (Alfonso-Loeches et al. 2010; Fernandez-Lizarbe et al. 2009; Pascual et al. 2011; Pascual et al. 2015; Wu et al. 2012). In addition to TLR4, high mobility group box-1 (HMGB1), another important inflammasome priming signal, has been found released from damaged neuron (Wang et al. 2015; Zou and Crews 2014). Further studies indicate that alcohol induces the acetylation and phosphorylation on HMGB1, which is highly associated with its release

(Bonaldi et al. 2003; Lippai et al. 2013; Youn and Shin 2006). Correspondingly, the expressional level of receptors (TLR2, TLR4, TLR9, and RAGE) of HMGB1 was also found elevated in response to alcohol consumption (Lippai et al. 2013). There is evidence indicating that NLRP3 activation in macrophage/monocyte stimulated the release of HMGB1 to amplify the inflammatory signaling (Willingham et al. 2009).

However, whether such vicious cycle exists in alcohol-induced neuroinflammation has not been determined. As the second activation signal of the inflammasome, previous studies suggest that alcohol-induced mitochondrial dysfunction and oxidative stress contribute to the inflammasome activation in astrocyte and neuronal progenitor cells

(Alfonso-Loeches et al. 2014; De Filippis et al. 2016). Taken together, both the first priming signal and the second activation signal can be induced by alcohol. To characterize the inflammasome activation in alcohol-induced neuroinflammation, a study was performed on mice with both pharmacological and genetic manipulation of an essential component of the NLRP3 inflammasome. After administrated with 5% ethanol for five weeks, the expressional levels of NLRP1, NLRP3, ASC, and proinflammatory cytokines increased in control group, suggesting the functional role of alcohol as the first priming signal. On the other hand, the increased caspase-1 activity and IL-1β release proved alcohol could also work as the second signal and complete the NLRP3 inflammasome activation (Lippai et al. 2013). In the mice genetically deleted with TLR4,

NLRP3, or ASC less, ethanol administration failed to induce the activation of caspase-1 and production of IL-1β. Furthermore, the caspae-1 activity and IL-1β were found not increased in NLRP3-KO OR ASC-KO mice (Lippai et al. 2013). Based on the available evidence, inflammasome-IL-1β signaling cascade plays a critical role in alcohol-induced neuroinflammation. Following studies focused on the inflammasome activation and its association with alcohol-induced neuronal damage and function impairments. The ethanol-induced inflammasome activation impairs the differentiation of neuronal progenitor cells into the mature neuron and thus, inhibits the neurogenesis in the hippocampal area (De Filippis et al. 2016; Nixon and Crews 2002; Zou and Crews

2012). By pharmacological blockade of inflammasome activation, the alcohol-induced impairment of neurogenesis could be reversed (Zou and Crews 2012). Although rIL-1ra successfully prevented the alcohol-induced inflammasome activation and proinflammatory cytokines release, its protective role in neurogenesis is still largely unknown. In future, more experiments should be tested on NLRP3-KO or ASC-KO mice to demonstrate whether inhibition of inflammasome could reduce the drink of alcohol or reverse the neuroinflammation-associated neuronal damage.

Gut-liver-brain axis is another promising but less studied area associated with inflammasome and alcohol abuse. It has been hypothesized that the peripheral endotoxin could cross into the brain due to the impairment of BBB integrity by alcohol consumption (Haorah et al. 2007; Haorah et al. 2005). Furthermore, it is well-accepted that alcohol disrupts the intestinal epithelium integrity, which promotes the translocation of intestinal microbiome (Draper et al. 1983; Fouts et al. 2012; Hartmann et al. 2012).

Thus, it is reasonable to assume that alcohol can increase the endotoxin circulation and further enter into the brain tissue. However, recent evidence suggests that endotoxin level does not change after five weeks of alcohol administration, which ruled out this possibility (Lippai et al. 2013). It might be explained by the fact that, in a healthy individual, the circulated endotoxin would be kept in check by the interaction of multiple organs (Wang et al. 2010). However, it is still unknown that if translocated endotoxin could escape the surveillance of immune system in the late stage of alcohol abuse, which is featured by a persistent systemic inflammation and liver detoxification impairment. In that case, it is still possible that gut-derived endotoxin will cross into the brain and prime the alcohol-induced inflammasome activation. Another potential connection between gut and brain in the context of alcohol abuse might be mediated by the liver damage. It has been found that alcohol-induced translocation of endotoxin can initiate the inflammasome-related proinflammatory response in liver (Ganz et al. 2011).

The activation of inflammasome not only plays a central role in alcoholic liver disease but also promotes the liver DAMPs release into the blood (Hritz et al. 2008; Petrasek et al. 2012). Liver is considered as a major source of inflammatory cytokines releasing into the serum (Szabo et al. 2011), thereby inflammasome activation in liver induced by the gut-derived endotoxin could further amplify the systematic inflammation. Once these circulated proinflammatory cytokines enter the brain tissue and activate the NF-κB pathway, the CNS inflammasome is primed to be activated (D'Mello and Swain 2017).

Finally, whether gut-liver-brain axis was achieved by the liver-potentiated generalized immune response and subsequent neuroinflammation needs to be further investigated.

The inflammasome activation in both liver and brain might serve as a converging signaling that provides a promising therapeutic target to dampen the inflammatory cross-interaction in systematic level.

7.3. Inflammasome in stimulants abuse

Cocaine and methamphetamine (Meth) are two primary abused psychostimulants in the United States. Cocaine is a psychostimulant drug that binds to dopamine transporter and inhibits its reuptake of synaptic dopamine. Compared to cocaine, methamphetamine not only blocks the reuptake but also promotes the dopamine release. They are both highly addictive and neurotoxic after long-term abuse.

Accumulating evidence suggest that neuroinflammation underlies the mechanisms of neurotoxicity and provides a promising target. To better understand the mechanisms of cocaine- and Meth-related neuroinflammation, latest researches on inflammasome activation were reviewed. Also, whether inflammasome-related signaling serves as a converging pathway mediating the synergistic neurotoxic effect of HIV-1 infection with stimulant abuse was also discussed.

In mice administrated with cocaine, NF-κB signaling was found to be activated and associated with structural changes that mediate the drug reward-based learning (Ang et al. 2001; Russo et al. 2009). In consistent with this, the upregulation of multiple proinflammatory mediators and microglia activation were detected in cocaine abused animal model (Blanco-Calvo et al. 2014; Piechota et al. 2010; Renthal et al. 2009). It has been suggested that the proinflammatory signaling activation is attributed to the dysregulated redox status after cocaine administration (Dietrich et al. 2005; Muriach et al. 2010; Uys et al. 2011). Although with the activation of the first signal (NF-κB

signaling) and the second signal (oxidative stress), there is little evidence indicating the inflammasome activation in the brain after exposure to the cocaine. In cultured microglia, cocaine stimulates the proinflammatory release mediated through the ER stress-autophagy and ER stress-TLR2 axes (Costa et al. 2013; Guo et al. 2015b).

However, given the fact that ER stress is also a well-accepted activating signal for

NLRP3 inflammasome activation (Lebeaupin et al. 2015; Menu et al. 2012), there is no further study performed to investigate the role of the inflammasome in cocaine-induced microglial activation.

On the other hand, cocaine downregulates the expression of tight junction while upregulates the brain endothelial adhesion molecule and the expression of CCL2

(Dhillon et al. 2008; Gan et al. 1999). Therefore, cocaine not only impairs the integrity of

BBB but also promotes the transmigration of monocyte. In HIV-1 infected patients, cocaine abuse enhances the transmigration of HIV-1 infected monocyte/macrophage

(Zhang et al. 1998). Emerging evidence suggest that HIV-1 virus proteins could prime and activate the NLRP3 inflammasome (Chivero et al. 2017; Guo et al. 2014; Walsh et al. 2014b). For this reason, it is worth investigating whether cocaine could potentiate the

HIV-1-induced inflammasome activation. Current evidence indicates that cocaine potentiates the ROS activation in HIV-1-infected macrophage, which is accompanied by the upregulation of inflammasome forming genes (Atluri et al. 2016). It also suggests

NLRP1-caspase-5 axis may also contribute to the synergistic effect induced by the cocaine and HIV-1 infection. The potentiated activation of ROS signal was considered as a converging point that activated the NLRP3 inflammasome. However, the caspase-

1 activity and processing of IL-1β were not detected, which were critical to evaluate the inflammasome activation. Taken together, current evidence suggests that inflammasome may serve as a converging signaling that mediates the synergistic

proinflammatory effect in the macrophage. In future, whether cocaine-induced ER stress also potentiates the HIV-1-induced inflammasome activation in microglia is worthy of further investigation.

Chronic abuse of Meth is a feature with neurotoxicity marked by diminished dopamine concentration, low level of the dopamine transporter, and neuroinflammation

(Bowyer et al. 2008; Schwendt et al. 2009; Seiden and Sabol 1996; Sekine et al. 2008;

Volkow et al. 2001b). Although with abundant evidence against Meth-induced microglial activation as reviewed above, little study performed on microglia focuses on inflammasome. However, HMGB1 was found to be upregulated in Meth-administrated rat and induced the IL-1β production (Frank et al. 2016). The author proposed that

HMGB1 be released from the Meth-stressed neuron. It has been demonstrated that neuron-derived HMGB1 is recognized by microglia as the danger-associated signal and primes the NLRP3 inflammasome (Weber et al. 2015). As for the second activation signal, there are accumulating evidence suggesting that Meth application induces the mitochondrial damage and ROS production (Brown et al. 2005; Brown and Yamamoto

2003; Mashayekhi et al. 2014; Tian et al. 2009). Therefore, after priming with neuronal released HMGB1 in vivo, Meth is potential to activate the microglial NLRP3 inflammasome via the mitochondrial ROS pathway (Zhou et al. 2011).

7.4. Inflammasome in Morphine abuse

Morphine is originally isolated from poppy straw or opium poppy (Hagel and

Facchini 2010). It is on the World health organization’s list of Essential Medicines as an effective analgesic medicine (Organization 2010). Up to now, morphine is still one of the most effective drugs to treat both acute and chronic severe pain. However, the clinical application of morphine is limited by its long-term deleterious effects including addiction and withdrawal symptoms (Martin and Fraser 1961; Tompkins et al. 2009; Wesson and

Ling 2003). In chronic pain management, the tolerance of morphine will occur on the repeated uses of the drug, which increase the dose of the drug to achieve the equal extent of pain relief (Nestler 1996). The increased amount of drug, in turn, promotes the development of drug dependence. To prevent the diminished morphine responsiveness in patient, multiple hypotheses were raised including µ-opioid receptor desensitization, synaptic plasticity changes, morphine-induced microglial activation and proinflammatory cytokines release (Appleyard et al. 1999; Bohn et al. 2000; Mao 2004; Mao et al. 2002;

Raghavendra et al. 2002). In this review, we primarily focus on the role of inflammasome-induced cytokines release in morphine tolerance.

Traditionally, morphine was defined as an immunosuppressive drug that subjects prolonged abuse under susceptibility of various infectious diseases (Vallejo et al. 2004;

Wei et al. 2003). Although many basic functions of both innate and adaptive immune system such as phagocytic activities of macrophages or T, B-cell antibody responses were inhibited by morphine, the production of proinflammatory cytokines is, in general, increased by morphine administration (Odunayo et al. 2010). Further studies discovered that morphine promoted the translocation of intestinal bacteria that induced the sepsis in mice (Frenklakh et al. 2006; Hilburger et al. 1997; Mellon and Bayer 1998).

Priming with morphine significantly potentiated proinflammatory responses in LPS administrated rats and promoted the progression of sepsis to septic shock (Ocasio et al.

2004). All these evidence provide an alternative explanation for morphine-induced vulnerability to infection, which is achieved by impairing the pathogen clearance capability while augmenting the pathogenic inflammatory response. Taken together, the immune modulation of morphine is much more complicated than we thought.

Recently, emerging evidence indicate that, even though the systemic administration of morphine dampens the activation of the peripheral immune system, local CNS

resident immune cell-microglial, is over-activated (Ferrini et al. 2013; Matsushita et al.

2013; Wang et al. 2012). Because cytokines released from activated microglia might alter the neuronal adaption to morphine (Kawasaki et al. 2008; Zhang et al. 2011a), the role of neuroinflammation in the development of morphine tolerance was extensively investigated. Further studies confirmed that morphine could non-stereoselectively bind to the accessory protein of TLR4, triggering the TLR4 oligomerization and proinflammation (Wang et al. 2012). It has been demonstrated that, while µ-opioid receptor expressed on the microglia-mediated analgesic effect of morphine (Ferrini et al.

2013), the microglial TLR4 is critical to the development of morphine tolerance (Eidson and Murphy 2013; Hutchinson et al. 2010). Inspired by this, following studies targeting microglial activation and subsequent cytokines release successfully block the chronic morphine-induced tolerance (Jiang et al. 2015; Mika et al. 2009). Among those morphine-induced cytokines, IL-1β stands out for its strong anti-analgesic effects against morphine and important role in morphine tolerance (Johnston et al. 2004; Shavit et al. 2005). Administration of morphine significantly stimulated the IL-1β release that is mediated by the TLR4 signaling, and IL-1 receptor antagonism, substantially reversed the morphine tolerance (Liang et al. 2016; Raghavendra et al. 2002). Because NLRP3 activation is primed by TLR4 activation and causes the release of IL-1β, the NLRP3 inflammasome is receiving more and more attention in the research field of morphine tolerance.

After chronic administration of morphine (10 mg/kg, twice a day for 7 days), the

Western blot analysis of spinal cord indicated an increase of IL-1β processing, while the pro-IL-1β was not significantly changed. In consistent with this, the activation form of caspase-1 was also significantly elevated while its pro-enzyme form remained constant

(Cai et al. 2016). To confirm the morphine-induced NLRP3 inflammasome activation in

vitro, the experiments in BV-2, a cell-line of microglia, were also performed. After treated with 200 µM of morphine, with or without the additional ATP signal, both the

NLRP3 and pro-IL-1β were significantly upregulated. Combined with the second activation signal provided by ATP, potent processing and release of IL-1β and caspase-

1 were observed in supernatant (Cai et al. 2016). All results indicated that morphine could work as the first priming signal, which allowed the microglia response to the second neuronal danger-associated signal (ATP). In addition to this, they also found morphine induced a strong production of mitochondrial ROS that might work as the second activation signal. After pre-administration of procyanidins, a potent free radical scavenger, both the morphine-induced activation of mitochondrial ROS and NLRP3 inflammasome were blocked (Cai et al. 2016). According to this study, it seemed morphine could both prime and activate the NLRP3 inflammasome simultaneously. In contrast to this, a study focused on spinal dorsal horn microglia indicated morphine only primed the NLRP3 inflammasome through the TLR4-NF-κB pathway, while the second signal was induced by DAMPs release from injured neuron (Grace et al. 2016). In the rat model with priming of a short course of morphine, a second prolonged enduration of chronic constriction injury (CCI)-allodynia was found mediated by microglial NLRP3 inflammasome. In a combination of morphine and CCI, the level of microRNA-223 that negatively regulated the expression of NLR3P was significantly decreased. The other essential components of NLRP3 inflammasome (TLR4, NLRP3, pro-Casepae-1, pro-IL-

1β) were all upregulated. As for the P2X7R, the critical receptor sensing the ATP released from an injured neuron was also elevated. Taken together, morphine induced a persistent sensitization status of microglia that prone to activate in response to neuronal DAMPs. After the introduction of P2X7R selective inhibitor, the morphine- induced development of sensitization was reversed. It indicated that morphine itself

might not be enough to elicit the activation of the NLRP3 inflammasome, but rather potentiate the pre-existing sterile neuroinflammation.

In future, whether the NLRP3 inflammasome selective antagonist could prevent the development morphine tolerance is highly clinically relevant. It will provide a novel strategy that maintains the strong analgesic effect of morphine while eliminating its chronic side-effects.

8. Summary

Traditionally, inflammation has four common cellular and molecular hallmarks: upregulation of proinflammatory cytokines and chemokines, activation macrophages

(and brain microglia), recruitment of leukocytes and tissue damage (Estes and

McAllister 2014). In the study of Meth-induced neuroinflammation, the solid evidence demonstrates that administration of Meth could induce a substantial inflammatory response (McConnell et al. 2015). Upregulated inflammatory mediators and proliferation of microglia was found highly correlated with subsequent Meth-induced neurotoxicity

(Kuhn et al. 2006; LaVoie et al. 2004). The Meth-induced microglial activation temporally precedes the neuronal damage and response to Meth application in a dose- dependent manner, suggesting a specific causal relationship with Meth-induced neurotoxicity (LaVoie et al. 2004; Thomas et al. 2004b). In human abusers, overactivated microglia has been found in multiple brain areas, which exist for years even in Meth abstainers (Sekine et al. 2008). Taking together, these results indicate neuroinflammation is a potential target for Meth-induced neurotoxicity. Multiple pharmacotherapies targeting neuroinflammation have been carried out in experimental animals and demonstrated neuroprotective effects against the Meth-induced neurotoxicity. However, because of the limited information on Meth’s target receptors in microglia, the precise molecular mechanism(s) for these protective drugs are still not fully understood. Given the possibility that the inflammatory responses of microglia after

Meth stimulation in vivo are a secondary response to the neuronal DAMPs (i.e.,

HMGB1 or dopamine-quinone), it is worth to investigate whether Meth could interact with other neuronal toxic factors to enhance the microglial activation in a synergistic manner. All in all, identification of Meth specific molecular receptor(s) in microglia will

allow us to develop more specific therapeutic strategies for not only eventually helping abusers quit from Meth, but also preventing or ameliorating Meth-induced neurotoxicity.

Currently, accumulating evidence mentioned above shows that inflammasome activation in the context of drug abuse plays a pivotal role in microglia-mediated neurotoxicity (Atluri et al. 2016; Cai et al. 2016; De Filippis et al. 2016). It worth noting that NLRP3 inflammasome is also an intersection point between HIV-1 infection and its

CNS comorbidities such as drug abuse (Atluri et al. 2016; Guo et al. 2014; Hernandez et al. 2014). It is well-accepted that Meth and cocaine abuse potentiated the neurological impairment of HIV-1 infection (Dhillon et al. 2008; Hoefer et al. 2015;

Kesby et al. 2015a; Kesby et al. 2015b). There are evidence indicate that HIV-1 virus proteins could activate the NLRP3 inflammasome in cultured microglia (Mamik et al.

2016; Walsh et al. 2014b). However, further experiments are needed to clarify their role in complicated activation processes of the NLRP3 inflammasome. In pathological relevant concentrations, whether HIV-1 proteins could serve both priming and activation signals is still not fully understand. In another hand, the role of drug abuse played in

NLRP3 inflammasome activation is also complicated (alcohol influences both the priming and activation steps; Morphine primarily target the first priming signal; cocaine induces only the second activation signal; limited information regarding Meth abuse).

Taken together, further dissecting the underlying mechanism regarding potentiation effects of drug abuse on HIV-1-induced neuroinflammation is desperately needed. The better understanding of inflammasome as a specific intersecting molecular platform converging danger-associated signals derived both from HIV-1, and drug abuse could help us determine the optimized interventional strategy blocking their synergistic neurotoxic effects.

Chapter 2

Inflammasome activation by Meth potentiates lipopolysaccharide (LPS) stimulation of IL-1β

production in microglia

Abstract:

Methamphetamine (Meth) is an addictive psychostimulant that is widely abused all around the world. Ample evidence indicates that chronic abuse of Meth induces the neurotoxicity that mediated by microglia-mediated neuroinflammation. Although the activated microglia were observed both in the Meth-administrated animal model and human abuse patient, the exact mechanism of Meth-induced microglia activation is still largely unknown. Because anti-neuroinflammation has been developed as a promising therapeutic strategy against Meth dependence, it is important to investigate inflammatory pathways that are specifically tied to Meth-induced neurotoxicity. NLRP3 is a well-studied cytosolic pattern recognition receptor (PRR), which promotes the assembl of the inflammasome in response to the danger associated molecular patterns

(DAMPs). We hypothesized that Meth abuse could activate NLRP3 inflammasome in microglia and promote the processing and release of interleukin (IL)-1β, which mediate the Meth-induced neurotoxicity. Rat cortical microglia and brain slices were cultured with physiologically relevant concentrations of Meth before LPS stimulation, and IL-1β maturation and release were found to be increased. The treatment potentiated aggregation of inflammasome adaptor apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and activation of the IL-1β converting enzyme caspase-1. Inhibition of capase-1 totally reversed the priming effect of Meth, thus further confirming the involvement of the inflammasome.

1. Introduction

Methamphetamine (Meth) is a highly addictive psychostimulant and is the second most widely used illicit drug worldwide, after cannabis (Degenhardt et al. 2010).

Neurotoxic effects of chronic Meth abuse are well established by neuroimaging studies and psychological tests (Chang et al. 2007; Sekine et al. 2001). Chronic use of Meth leads to damage in the dopamine and serotonin nerve terminals, which is associated with neuropsychiatric dysfunction including deficits in episodic memory and decision making as well as with mental illnesses like anxiety, depression, and psychosis

(Glasner-Edwards et al. ; Hoffman et al. 2008; Meredith et al. 2005; Newton et al. 2004;

Scott et al. 2007). How chronic Meth abuse causes neuropsychiatric dysfunction is not fully understood. Studies have shown that Meth-mediated neurotoxicity is associated with upregulation of pro-inflammatory cytokines and chemokines (Cadet and Krasnova

2009; Krasnova and Cadet 2009; Loftis and Janowsky 2014). It has been shown that

Meth treatment upregulated the expression of interleukin (IL)-1β, tumor necrosis factor

α (TNF-α) and CCL2 in several brain regions including the striatum and frontal cortex

(Flora et al. 2003; Flora et al. 2002; Kelly et al. 2012; Nakajima et al. 2004; Yamaguchi et al. 1991a; Yamaguchi et al. 1991b). Because of persistent neuroinflammation- induced neurotoxicity (Gonzalez et al. 2014; Qin et al. 2007), the recovery often takes years. Previous studies indicated that suppressing the neuroinflammation significantly reduced the amount of drug that mice self-administrated and attenuated prime- and stress-induced relapse (Beardsley et al. 2010; Snider et al. 2013). Although accumulating evidence suggests that neuroinflammation could be a promising target for

Meth abuse, the mechanisms by which Meth initiates the resident proinflammatory cascade are still largely unknown.

Because microglia are key cellular mediators of neuroinflammatory processes, many early events of neuronal degeneration are accompanied by microglial activation

(Heneka et al. ; Perry and Holmes ; Thomas et al. 2004b). Thus, the roles of microglial activation in Meth-induced neurotoxicity have been intensively studied (Friend and

Keefe 2013; LaVoie et al. 2004; Sekine et al. 2008; Thomas et al. 2004b). Sustained and repeated application of Meth-induced a significant microglial activation in both animal models and human abusers (Sekine et al. 2008; Thomas et al. 2004b). Further studies demonstrate that blocking microglial activation effectively reduces the Meth- associated neuronal damage and drug-seeking behavior (Attarzadeh-Yazdi et al. 2014;

Beardsley et al. 2010; Hashimoto et al. 2007; Tanibuchi et al. 2010; Thomas and Kuhn

2005a; Thomas and Kuhn 2005c; Zhang et al. 2006). However, although with the preponderance of evidence supporting the central role of microglia in Meth-induced neuroinflammation (Cadet et al. 2007; LaVoie et al. 2004; Sheng et al. 1994; Yue et al.), the precise mechanisms by which Meth activates microglia are still unclear. Early characterization of Meth-induced microglial activation has demonstrated that many direct pharmacological effects associated with Meth, such as inhibition of the dopamine transporter (DAT), increase release of dopamine (DA), and activation of D1 and/or D2

DA receptors, may not contribute to Meth-induced microglial activation (Thomas et al.

2004b). However, the time-course, dose-response and pharmacological profiles of

Meth-induced microglial activation suggest that microglia are not merely a secondary response to Meth-induced dopaminergic terminal damage (LaVoie et al. 2004; Thomas et al. 2004b). It should be noted that most experiments related to Meth-induced microglial activation were performed in vivo, in which it is extremely difficult to identify the key factor contributing to the sustained microglial activation. To better investigate the direct cellular mechanisms of Meth in microglia, an extended study on isolated microglia is clearly needed.

Until recently, one research group reported that Meth induces the release of damage-associated molecular patterns (DAMPs) such as high mobility group box-a

(HMGB1) that indirectly target the microglia to upregulate IL-1β expression. It is well accepted that over-activated microglia produce and release proinflammatory cytokines, thus inducing collateral damage to neurons (Colton 2009). The most prominent feature of neurotoxic microglia is their secretion of proinflammatory cytokines like IL-1β (Block et al. 2007). IL-1β is a well-recognized cytokine in the orchestration of CNS inflammation and is associated with elevated risk of drug dependence (Basu et al. 2004;

Liu et al. 2011; Liu et al. 2009). In light of recent evidence, the priming roles of alarming signals such as HMGB1 in neuronal stress-induced neuroinflammatory responses were explored (Frank et al. 2015). The sensitization of proinflammatory responses by these endogenous danger signals is primarily achieved by priming the NLRP3 inflammasome for subsequent immune challenges (Weber et al. 2015). The inflammasome is a multiprotein complex consisting of NOD-like receptors (NLRs), apoptosis-associated speck-like protein containing a CARD (ASC) and Caspase-1 (Mariathasan and Monack

2007). Unlike other cytokines, IL-1β production is controlled both by NF-κB and inflammasomes at the transcriptional and post-translational level separately. The first signal upregulates the expression of pro-IL-1β, and the second signal leads to inflammasome activation, further processing and promoting the maturation of IL-1β.

The activation of the inflammasome must be primed with toll-like receptor (TLR) signaling, which not only keeps NLRs from being degraded but also increases the expression of pro-IL-1β. Caspase-1 is the converting enzyme that cleaves pro-IL-1β into the final active form (Walsh et al. 2014a). Multiple endogenous danger signals, including lysosome rupture, ROS activation, and low intracellular K+, may be required for this process (Schroder and Tschopp 2010).

Because neuroinflammation was marked by an increased local number of microglia accompanied by the shift of morphology from small cell body with fine, ramified processes into an activated state (Hanisch and Kettenmann 2007). After an injury in the CNS, the capability of migration and proliferation of microglia around damaged brain areas is increased (Streit et al. 1999). The activated microglia primarily damage the neuron by release neurotoxins such as proinflammatory cytokines (Luo and Chen 2012).

Thus, in our present study, whether the cellular morphology, cell number, migrating ability, and production of inflammatory-related cytokines were affected by Meth treatment alone was first examined in cultured microglia. Pathologically relevant concentrations of Meth fail to upregulate proinflammatory cytokines in mRNA levels but promote the maturation and release of IL-1β after LPS-induced pro-IL-1β increase. For this reason, isolated rat microglia and cultured brain slices were applied to investigate the primary effects of Meth on the production of IL-1β. The increased processing and release of IL-1β were detected in LPS-primed microglia after additional Meth treatment.

Blockade of caspase-1 reverse the Meth-mediated processing of IL-1β, which indicated the involvement of regulation of IL-1β in post-translational level. Thus, we hypothesized that Meth potentiates IL-1β secretion and its activity by activating the inflammasome.

2. Materials and Methods

2.1. Materials

(+)-Methamphetamine (D-Meth), (-)-Methamphetamine (L-Meth) and LPS (from

Escherichia coli 0111: B4) were purchased from Sigma-Aldrich (St. Louis, MO). Aliquots of LPS were kept as 1 mg/ml stock solution in a -80 °C freezer. Both the D-Meth and L-

Meth were prepared into 50 mM stock solution and kept in a -20 °C freezer. The stock solution was diluted to desired concentrations with DMEM 10 min before experiments.

All other chemicals, unless otherwise specified, were from Sigma-Aldrich.

2.2. Animals

Pregnant female Sprague-Dawley (SD) rats used for experiments were purchased from Charles River Laboratories (Wilmington, MA). Animals were housed at constant temperature (22°C) and relative humidity (50%) under a regular light-dark cycle (light on at 7:00 AM and off at 5:00 PM) with free access to food and water. All animal use procedures were strictly reviewed by the Institutional Animal Care and Use Committee

(IACUC) of the University of Nebraska Medical Center (IACUC No. 13-069-10-EP).

2.3. Isolation and culture of microglial and Neuronal cells

Microglia were prepared from the cerebral cortex of postnatal (0-1 day old) SD rats as described previously (Liu et al. 2012b). Briefly, rat cortical tissues were dissected out in cold Hank’s Balanced Salt Solution (HBSS: Mediatech, Inc. Manassas, VA). The physical dissociation was fulfilled by repeated titration with 5 ml pipet up to 5 times. The following chemical digestion was achieved with 0.25% trypsin and 200 Kunitz units/ml

DNase (Sigma-Aldrich, St. Louis, MO) at 37 °C for 15 min (neuronal culture) or 30 min

(microglial culture). The pellet was in a cloud shape and primarily contained with

dissociated cells with tissue debris. The digested tissues were then suspended in cold

HBSS and further dissociated by titration with 5 ml pipet up to 25 times. To remove the tissue debris, suspension medium was filtered through 100 μm and 40 µm pore cellular strainers (BD Bioscience, Durham, NC), respectively.

For the microglial culture, the isolated cells (25 x 106) were plated into T75 cm2 flasks in a high-glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 1x glutaMAX, 1% penicillin/streptomycin (Life Technologies,

Grand Island, NY), and 300 ng/ml macrophage colony-stimulating factor (M-CSF) supplied by the Culture Core facility of Department of Pharmacology and Experimental

Neuroscience, University of Nebraska Medical Center. The culturing medium was half- changed daily. After 10 days in culture, the flasks were gently shaken and detached cells were collected and seeded onto 35 mm2 (2.5x106 cells/dish) and 60 mm2 (7.5x106 cells /dish) culture dishes, and 12 well (1x106/well) or 96 well plates (0.4x106/well) based on the experimental requirements with M-CSF free DMEM. The suspensory glial cells (primary astrocytes) were removed 1 h after seeding by replacing the culture media. The resultant cultures were 98-100% microglia as determined by staining with anti-CD11b (Abcam, Cambridge, MA), a marker for microglia.

Primary cortical neurons were prepared from 19-day old embryonic rats. After physical dissociation, dissected cortical tissues were digested with 0.25% trypsin and

DNase (200 Kunitz) in 37 ºC for 15 min and then, generally dissociated with attached tissues by repeated titration (up to 15 times with 5 ml pipette). The dissociated cells were filtered through 100 and 40 µM pore cellular strainers. Isolates were seeded onto

6 poly-D-lysine-coated coverslips (0.2×10 cells/well in a 12-well plate) with plating medium (1% horse serum in neurobasal medium supplemented with 2% B27, 1% penicillin/streptomycin and 0.25% glutaMAX, all reagents from Life Technologies) for

the first 6 h and exchange to culture medium for 10-14 days (plating medium without 1% horse serum and further supplemented with 20 µM 5-fluorouracil to reduce non- neuronal cell growth). During this period, the culture medium were half-changed every two days.

2.4. Brain slices culture

Sprague-Dawley rats with 21-days postnatal were chosen to prepare for coronal brain slice cultures followed by instruction described by Liu, etc (Liu et al. 2017). The slices were cut by vibratome (Campden Instruments, MA752 Motorised advance vibroslice) that pre-sterilized by spray of 70% of ethanol followed by exposure to ultraviolet light within the biological safety cabinet for at least 1 h. The surgical instruments were sterilized by autoclave. To adjust the orientation of the isolated rat brain, we prepared the 2% of agarose gel with sterilized water and cut into the shape of the platform at an angle to help to position the brain. Whole cutting processes must be completed with the

Artificial cerebrospinal fluid (ACSF) that contained (in mM) NaCl 124.0, KCl 3.0, CaCl2

2.0, MgCl2 2.0, NaH2PO4 1.25, NaHCO3 26.0, and glucose 10.0. The pH was adjusted to 7.2, and the osmotic pressure was fall in the range between 290-310 mOsm, as measured by vapor pressure osmometor (WESCOR, Logan, UT). The purpose of

ACSF in brain slice cutting is to match the electrolyte concentrations, osmolality, and pH of the internal environment of the brain and thus, minimize the brain damage during the slice preparation. To achieve the sterilization standard, the ACSF solution were made with sterilized double stilled water and filtered by 0.22 µM strainer. The culture medium was made with 50% MEM (Gibco, Gaithersburg, MD), 25% horse serum

(Atlanta Biological, Flowery Branch, GA), 25% Hank’s solution (Invitrogen), and 6.5 mg/ml glucose. The inserts (30 mm diameter, sterile, 0.8 µM pore size, low-protein

binding membrane) were placed in to 6-well plate for brain slices. All plate with inserts were added with culture medium 1 h in advance and placed in an incubator.

Rats were anesthetized with isoflurane and decapitated by animal decapitator (Stoelting,

51330). A 10-cm dish with ice inside were covered with filter paper that moistening by cold ACSF. The whole brain was dissected out and placed in pre-cooled and oxygenated ACSF. After that, the brain was spooned out and further dissected out the cerebellum in a 10-cm dish with filter paper. The brain was then transferred to the vibratome stage with the cube of an agarose gel on the top of it. Super glue was used to fix the brain on the stage against the cube of agarose gel. The stage was placed in cutting chamber filled with ACSF. It worth noting that the oxygen must be provided to

ACSF through the whole cutting procedure. Each slice was 400 µM and separated by soft little brushes. The cut slices were carefully transferred to the culture insert in 6-well plate with 1 ml/well of culture medium below. There is only one side of the slice was faced with medium while another one side was not. Before placing the plate back to the incubator with 37 ºC, 5% CO2, it was important to make sure there was no bubble that potentially block the contact of brain slice with the medium.

Figure 3. Illustration of brain slice cutting and culturing technique.

2.5. Quantitative PCR analysis

The mRNA level of microglial pro-inflammatory cytokines were examined. Microglia was incubated with indicated concentrations of Meth for 12 h, with or without co- administration of LPS (100 ng/ml). Total RNA from microglia was extracted with TRIzol

(Ambion, Carlsbad, CA). Two micrograms of RNA were reverse transcribed into complementary DNA (cDNA) with the SuperScript IV First-Strand Synthesis System

(Invitrogen, Carlsbad, CA). cDNAs were amplified and quantified with the SYBR Green

PCR Master Mix system (Applied Biosystems, Warrington, UK) and results normalized to GAPDH internal controls. PCR primers used were listed as follows: forward IL-1β

GAC CTG TTC TTT GAG GCT GAC A; reverse IL-1β AGT CAA GGG CTT GGA AGC

AA; forward TNFα CGC CAC CGG CAA GGA; reverse TNFα GAC ATT CCG GGA

TCC AGT GA.

2.6. Total protein extraction and concentration determination

After priming with LPS for 6 h, the processing of pro-IL-1β and activation of capase-

1 were analyzed after 12 h of Meth treatment. Microglia treated with a combination of

Meth and LPS were lysed using RIPA buffer (Bio-Rad, Hercules, CA). In details, after treatment of Meth, cultured microglia were washed with ice-cold PBS (5 min x 3 times).

For each well in 6 well-plate, 150 µl RIPA buffer supplemented with protease inhibitor was added. The attached cells were scraped out and kept incubated in ice-cold environment for another 30 min. The cellular lysates were further centrifuged (RCF

8000 g) for 15 min and supernatant were transferred.

The BCA protein assay kit (ThermoFisher Scientific, Waltham, MA) was used to quantify the sample concentration. The procedure was followed by manufactural instruction and the sample concentration was determined by standard curves. For each

sample, 10 µl of supernatant was added to 90 µl of BCA working solution. After 30-min incubation in 37 ºC, the microplate reader was used to detect the absorbance of all the samples at the testing wavelength at 560 nm. Finally, the rest of samples were denatured and ready for the western blotting.

2.7. Western blotting

Twenty micrograms for total proteins was separated by 12% gel and transferred to nitrocellulose polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 3% BSA in Tris-buffered saline (TBS) and incubated overnight at 4 °C with either mouse monoclonal antibody to IL-1β at a 1:500 dilution (R&D system, MN, USA), rabbit polyclonal antibody to caspase-1 at a 1:200 dilution (Santa Cruz, Dallas, TX) or anti-mouse β-actin monoclonal antibody (1:5000, Sigma-Aldrich). Washing buffer was

TBS with 0.2% Tween (TBS-T). The secondary antibody was horseradish peroxidase

(HRP)-conjugated anti-rabbit or anti-mouse antibody (1:10000, Jackson

ImmunoResearch Laboratories, PA). Labeled proteins were shown by the Pierce enhanced chemiluminescence (ECL) system (Thermo Fisher Scientific, Waltham, MA).

2.8. Immunofluorescences

Immunocytochemistry was conducted to quantify ASC aggregation as a readout of inflammasome activation. Microglia were seeded on coverslips in a 12-well plate at a density of 0.5 × 106 cells per well. After priming with LPS (100 ng/ml), antagonists were applied 1 h before Meth treatment. The cells were fixed with 4% paraformaldehyde

(PFA) for 20 min at room temperature. Then, the cells were blocked and permeabilized in PBS with 10% goat serum and 0.1% Triton X-100 for 30 min.

The primary antibodies used here including rabbit polyclonal antibody to ASC

(Santa Cruz, CA, USA) at 1:100 dilution. The microglia were identified by mouse

monoclonal antibody CD11b in 1:500 dilution (Abcam, Cambridge, MA). The secondary antibodies used here were goat anti-rabbit Alexa 488 (1:1000) and Alexa 594 (1:1000) from ThermoFisher Scientific (Waltham, MA).

2.9. MTT assay

The cellular viability was assessed by MTT assay. To minimize the diversity between replicates, the microglia were cultured in 48-well plates with same density. Sterile PBS was used to make stocking solution of 3-(4,5-Dimethylthiazol-2-yl)-2,5-

Diphenyltetrazolium Bromide (MTT) at the concentration of 5 mg/ml (10x). The working solution was diluted 10 times with DMEM and pre-warmed before applied to cells. The pre-treated microglia were incubated with working solution of MTT in 37 ºC, 5% CO2 for

2 h. After that, the MTT solution in each wells were discarded and attached cells were dissolved in 200 µl of dimethyl sphingosine (DMSO). Additional 30 min were needed for full lysis. The dissolved solution was then transferred to 96-well plate. The final absorbance at 560 nm was determined by microplate reader.

2.10. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)

staining

Neuronal apoptosis was assessed by In Situ Cell Death Detection Kit, Fluorescein

(Roche Applied Science, Indianapolis, IN). The media from treated microglia were diluted 5 times by basal neuronal medium and were transferred to the cultured neuron for 24 h. After fixing (4% paraformaldehyde) and permeabilizing (0.1% Triton X-100), neurons were then processed for TUNEL staining for 1 h at 37 °C and mounted using

ProLong Gold antifade reagent with DAPI (Molecular Probes, Eugene, OR).

2.11. Cytokine analysis by ELISA

In the experiments measuring secretion of IL-1β, microglia were primed with LPS and then stimulated with indicated concentration of Meth. After first 6 h priming with

LPS (100 ng/ml), microglia were washed three times before further treatment of Meth.

For detection of IL-1β release, the supernatant of Meth-treated microglia collected at 6 h, 12 h, and 24 h. The IL-1β in supernatants were detected using the IL-1β ELISA kit

(R&D system, MN, USA). The experiments were performed following the manufacturer’s instructions. Briefly, the plates were coated with capture antibody overnight at room temperature, and then, the filtered (0.2-µm strainer) BSA (0.1%) was used as the blocking reagent. The capture antibody-coated 96-well plate were incubated with collected supernatants for at least 2 h in room temperature. It followed by 2-h application of the detection antibody. Finally, the Streptavidin-HRP working solution was incubated for 20 min before substrate solution added to each well.

2.12. Migration assay

Migration of microglia in response to CCL2 and CX3CL1 was assessed by using 8 µM- pore 24-well Trans well insert with a polyethylene terephthalate tracketched membrane

(BD Bioscience, Franklin Lakes, NJ). To ensure the cell adhesion that is necessary for migration, each insert membrane was coated with 10 µg/ml of fibronectin (Sigma, St.

Louis, MO). The minimal volume of coating solution was 400 µl of each 24 well to make sure insert membrane was evenly coated. Bubbles under the insert membrane were needed to be carefully excluded before placing into the 4 ºC overnight. For sterilization purpose, the 24-well plate needs to be well sealed by Parafilm (BEMIS, Neenah, WI).

Microglia were washed with PBS and pretreated with Meth 50 µM or not in serum- free DMEM (37 ºC). After 2-h of incubation, microglia were resuspended and counted by trypan blue. Each upper chamber of the fibronectin-coated insert were placed with microglia at a density of 5 x 105 cells/ml. The principle of adding medium in both upper

and lower chamber was determined by matching the medium surface level between inner and outside the chamber, which prevented the exchange flow disrupting the migration. CCL2 (25 ng/ml) and CX3CL1 (100 ng/ml) were added separately in the lower chamber as positive control while the serum-free DMEM were loaded as negative control. After 2-h or 6-h incubation at 37 ºC, the transmembrane microglia were fixed and stained with HEMA-3 solution (Fisher Scientific, Kalamazoo, MI). The microglia left on the upper chamber was gently removed with cotton swabs and PBS. The transwell membranes with microglia were removed from insert and numbers of stained microglia were counted with 6 randomly selected scope-fileds per well.

2.13. Colorimetric assay for Caspase-1 activity

Caspase-1 activity was measured by its colorimetric assay kit (RayBiotech,

Norcross, GA). The labeled YVAD-p-nitroanilide (pNA) is the substrate of enzyme caspase-1, which allows this assay to detect the change of caspase-1 activity after treatment. The performance was followed by the manufacturer’s instructions. After cleavage from YVAD by the caspase-1 within cellular lysis of pretreated microglia, the light emission of chromophore pNA was quantified by a spectrophotometer at 405 nm.

2.14. Data analyses

All data are expressed as mean ± S.D. unless otherwise indicated. Statistical analyses were performed by one-way ANOVA followed by post hoc analysis (indicated in each figure legend) in OriginPro 8.0 (OriginLab, Northampton, MA). A minimum p- value of 0.05 was chosen as the significance level for all tests. The densities of target western blot bands were quantified by AlphaView (ProteinSimple, San Jose, CA) and standardized by β-actin band density. In the section using a specific fluorescent probe against lysosomes (LysoTracker Red DND-99), 9 fields under the fluorescent

microscope were taken, and all cellular fluorescent intensities were quantified by Image

J (Schneider et al. 2012). The intensities of all individual cells in each field were averaged and transformed to fold changes against a control group. As for the activity of caspase-1 and NO production assay, the arbitrary absorbance of treated groups was also displayed as fold change against the control group. To quantify the neuronal apoptosis induced by Meth, 6 different fields under 40X objective were captured for each treatment group. The percentage of apoptotic neurons was analyzed by the number of TUNEL-positive cells normalized by the total DAPI signals. All experiments were performed in triplicate unless otherwise specified.

3. Results

3.1. Microglial morphologic, migration, and proliferation are not affected by Meth

treatment alone

Activated morphological change is a highly characteristic feature of microglial activation. However, our result in Fig 5 failed to observe the morphological transition from resting morphology, a small rod-shaped somata with numerous thin and ramified processes, to activated morphology with the amoeboid shape of the cellular body and thicker processes. Because the CX3CL1-CX3CR1 signaling axis plays a pivotal role in controlling the neurotoxic activation of microglia, the expression level of CX3CR1 was detected in our experiments. However, there was no obvious the protein level alteration of CX3CR1 within the Meth-treated microglia.

Recruitment of microglia to damaged brain areas is a key cellular event, so whether

Meth treatment altered the migration ability of microglia was tested. CCL2 and CX3CL1 are two major microglial chemotactic factors. In this study, we measured the chemotaxis of cultured microglia with CCL2 and CXCL1. As shown in Fig 6A, after 2-h incubation with CCL2 and CX3CL1 in the lower chamber, only latter one significantly increased the number of attracted microglia. In the group of 6-h incubation, both CCL2 and CX3CL1 significantly promoted the migration while the chemotactic effect of

CX3CL1 was still stronger than the CCL2. For this reason, CX3CL1 was applied for the following study as microglial chemotactic factors. In Fig 6B, microglia were either pretreated 2 h with or without 50 µM Meth. While the migration of microglia toward

CX3CL1 was increased in a dose-dependent manner, the Meth pretreatment did not further differentiate the chemoattractant capability of CX3CL1.

Except for the increased recruitment and morphological change of microglia, proliferation is another important feature of microglia-mediated neuroinflammation. Thus, the viability of Meth-treated microglia was determined after different incubation time. As

shown in Fig 7, the number of live microglia was not altered by 24-h and 48-h Meth treatment. However, the highest concentration of Meth (450 µM) significantly decreased the viability of microglia after 72-h incubation (from 1.00 ± 0.11 to 0.68 ± 0.08, fold change to control).

Figure 4. Meth did not increase the activated morphological change in cultured microglia

The cultured microglia were treated with different concentrations of Meth for 12 h.

Immunofluorescent staining with specific antibody againstCD11b and CX3CR1 were performed to display the morphology of microglia. All pictures were taken at 40X magnification.

Figure 5. Meth did not increase the activated morphological change in cultured microglia. A: After incubation with CCL2 25 ng/ml and CX3CL1 100 ng/ml, the microglia were stained and counted. The quantifications of 2-h and 6-h number of microglia chemotaxis was shown in bar graph respectively. B: CX3CL1s (25 ng/ml and 100 ng/ml) were chosen as positive controls to assess the influence of Meth treatment on microglial migration. Compared to the groups following the same procedure without

Meth treatment, microglia pre-treated with 50 µM Meth were incubated with CX3CL1 for

6 h. The quantification results were shown in associated bar graph. * p < .05, ** p < .01,

*** p < .001 vs Ctrl. All images were taken at 20X magnification.

Figure 6. Meth did not induce the proliferation of microglia. After seeding into the 96- well plate, microglia were treated with different concentrations of Meth. The viability of microglia was measured by MTT assay at three-time points. * p < .05 vs Ctrl.

3.2. Analyses of Meth-induced change of proinflammatory cytokines

The cytokine-mediated inflammatory response is critical to fight insults in the CNS.

Thus, the balance of pro- and anti-inflammatory cytokines is critical to resolve the detrimental neuroinflammation. We measured the transcriptional levels of IL-1β and

TNFα (the most prominent neurotoxic proinflammatory cytokine) in Meth-treated microglia. In the meantime, the supernatant was collected to detect the inflammatory- related cytokines.

To investigate if proinflammatory cytokines were upregulated in isolated microglia treated with Meth, we analyzed the production and secretion of IL-1β and TNFα via qPCR and ELISA. After treatment with different concentrations of Meth, there was no significant increase of proinflammatory cytokines (IL-1β and TNFα) detected in transcriptional levels (Fig. 8A, B). Consistent with this, the secreted IL-1β and TNFα in collected supernatants of Meth-treated microglia (both in dextrorotary and levorotatory form) were undetectable. while they were upregulated in supernatants of the LPS- treated group (Table.1). We also measured the secretion of other pro- and anti- inflammatory cytokines, such as IL-4, IL-6, and IL-10 (Table.1). The results showed that the release of these cytokines was not affected by Meth treatment alone.

A B

Figure 7. Meth did not stimulate the production of proinflammatory cytokines in transcriptional level. The transcriptional levels of IL-1β and TNFα were analyzed via qPCR after Meth treatment on isolated microglia (A and B).

Table. 1 Release of inflammatory-related cytokines in microglia after Meth treatment.

Microglial supernatant was collected after treatment of Meth. LPS (100 ng/ml) was added as a positive control. The concentrations of inflammatory-related cytokines were measured by ELISA.

3.3. Meth potentiate LPS-induced microglia activation and neurotoxicity

To test whether Meth treatment could potentiate pre-existing neuroinflammation, we primed microglia 6 h with 100 ng/ml LPS and then stimulated with Meth for additional 12 h. The activated morphological change was observed after Meth administration to LPS- primed microglia (Fig. 8). The number of processes was decreased by Meth application in a dose-dependent manner. Also, the cellular body adapted to a rounded/amoeboid shape. Meth treatment shortened the processes and converted these microglia into a larger soma.

After the successful detection of potentiated microglia by Meth, we collected the supernatant of microglia and transferred to the neurons. Increased neuronal apoptosis was delectated in groups treated with both LPS and Meth. After Meth treatment on

LPS-primed microglia, the supernatant was collected and diluted by pre-warmed neuronal basal medium (1:5). The complete neuronal medium was removed from the cultured neuron and replaced by the diluted microglia culture medium. To detect the apoptotic neuron, TUNEL staining was applied after 12-h treatment. Because previous studies indicated that Meth alone could induce neuronal apoptosis, we included the group of Meth alone as a comparison (Jayanthi et al. 2004). The percentage of TUNEL- positive cells was significantly increased after Meth treatment (Fig. 9). Although the administration of LPS alone and Meth alone (50 µM and 150 µM) also increased the percentage of the apoptotic neuron, the further application of Meth still significantly potentiated the proapoptotic effects in comparison to both reagents (Fig. 9). The maximal potentiation effect was reached with combined treatment with 50 µM Meth (Fig.

9). There was a 1.8-fold increase compared to LPS alone and a 1.5-fold increase compared to the same concentration of Meth alone.

The expression and release of inflammatory cytokines were analyzed using qPCR

(Fig. 10) and ELISA (Table. 2). As the results showed in microglial group treated with

Meth alone, there were no significant changes of IL-1β and TNFα in transcriptional levels (Fig 10A and B). In supernatant, more inflammatory-related cytokines were detected including TNFα, IL-6, IL-4, and IL-10 (Table. 2). However, there was no significant potentiation effect detected after Meth application in Meth-primed microglia.

Because our study primarily focused on the inflammasome-induced IL-1β signaling pathway, we further examined IL-1β production at different time points. As shown in Fig.

10C, the potentiation effect of Meth on LPS-induced IL-1β production was close to statistically significant (p=0.07) at 6 h, but is insignificant at 12 and 24 h. Taken together,

Meth treatment, either used alone or combined with LPS stimulation, did not alter the expression of proinflammatory cytokines.

Figure 8. Potentiation of the LPS-induced morphological change by Meth. Microglia were primed with 100 ng/ml LPS for 6 h. Different concentrations of Meth were then applied to cultured microglia and harvested after 12-h incubation. The morphology was displayed via immunofluorescent staining with CD11b and counterstained with DAPI. All pictures were taken at 60X magnification.

Figure 9. Potentiation of the LPS-induced neurotoxicity by Meth. Neuronal apoptosis was analyzed by combined TUNEL/DAPI staining, and quantification of apoptotic neurons was performed by normalization of TUNEL-positive cells to a total number of

DAPI-positive cells. The apoptosis induced by incubation with supernatant of cultured microglia treated with indicated concentrations of Meth. The increased apoptotic neurons were visualized by green TUNEL-positive signals that were notably observed in LPS + Meth treatment group (A). The percentage of apoptotic neurons were counted and statistically analyzed. The quantified results were exhibited in the bar graph of panel A. * p < .05, ** p < .01, *** p < .001 vs Ctrl. # p < .05, ## p < .01, ### p < .001 vs

LPS. All images were taken at 40X original magnification.

A B

Figure 10. Meth did not potentiate the expression of proinflammatory cytokines induced by LPS. After priming with 100 ng/ml LPS for 6 h, the transcriptional levels of IL-1β (A) and TNFα (B) were detected by qPCR.

Table 2. Meth did not alter the LPS-induced release of TNF-α, IL-6, IL-4, and IL-10.

Microglia primed with LPS were treated with different concentrations of Meth. The supernatant was collected after 12 h and the secretion of inflammatory-related cytokines was measured by specific ELISA kit.

3.4. Enhancement of processing and release of IL-1β by Meth treatment

IL-1β was also regulated by the inflammasome signaling pathway in processing and release. It is well-documented that full activation of the inflammasome requires the first priming signal to upregulate their major components and the primary substrate. As the results in Fig. 7B indicate that the Meth alone does not fit in the role as the first activation signal of the inflammasome. However, it is still unknown if Meth could work as the second activation signal for inflammasome and thus stimulate the processing and release of IL-1β. For this reason, the maturation of IL-1β indicated by the increase of its smaller-sized activation form and its subsequent release were detected in Meth- treated microglia. After priming with LPS (classical first signal for inflammasome activation) for 6 h, the secretion of IL-1β was significantly upregulated along with the increasing concentrations of Meth (Fig. 11A). To distinguish if this potentiation effect was receptor-mediated or not, a levorotary form of Meth (L-Meth) was introduced into experiments as a comparison. The potentiation effect of the dextrorotary form of Meth

(D-Meth) started at the concentration of 6 μM and peaked at 50 μM for a 2.6-fold increase after priming with 100 ng/ml LPS (Fig. 11A). Unlike D-Meth, the L-Meth was less potent, with a significant increase of IL-1β release only detected in 50 and 150 µM of Meth administration and peaking at 50 µM for the 1.5-fold increase (Fig. 11B).

However, both enantiomers showed a dose-dependent manner of potentiation on LPS- induced IL-1β release (Fig. 11A and B). To better characterize the potentiation effect of

Meth on LPS-induced IL-1β release, the time course experiment of D-Meth was performed to determine the best time point for the subsequent studies. As shown in Fig.

11C, the maximal effect ( a 2.1-fold increase compared to the LPS group) of D-Meth was reached at 12 h. Thus, this time point was selected for the investigation on Meth- induced inflammasome activation. The potentiation of LPS-induced IL-1β release was also evidenced in cultured brain slices treated with Meth (Fig. 11D).

The following studies on IL-1β maturation also confirmed that Meth promoted the cleavage of pro-IL-1β into its smaller-sized activation form in a dose-dependent manner

(Fig 12). While the production of pro-IL-1β was not significantly changed after 12 h treatment of Meth, the 17-kD form of IL-1β was significantly increased. Consistent with the ELISA result, Meth promoted the processing of IL-1β starting from 6 µM and reached the maximal effect at 50 µM (1.7-fold increase compared to control). Based on these results, it is concluded that the processing and release of IL-1β were promoted by

Meth administration after priming with LPS, which indicated a possible role of inflammasome activation in Meth-induced potentiation effects.

To investigate whether Meth-promoted IL-1β maturation was achieved by activation of the inflammasome, the measurement of the processing of pro-caspase-1 by western blot were chosen to be the primary readouts for Meth-induced inflammasome activation.

Also, the caspase-1 inhibitor was introduced to prove that the Meth-induced IL-1β activation was specifically mediated by caspase-1 activation. The functional mature form of caspase-1 (10-kD band in Fig. 13A) was found to be significantly upregulated with Meth treatment while the expressional level of pro-caspase-1 did not change (Fig.

13A). The functional cleaved form of caspase-1 was significantly increased with 20 µM

Meth treatment and reached 1.8-fold increase with 50 µM Meth treatment.

Correspondingly, the colorimetric assay indicated a significant increase in caspase-1 activity in the same pattern (Fig. 13B). Consistent with the results of IL-1β processing, the maturation of IL-1β and caspase-1 were not maximal at the highest concentration of

Meth treatment (150 µM). For this reason, 50 µM as the concentration with peak potentiation effect was chosen for the following studies. To ensure that the Meth- induced IL-1β maturation was specifically mediated by inflammasome activation, the caspase-1 inhibitor Ac-YVAD-CMK at 10 µM (Walker et al. 1994) was applied 1 h before Meth treatment. As shown in Fig 14, the pretreatment of caspase-1 inhibitor

significantly reduced the Meth-induced processing of IL-1β while the expressional level of pro-IL-1β was not affected.

A B

LPS 100 ng/ml LPS 100 ng/ml C D

Figure 11. Enhancement of IL-1β release in cultured microglia and brain slice after

Meth treatment. Microglia were pretreated 6 h with LPS (100 ng/ml) and then incubated

12 h with different concentrations of D-Meth (A) or L-Meth (B). Alternatively, after 6 h pretreatment of LPS, cells were incubated with Meth (50 µM) for indicated times (C).

The supernatants were collected, and concentrations of secreted IL-1β were measured by ELISA. The concentrations of IL-1β in supernatants from brain slices were shown in panel D. * p < .05, ** p < .01, *** p < .001 vs LPS.

Figure 12. Meth promoted the processing of IL-1β in cultured microglia. The optimized time point (12 h) and concentration (50 µM) of Meth treatment were applied on cultured microglia. The production and processing of IL-1β were detected by western blot. After standardized with β-actin, the densitometry of bands was analyzed and quantified, as shown in bar graphs (D). * p < .05, ** p < .01, *** p < .001 vs LPS.

Figure 13. Meth activated the caspase-1 in culture microglia. The cleavage and activation of caspase-1 were measured by western blot and quantified by colorimetric assay. The cultured microglia were primed with LPS 100 ng/ml for 6 h and treated with different concentrations of Meth for another 12 h. To evaluate the magnitude difference of the precursor and cleavage form of caspase-1, the densitometry of 45 kD pro- caspase-1 and 10 kD caspase-1 were quantified separately, which are shown in associated bar graphs (A). The enzyme activity of caspase-1 after incubation of Meth was measured by caspase-1 colorimetric assay (B).

Figure 14. Blockade of caspase-1 activation attenuated the Meth-mediated processing of IL-1β. After 6 h priming of LPS, the caspase-1 inhibitor Ac-YVAD-cmk (10 µM) was applied 1 h before Meth treatment and the processing of IL-1β was measured after another 12 h of Meth (50 µM) treatment. *p < .05, ** p < .01, *** p < .001 vs LPS. # p

< .05, ### p < .001 vs Ctrl. $$ p < .01 vs LPS + Meth 50 µM.

3.5. Activation of inflammasome after Meth treatment in LPS-primed microglia

The inflammasome is a molecular platform that is comprised of the sensing molecule NLRP3, the adaptor ASC protein, and the executive enzyme caspase-1. The primary direct evidence against inflammasome activation was oligomerization of the

ASC protein and auto-cleavage activation of caspase-1. The previous results in Fig. 13 have been demonstrated the specific activation of caspase-1 in response to Meth application. We further performed the immunofluorescent staining target ASC protein to confirm the activation of the NLRP3 inflammasome in the context of Meth stimulation.

As a pivotal component of the inflammasome, ASC proteins aggregate after

NLRP3 activation to help assemble the inflammasome platform. In our experiments, the

ASC protein formed aggregates that appear as “speck”-like spots near the nuclear periphery, indicating inflammasome formation after Meth treatment (marked by arrows in Fig. 3C). No ASC aggregation was detected in either the control or LPS groups; rather, ASC aggregated only after treatment with Meth. The number of ASC aggregates also reached its highest level in the 50 µM Meth treatment group.

As the sensor of the inflammasome, the NLRP3 co-localized with pro-caspase-1 to assemble the platform, which promotes the autoproteolysis-mediated caspase-1 activation. In our current experiments, the distribution of both NLRP3 and pro-caspase-

1 were measured by specific immunofluorescent staining at different time points. As shown in the Fig. 16, the NLRP3 and caspase-1 were evenly distributed all around the cellular body. Most of the fluorescent signals were not overlapped before application of

Meth. The NLRP3 started to aggregate and co-localize with caspase-1 after 6-h treatment of Meth. The increased double-positive signals were increased and lasted at least 24 h.

Figure 15. Aggregation of ASC protein in Meth-treated microglia. As an indicator of inflammasome activation, the change of distribution of ASC protein was visualized by immunofluorescent microscope. The formation of ASC oligomers was detected in the cytoplasm and marked by red arrows. All pictures were taken at 60X magnification.

Figure 16. Co-localization of NLRP3 and caspase-1 in Meth-treated microglia. The co- localization of NLRP3 and caspase-1 were assessed in cultured microglia treated with

LPS and Meth at indicated concentration. Microglia were fixed at different time points.

The distribution of NLRPR3 and caspase-1 were detected by specific immunofluorescent staining and overlapped fluorescent signal were considered as the sign of co-localization.

4. Discussion

Although neuroinflammatory responses marked by microgliosis and astrogliosis are well-documented in Methamphetamine (Meth) abusers (Robson et al. 2014; Sekine et al. 2008), the mechanisms by which Meth directly contributes to the initiation and progression of the microglia-mediated neuroinflammation is still not fully understood. As a strong addictive psychostimulant, Meth abuse induces not only a strong euphoric effect leading to addiction but also affects innate and adaptive immunity thereby causing neurotoxicity (Loftis and Janowsky 2014). Direct effects of Meth were investigated by the evaluation of Meth-induced proinflammatory cytokine levels on isolated microglia. The results suggest that Meth stimulation activates endogenous danger signal pathways, therefore promoting inflammasome-mediated IL-1β maturation and secretion. These data suggest that Meth administration potentiates the microglia- associated neurotoxicity by activating the production and release of inflammasome- mediated IL-1β.

Microglial activation was featured by increase capability of migration, morphological change to amoebae/rounded shape with fewer processes, high rates of proliferation, and secretion of neurotoxins (Lull and Block 2010). Our current results indicate that

Meth treatment alone does not shift the resting microglia into proinflammatory phenotype. This is consistent with previous studies that also failed to detect proinflammatory cytokines production in isolated microglia (Frank et al. 2016). In the concentration of 450 µM, Meth decreased the cellular viability after 72-h incubation.

This result also matches with the previous study on Meth-induced apoptosis of microglia (Coelho-Santos et al. 2012). To maximally mimic the in vivo concentrations of

Meth under pathophysiological conditions, the dosing range of Meth was referred to via the biodistribution (BD) profiles from previous studies. The Meth level in the circulating blood of an abuser is reported to be 2.0 µM on average but as high as 11.1 µM (Melega

et al. 2007). The Meth level in the brain of an abuser is further estimated according to the tissue-to-serum ratios of Meth in rats, which is 9.7 for brain distribution (Riviere et al.

2000). Based on the above references, the dosing range of Meth was determined to be

6-150 µM.

Proinflammatory cytokines are important mediators of microglia-induced neurotoxicity in many neurodegenerative diseases (Lull and Block 2010). To demonstrate the Meth effects on the transcription of proinflammatory cytokines, the mRNA levels of IL-1β and TNFα were measured after Meth administration at different concentrations and time courses. LPS stimulation was used as a positive control to rule out the possibility of microglial anergy. After Meth treatment alone, there is no significant up-regulation of IL-1β and TNFα observed in cultured rat microglia. This result is consistent with the previous studies which also failed to detect any Meth- induced proinflammatory responses in the cultured microglia. Nevertheless, it is worth noting that up-regulated proinflammatory signals were observed in vivo after Meth administration (Frank et al. 2016). The possible explanation is that Meth induces neuronal damage and thus promotes the alarming signals released from neuron, which ultimately upregulates the proinflammatory signaling in microglia. Yet, we cannot rule out the possibility that Meth potentiates the inflammatory signaling in comorbid diseases; i.e., in HIV-associated neurological disorder (HAND). Meth abuse was also found to potentiate HIV-1 infection-induced microglial activation and to mediate the excessive release of proinflammatory cytokines (Flora et al. 2003; Langford et al. 2003). In mice administrated with HIV-1 Tat plus Meth, the expression of TNF-α and IL-1β was elevated in the striatum (Flora et al. 2003). For this reason, we performed experiments to investigate whether Meth treatment could enhance the pre-existing inflammatory stimulation in cultured microglia. While the LPS-induced release of other inflammatory- related cytokines were not altered by Meth treatment, the secreted IL-1β was

significantly potentiated. The results show that Meth treatment enhanced the LPS- induced IL-1β secretion in both dose- and time-dependent manners, although the transcriptional levels of TNFα and IL-1β were not significantly potentiated.

This dissociation between the transcriptional induction and release strongly suggests an involvement of a post-translational regulatory mechanism (Davis et al.

2011; Lopez-Castejon and Brough 2011). The IL-1β is upregulated as a proprotein (pro-

IL-1β) that needs to be further processed into its active form proteolytically (Black et al.

1988). In microglia, the processing of pro-IL-1β is mediated by the NLRP3 inflammasome at the post-translational level (Walsh et al. 2014a). The inflammasome is a multiprotein complex, which serves as a caspase-1 activation platform (Franchi et al.

2009). Active caspase-1 is the cleaving enzyme for processing pro-IL-1β into its mature, biologically active forms. Typically, two independent signals are required to fully activate the NLRP3 inflammasome (Hanamsagar et al. 2012). The first signal is to upregulate the major substrate (pro-IL-1β) and essential components (NLRP3) required for inflammasome activation. Because pro-IL-1β and NLRP3 are not constitutively expressed in microglia, additional factors are needed for transcriptional induction (Burm et al. 2015; Dostert et al. 2008; Halle et al. 2008). Although our goal is to investigate the role of the inflammasome-mediated proinflammatory signal in Meth-potentiated neurotoxicity HIV-1 infection, little is known about HIV-1 protein as the first signal for the inflammasome activation. In addition, LPS is typically used as the first priming signal on microglia for inflammasome studies (Halle et al. 2008; Parajuli et al. 2013; Sanz and Di

Virgilio 2000; Shi et al. 2012). The process of the LPS-primed NLRP3 inflammasome is well characterized, involved with the transcriptional induction (pro-IL-1β and NLRP3 induction) and with multiple posttranslational events (extracellular signal-regulated kinase 1 phosphorylation, proteasome inhibition, and NLRP3 deubiquitination)

(Ghonime et al. 2014; Juliana et al. 2012). In the CNS system, administration of LPS

induces neurodegeneration, which is associated with microglial activation and subsequent release of IL-1β (Arai et al. 2004). Based on these reasons, LPS was chosen as the stimulating factor in this study to mimic the priming stimulation of neuronal DAMPs or of the HIV proteins in the virus-bearing brain (Garvey et al. 2014).

Currently, the role of Meth on inflammasome activation is not fully depicted. To investigate whether the inflammasome is the molecular target that responds to Meth stimuli, we systematically studied the inflammasome activation as a common downstream receptor locus for convergence of signals from multiple danger-associated pathways (Tschopp and Schroder 2010). The inflammasome consists of NLRP3

(sensor receptor), ASC (bridge protein), and caspase-1 (IL-1β converting enzyme) and activates in response to various upstream cellular danger-associated signals (Latz et al.

2013). As the primary mediator of maturation of pro-IL-1β, NLRP3 inflammasome activation is always accompanied by processing of pro-IL-1β, cleavage of caspase-1 and ASC protein aggregation. After sequential stimulation of LPS and Meth, IL-1β and caspase-1 are cleaved to their activated forms, which exhibit lower molecular weight.

Aggregated ASC proteins were also found to be increased in co-treated groups. This matches the classical cellular pattern of inflammasome activation, in which principal components redistribute from dispersed to clustered and further promote restoration of caspase-1 enzyme activity after the cross-cleavage process (Franchi et al. 2009). As expected, the activity of caspase-1 was elevated along with increased concentration of

Meth, and inhibition of caspase-1 totally reversed the enhancement of LPS-induced IL-

1β processing and release. This result supports the specific role of inflammasome activation in the Meth-induced potentiation effect.

Together with results that Meth alone did not upregulate pro-IL-1β, we conclude that

Meth serves as a second activating signal for inflammasome activation. However, it is still unclear whether Meth-induced inflammasome activation is a receptor-mediated

event or not. To address this question, the levorotatory form of Meth (L-Meth) was introduced. Meth is well-known to bind with plasma and vesicular monoamine transporters. In contrast, L-Meth has been proven to have a much less binding affinity with these monoamine transporters and thus is also less potent than the dextrorotatory form of Meth (Melega et al. 1999). The ELISA result demonstrated that, without the LPS priming signal, none of the enantiomeric forms of Meth could induce the detectable IL-

1β release. However, treatment of Meth (both D- and L- forms) after the introduction of the priming signal LPS indeed significantly promoted the secretion of IL-1β in a dose- dependent manner. Thus, monoamine transporters are very unlikely to be the candidate receptors that mediate the potentiation effect of Meth. Due to the limited information on the differential binding affinity of the two enantiomers on other Meth-target receptors such as sigma-1 and trace amine-associated receptor 1, it is still hard to conclude whether Meth-potentiated IL-1β release is receptor-mediated or not.

Chapter 3

Mitochondrial ROS production and lysosomal membrane permeabilization are

molecular mechanisms of Meth-induced

inflammasome activation

Abstract:

In Chapter 2, we demonstrated that Meth potentiated the LPS-induced IL-1β release.

After priming with LPS, the Meth treatment activated the NLRP3 inflammasome that promoted the processing and release of IL-1β. However, the underlying mechanisms of

Meth-induced NLRP3 inflammasome are still largely unknown. In this chapter, the specific inhibitors targeting the NLRP3 upstream danger-associated signaling pathways were applied in Meth-treated microglia. Specific molecular probes were also used to evaluate the Meth-associated cellular impairments that might contribute to the activation of the inflammasome.

Both lysosome and mitochondria impairment were found after Meth administration, and their specific inhibitors blocked the Meth-induced inflammasome activation. The

Meth-induced mitochondrial ROS and cathepsin B release were confirmed by specific molecular probes or fluorescent staining. The LPS-induced neurotoxicity was significantly potentiated by further Meth treatment and could be reversed by mitochondrial ROS and cathepsin B-specific inhibition. Together these results suggest that Meth triggers the activation of the inflammasome in a manner dependent on both mitochondrial and lysosomal danger-signaling pathways.

1. Introduction

The inflammasome is cytosolic pattern recognition receptors which are activated by multiple dangerous-associated molecular patterns (DAMP) pathways (Hanamsagar et al. 2012). The well-accepted model for NLRP3 activation is a two-step process including initial signaling from membrane-bound TLRs for inducing pro-IL-1β and secondary signaling from cytosolic NLRs (Hanamsagar et al. 2012). NLRP3 could respond to numerous structurally distinct “danger” signals including ROS activation, lysosomal cathepsin B release, K+ efflux and mitochondrial damage. Upon sensing the activation signal, NLRP3 will be oligomerized by additional translocation of adaptor protein ASC. The complete oligomerization forms a platform for caspase-1 recruitment.

The proximity of procaspase-1 induces an autoproteolytic process which converts the proenzyme into active from (Guo et al. 2015a).

Meth was associated with mitochondrial and lysosomal damage in both immune and non-immune cells (Nara et al. 2012; Potula et al. 2010; Wu et al. 2007). In isolated mitochondrial treated with Meth, increased ROS was detected accompanied with the collapse of mitochondrial membrane potential (Nara et al. 2012). Mitochondrial ROS production was also considered as the key cellular event leading to NLRP3 inflammasome activation (Tschopp and Schroder 2010). For this reason, we measured the microglial mitochondrial ROS production after Meth treatment. Our results indicate that Meth could activate the mitochondrial ROS in a both dose- and time-dependent manner.

On the other hand, the Meth-induced dysfunction of lysosomal was also reported

(Nara et al. 2012; Talloczy et al. 2008). Acidotropic uptake of Meth will produce osmotic swelling after accumulating in acidic organelles, particularly endosomes and lysosomes

(Cubells et al. 1994). Our results suggest that Meth treatment impaired the integrity of

lysosomal membrane, which promotes the translocation of cathepsin B into cytosol and release.

By specific inhibition of mitochondrial ROS and cathepsin B, we successfully block the Meth-potentiated neurotoxicity. NLRP3 inflammasome is a promising target to prevent the neuroinflammation-induced neurotoxicity in the context of Meth abuse.

2. Materials and methods

2.1. Immunofluorescent staining

The general procedure is in accordance with the description in Chapter 1. What is worth noting is that the step of permeabilization was separated with blocking in this chapter. The fixed microglia were permeabilized by 0.1% Tween in PBS for only 5 min.

The first antibody used here is Cathepsin B (Sana Cruz, Dallas, TX) at 1:100 dilution.

2.2. ELISA

After priming with LPS 100 ng/ml for the first 6 h, the inhibitors were applied 1 h before Meth treatment. The microglial supernatants were collected after 12-h incubation, and the concentrations of IL-1β were determined by specific ELISA kit (R&D system,

MN, USA).

2.3. Fluorescent dye loading and imaging

Fluorescent probes against ROS production, namely MitoSOX Red and H2DCFDA, were loaded onto the microglia that were treated with Meth for an additional 6 h after priming with LPS. MitoSOX Red and 5-(and-6)-chloromethyl-2’,7’- dichlorodihydrofluorescein diacetate (CM-H2DCFDA) (Life Technologies, Eugene, OR) were deployed to examine the intracellular ROS production. CM-H2DCFDA enters cells passively, and the acetate groups of the probe were cleaved by intracellular esterase leading to better cellular retention. After oxidation by reactive oxygen intermediates generated in response to Meth, the non-fluorescent H2DCFDA is converted to the highly fluorescent 2’,7’-dichlorofluorescein (DCF). The MitoSOX Red is a fluorogenic dye that is a highly selective indicator of ROS derived from mitochondrial in live cells. After treating with Meth for 6 h, the medium was removed and washed 3 times with pre- warmed PBS. The MitoSOX (2.5 μM) and CM-H2DCFDA (5 μM) working solutions were freshly made with pre-warmed DMEM medium and incubated on treated microglia at

37 °C for 30 min. The cells were then fixed with ice-cold 4% paraformaldehyde in PBS

for 10 min and counterstained with DAPI. To quantify the results, the microglia were seeded onto a 96-well black plate in a density of 0.12 x 106/well and the treatment procedure mentioned above was then repeated. After loading with MitoSOX or CM-

H2DCFDA, the intensities of specific fluorescent signals were evaluated by the microplate reader.

To assess the disruption of the lysosomal integrity of after 24 h of Meth administration, the specific molecular probe (LysoTracker) was applied onto microglia.

The microglia seeded in a 12-well plate with coverslip were loaded with 50 nM

LysoTracker Red DND-99 (Life Technology, Eugene, OR) in pre-warmed DMEM for 30 min. Cells were then washed 3 times and visualized by fluorescent microscopy.

2.4. Measurement of nitric oxide (NO) production

The concentration of nitrite was measured by the Griess Reagent System according to the manufacturer’s instruction (Promega, Madison, WI). After treatment of LPS and

Meth, 50 µl aliquots of supernatant were collected from each group. Equal amounts of sulfanilamide solution and NED solution were added with collected supernatants for 10 min and 30 min separately. The absorbances of the final samples were measured on a plate reader with filters at 520 nm and 540 nm.

2.5. Tunel staining

To test whether inhibition of inflammasome activation could block the Meth potentiated neurotoxicity, the caspase-1 specific inhibitor was applied 1 h before Meth treatment. Also, the specific mitochondrial ROS and cathepsin B inhibitors were also added to confirm the involvement of mitochondrial and lysosomal impairment in Meth- induced inflammasome activation. The procedure is followed with instruction manual of

Tunel cellular apoptosis detection kit (Roche Applied Science, Indianapolis, IN). In the step of quantification, only the Tunel positive signals that overlapped with DAPI were counted.

2.6. Statistical analyses

All experimental data were statistical analyzed by OriginPro 8.0 (OriginLab,

Northampton, MA). One-way ANNOVA followed with Turkey test was performed for multiple groups comparison. For quantification of imaging results, the mean fluorescent intensities of 6 fields in one group were analyzed in comparison to control.

3. Results

3.1. Identification of danger signaling-associated molecular pathways leading to

Meth-induced inflammasome activation

The NLRP3 inflammasome activation may occur through multiple cellular danger signaling pathways including the induction of ROS, lysosomal disruption and cathepsin

B release, potassium efflux, and calcium mobilization. Accumulating evidence suggest that Meth application could activate the mitochondrial ROS production and disrupt the normal lysosomal function (Cubells et al. 1994; Funakoshi-Hirose et al. 2013;

Mashayekhi et al. 2014; Potula et al. 2010; Shiba et al. 2011; Tian et al. 2009; Wu et al.

2007). To identify the inflammasome-related upstream molecular events induced by

Meth, specific inhibitors against mitochondrial ROS and lysosomal pathways were introduced to test if Meth-induced IL-1β release was blocked. After pretreatment of inhibitors, immunofluorescent staining of the ASC protein and measurement of secreted

IL-1β were performed on Meth-treated microglia. As shown in Fig. 19, the cathepsin B inhibitor CA-074Me at 10 µM (Hill et al. 1994) totally reversed the Meth-induced ASC protein oligomerization. However, aggregation of the ASC protein was only partially blocked by pretreatment of the mitochondrial ROS inhibitor Mito-TEMPO at 100 µM

(Liang et al. 2009) (Fig. 19). To ensure the specificity of these inhibitors, the dose effects of all inhibitors were examined by a Meth-induced IL-1β release experiment. By increasing the concentrations of inhibitors applied, the blocking effects of all the inhibitors on the potentiation of IL-1β secretion were nicely in a dose-dependent manner (Fig. 18). Finally, to examine if Meth potentiates IL-1β through upregulation of major components of the inflammasome, we measured the transcriptional level of

NLRP3 after application of LPS and Meth. LPS, as the classical first signal, successfully upregulated the expression of NLRP3, while Meth application failed to alter the production of NLRP3 (Fig. 17).

Figure 17. Meth did not regulate the expression of NLRP3. The transcriptional level of

NLRP3 was measured by qPCR on Meth-treated microglia with or without priming signal.

Figure 18. Involvement of Mitochondrial ROS and lysosomal cathepsin B in Meth- induced IL-1β release. Isolated microglia were pretreated with different concentrations of inhibitors before LPS and Meth application; the secreted IL-1β levels were quantified by ELISA. *** p < .001 vs LPS. # p < .05, ## p<.01, ### p < .001 vs LPS + Meth 50 µM.

CD11b/ASC

Figure 19. Blockade of mitochondrial ROS and lysosomal cathepsin B prevented the

Meth-induced ASC protein aggregation. Punctate ASC immunoreactivity was detected in the cytoplasm of Meth (18 μM) treated microglia (indicated by red arrows). Microglia pretreated with lysosomal cathepsin B inhibitor (CA-074Me 10 μM), and mitochondrial

ROS inhibitor (Mito-TEMPO 100 μM) exhibited minimal ASC immunoreactivity.

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3.2. Production of mitochondrial ROS after Meth treatment

Mitochondria are a major source of inflammasome-activating ROS (Zhou et al.

2011). To investigate if Meth-induced ROS formation primarily locates at the mitochondria, a molecular probe that distinguishes the source of ROS was applied.

After loading with the specific molecular probe, the fluorescent microscope was used to take images, and the microplate reader was used to quantify the results. As a total endogenous ROS indicator, H2DCFDA fluorescent intensities increased after LPS stimulation. Meth application further activated the endogenous ROS system evidenced by elevated H2DCFDA fluorescence in a dose-dependent manner (Fig. 20A).

Consistently, the mitochondria-specific probe Mito-SOX also indicated a further increase of mitochondrial ROS after Meth treatment (Fig. 20B). The accumulated red signals indicating mitochondrial ROS are primarily localized in the cytosol but not in the nucleus as marked by DAPI, suggesting that the probes target the mitochondria. The peak effect of Meth on total ROS formation reach a 1.7-fold increase compared with the

LPS group. Matching this magnitude, the Meth-induced generation of mitochondrial

ROS peaks at 1.6-fold compared to the LPS group.

Time course study of Meth-induced mitochondrial ROS production indicates that the effect of Meth started as early as 2 h and lasted 24 h (Fig. 22).

In addition to ROS, reactive nitrogen species (RNS) are also found mediating the

Meth-induced neurotoxicity (Quinton and Yamamoto 2006). To test if the activation of

RNS is also incorporated into the Meth-induced cellular stress system, the nitrites secreted in the supernatants of Meth-treated microglia were measured and quantified as shown in Fig. 21. There is no significant change of the nitrite level after Meth treatment.

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Figure 20. Meth increased the production of total ROS and mitochondrial ROS. The total ROS and mitochondrial ROS are detected by the specific fluorescent molecular probe. The change of the fluorescent intensity was visualized by microscope and quantified by the microplate reader. Isolated microglia were primed with LPS 6 h and then incubated with indicated concentrations of Meth. Fluorescent images of DCFDA green signals (A) and MitoSOX Red signals (B) were taken in microglia after 12 h of

Meth treatment. The quantitative analyses of DCFDA and MitoSOX signals were exhibited below. * p < .05, ** p < .01, *** p < .001 vs Ctrl. # p < .05, ## p < .01, ### p

< .001 vs LPS. All images were taken at 40X magnification.

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Figure 21. Meth did not increase the NO in the microglial supernatant. NO productions in harvested supernatants were measured using Griess reagent system.

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Figure 22. Time-dependent activation of mitochondrial ROS after Meth treatment. After priming with LPS, microglia were treated Meth with different times. MitoSOX was used to detect the production of mitochondrial ROS. All pictures were taken at 10X magnification.

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3.3. Permeabilization of lysosomal membrane in Meth-treated microglia

Another inflammasome-related pathway identified is the Meth-induced cathepsin B release. To further investigate the mechanism of lysosome disruption and subsequent content release after Meth application, lysosomal membrane permeabilization (LMP) was assessed in this cellular model. The assessment of lysosomal integrity was achieved by measuring the retention rate of LysoTracker and detecting the intracellular distribution of lysosomal protease cathepsin B. LysoTracker-labeled lysosome in the control group revealed a cytoplasmic punctuate structure, but with increasing concentrations of Meth, fluorescence intensity and the number of punctuates apparently decreased (Fig. 23). LPS administration significantly increased the signals of

LysoTracker while additional treatment of Meth reduces the signals in a dose- dependent manner (Fig. 23). Another sign of LMP is the translocation of soluble lysosomal protease from the lysosomal lumen to the cytosol. Immunofluorescence staining reveals a redistribution pattern of cathepsin B after Meth application, marked by the loss of punctuating shape and diffusion of stain throughout the cellular cytosol (Fig.

24). However, as the cellular marker of the lysosome, the fluorescent intensities of lysosomal-associated membrane protein 1 (LAMP-1) did not change with Meth treatment (Fig. 24).

The time course study of Meth-induced cathepsin B leakage and release indicated that LMP was disrupted at least 6 h after Meth incubation. The puncta shape of cathepsin B was decreased after administration of Meth. The cathepsin B-specific fluorescent intensity was also diminished.

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Figure 23. Increased permeabilization of lysosomal membrane in Meth-treated microglia. Lysosomal integrity was measured by LysoTracker. The fluorescent intensity was quantified and exhibited in the form of bra graph. *** p < .001 vs Ctrl; ### p < .001 vs

LPS. Images in panel A were taken at 40X magnification;

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Figure 24. The release of cathepsin B in Meth-treated microglia. Colocalization of cathepsin B and lamp-1 was assessed by fluorescent microscopy. Images in panel B were taken at 630X magnification.

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Figure 25. Time-dependent disruption of lysosomal membrane and cathepsin B release after Meth treatment. The LPS-primed microglia were treated with Meth and harvested and fixed at different time points. Specific immunofluorescent staining exhibited the distribution of cathepsin B along the time courses.

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3.4. Inhibition of inflammasome activation block the Meth-potentiated

neurotoxicity

Inhibitors against Meth-induced inflammasome activation were applied to determine if blocking of inflammasome activation could rescue the Meth-enhanced neurotoxicity.

The cultured microglia were pretreated with inhibitors against caspase-1 activity (YVAD), cathepsin B (CA-074Me), and mitochondrial ROS (Mito-TEMPO). All inhibitors significantly reduced the percentage of apoptotic neuron in comparison to the LPS +

Meth 50 µM group (Fig. 26). However, the inhibitors did not totally reverse the apoptotic effect. The percentage of apoptotic neurons in the group with inhibitors were still significantly higher than the control (Fig. 26). Taken together, Meth significantly potentiated the LPS-induced neuronal apoptosis, and it was blocked by inflammasome- related inhibitors.

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Figure 26. Inhibition of mitochondrial ROS and lysosomal cathepsin B attenuated the

Meth-potentiated neurotoxicity. The blockage effect of inflammasome inhibitors on

Meth-induced neuronal apoptosis were examined and quantified in panel B. After priming with LPS for first 6 h, the inhibitors were applied 1 h before additional Meth treatment. The collected supernatant was transferred to cultured neuron and TUNEL- staining were performed. * p < .05, ** p < .01, *** p < .001 vs Ctrl. # p < .05, ## p < .01,

### p < .001 vs LPS. && p < .01, &&& p < .001 vs LPS + Meth 50 µM. All images were taken at 40X original magnification.

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4. Discussion

Although it has been proven that the potentiation of Meth on LPS-induced IL-1β processing and release is mediated by the inflammasome, the mechanism is still unknown. Typically, it could be achieved by upregulation of the main components of the inflammasome or by the activation of upstream danger signaling pathways. We examined the expressional level of NLRP3, which was not significantly upregulated after Meth treatment. Consistent with that, the production of pro-caspase-1 was also not increased. Based on this evidence, it could be concluded that Meth does not regulate the production of inflammasome components. Otherwise, a considerable body of evidence indicates that Meth induces cellular damage and distress, particularly in mitochondria- and lysosome-related pathways (Cubells et al. 1994; Nara et al. 2012;

Potula et al. 2010). Thus, we hypothesized that Meth treatment could trigger endogenous stress signals in microglia and may directly contribute to Meth-induced inflammasome activation. As mentioned above, the primary cellular targets of Meth in peripheral immune cells are mitochondrial ROS activation and lysosome disruption, which are both well-accepted upstream pathways for inflammasome activation

(Hornung et al. 2008; Weber and Schilling 2014; Zhou et al. 2011). Specific pharmacological inhibitors were used to dissect the Meth-associated danger signaling pathways. The inhibitors targeting the mitochondrial ROS and lysosomal protease cathepsin B are both able to block the Meth-induced inflammasome activation in a dose-dependent manner. Therefore, Meth could activate the inflammasome through the above two signaling pathways. However, it is worth noting that the blocking effect of inhibitors applied here may also be due to an off-target effect. It has been reported that

RNA interference and cathepsin-B-/- cell lines fail to replicate CA-074Me results in some studies (Newman et al. 2009). For this reason, more experiments regarding these two subcellular pathways need to be done.

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Further studies were performed with the specific molecular probe and immunofluorescent staining to prove that the lysosomal disruption is specific to the

Meth potentiation effect. Compared to the low fluorescence levels in control and in the

LPS alone groups, there is a dose-dependent increase in both total ROS and mitochondrial ROS signals after treatment with different concentrations of Meth.

Because the mitochondrial ROS system also belongs to the total ROS system, the increased proportion of mitochondrial ROS is matched with the change of total ROS, which suggests that the mitochondria is the primary source of Meth-induced ROS production. Previous studies indicate that incubation of microglia with a high concentration (1 µg/ml, 12 h) of LPS could induce mitochondrial ROS activation (Park et al. 2015). However, the concentration of LPS we used here is much lower (100 ng/ml,

6 h), and a washing step was performed before further Meth administration. This may explain why we did not observe the elevation of mitochondrial ROS signal in the LPS treatment group.

On the other hand, to further assess the Meth-induced lysosomal membrane permeabilization (LMP), lysosomotropic fluorochromes that usually accumulate inside acidic organelles were applied in Meth-treated microglia (De Milito et al. 2007). Both the reduced fluorescence intensity of LysoTracker and the diffuse distribution pattern of cathepsin B were detected in Meth-treated microglia, which are signs of lysosomal dysfunction. The decreased LysoTacker fluorescence is not entirely specific for LMP, which may also reflect an increase in lysosomal pH (Boya et al. 2003b). However, the loss of pH gradient does not result in the translocation of cathepsin B. Therefore, the reduction of fluorescence intensity of LysoTracker is very likely to be an indicator of

LMP but not merely the loss of pH gradient. LAMP-1 is a glycoprotein constituent of the lysosomal membrane that is constitutively expressed to resist hydrolase digestion

(Eskelinen 2006). We did not observe a noticeable reduction of the LAMP-1 specific

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signal after Meth administration. The constant expression of LAMP-1 signal suggests that Meth does not completely collapse the lysosome, which is an inhibition signal for the inflammasome (Katsnelson et al. 2016). It needs to be noted that the degree of lysosome impairment is also a key element for determining the ultimate result. Recent evidence indicates that while partial lysosome rupture may lead to inflammasome activation, complete lysosome rupture is a route to necrosis but not to the inflammasome (Guicciardi and Gores 2013). The uncoupling of cathepsin B and LAMP-

1 signal indicates a low-level disruption of lysosomal membrane, but not an extensive rupture (Boya et al. 2003a). Based on these results, the lysosomal disruption may be another candidate mechanism for Meth-induced inflammasome activation.

These results indicate that both pathways may play a role in Meth-induced inflammasome activation. It is still unknown whether Meth pretreatment directly activates both pathways or not. Some studies suggest there is a convergence point merged from multiple danger signaling pathways that directly activates the NLRP3

(Munoz-Planillo et al. 2013; Subramanian et al. 2013; Tschopp and Schroder 2010). In the study of Aβ-induced microglial activation, both ROS activation and cathepsin B leakage are required for inflammasome activation (Taneo et al. 2015). However, because the recent evidence indicates a complicated interplay between mitochondria and lysosomes in initial NLRP3 activation, it needs to be noted that these two cellular danger-associated pathways induced by Meth are possibly associated (Heid et al.

2013). A recent investigation on the interaction between disruption of the mitochondria and the lysosome followed by NLRP3 inflammasome activation indicates that release of mitochondrial ROS might precede and cause lysosomal membrane permeabilization

(Heid et al. 2013). Therefore, the possibility of lysosomal damage as the subsequent result of Meth-induced mitochondrial ROS cannot be entirely ruled out.

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It is still not clear whether there are specific receptors expressed on lysosomes or mitochondria that directly interact with Meth at physiological concentrations. Increasing evidence demonstrates that the NLRP3 inflammasome assembles at the ER- mitochondrial interface (Misawa et al. 2013; Zhou et al. 2011), suggesting a direct interaction of mitochondria and newly formed inflammasome. Also, lysosomal membrane permeability is also regulated by ROS production (Boya and Kroemer 2008).

Therefore, the Meth-induced inflammasome activation is very likely governed by mitochondrial disruption, which is followed by lysosomal damage. Further studies need to be done to investigate whether a direct receptor of Meth is expressed on mitochondria while the lysosome functions as a downstream signal. Because the Meth- induced mitochondrial toxicity is confirmed on isolated mitochondria, Meth is a more likely direct target with mitochondria (Mashayekhi et al. 2014).

The time-course studies performed on Meth-induced mitochondrial ROS and cathepsin B release suggested that mitochondrial impairment happened earlier than the disruption of LMP. Considering that mitochondrial ROS can promote the permeabilization of lysosomal membrane, the primary target of Meth is very likely located in the microglial mitochondria.

Finally, the Meth potentiation effect on LPS-induced neurotoxicity is also examined.

Previous results have demonstrated that LPS significantly activates the microglia in a dose-dependent manner and thus induces a potent neurotoxic effect (Houdek et al.

2014). In our in vitro neuroinflammation model, LPS was applied before Meth treatment to mimic pre-existing inflammatory stimulation. After priming with LPS, the supernatants of Meth-treated microglia significantly increased the percentage of apoptotic neurons compared to LPS alone. Without the priming signal of LPS, Meth also induced apoptosis of neurons at high concentration, but with much less potent effect. To test if specific inhibition of Meth-induced inflammasome activation could block this pro-

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apoptotic effect, inhibitors were applied, and a reduced number apoptotic neurons were found after Meth treatment. Thus, preventing the inflammasome activation could be a promising strategy against Meth-induced neurotoxicity.

These findings provide a novel insight into Meth influence on the resident immune system within the CNS and a potential explanation of why Meth abusers suffer a more aggressive progression of many other neuroinflammatory neurodegenerative diseases than non-users (Callaghan et al. 2012; Nath et al. 2001; Rippeth et al. 2004). Because

LPS is a broad pro-inflammatory stimulus, it is possible that the same mechanism could also function in other aspects of Meth-associated morbidity. Because neuroinflammation itself contributes to many behavioral changes and cognitive impairment (Lee et al. 2008; Thrash et al. 2009; Wager-Smith and Markou 2011;

Zerrate et al. 2007), uncovering an association of Meth abuse and enhancement of pre- existing neuroinflammation could help prevent exacerbation of disease progression.

Treatments that target neuroinflammation may be a promising approach for Meth dependency, which is currently under investigation in a Phase 2 clinical trial (IBUD Ph II

NCT01860807).

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Chapter 4

Meth potentiation of HIV-1 gp120-induced

inflammasome activation

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Abstract: Methamphetamine (Meth) is an addictive psychostimulant and its abuse results in neurological and behavioral consequences (Rusyniak 2013). Clinical studies have demonstrated that a long-term Meth abuse exacerbates HIV-1-associated neurocognitive disorders (HAND) (Langford et al. 2003; Rippeth et al. 2004). Ample evidence indicates that persistent microglial activation is an important risk factor for neuropathological outcomes during chronic HIV brain infection (Garvey et al. 2014;

Glass and Wesselingh 2001; Lindl et al. 2010). As HIV-1 does not infect neurons, much of neural cell injury in HIV infected brain is mediated by neurotoxic factors released by infected microglia and macrophages (Dickson et al. 1993; Garden 2002; Yadav and

Collman 2009). One of the primary neurotoxins orchestrating neurotoxic effect is HIV-1 glycoprotein 120 (gp120). In addition to its direct toxic effect on neural cells in the CNS, gp120 can cause neural cell injury via an indirect mechanism, meaning gp120 induces microglia activation and resultant production of proinflammatory molecules leading to neural cell injury (Kaul et al. 2005). Meth abuse exacerbates gp120/microglia- associated neurotoxic activity (Kesby et al. 2015a; Samikkannu et al. 2015). While most studies have focused on the individual effect of Meth and gp120, few have been done on their intersecting/synergistic effects in the brain. Nevertheless, how Meth exacerbates gp120-induced microglial neurotoxic activity is not fully understood.

Increasing evidence indicates the inflammasome, a key signaling platform involved in

HIV-1-associated microglia activation and production of proinflammatory molecules

(Walsh et al. 2014b), may be involved in Meth/gp120-induced, microglia-mediated neurotoxic activity in the brain. The primary function of this multiprotein complex is to process IL-1β from an inactive precursor to its active form. It is our hypothesis that relevant pathophysiological concentrations of Meth could enhance gp120-induced IL-1β production by activation of the inflammasome and result in the potentiation of

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neuroinflammation-associated neurotoxicity. In our preliminary studies, we found Meth could potentiate gp120-stimulated microglial IL-1β processing and release. The gp120- indcued total ROS production was potentiated by Meth treatment.

1. Introduction

Methamphetamine (Meth) is a highly addictive psychostimulant that is the second most widely used illicit drug worldwide (after cannabis) (Degenhardt et al. 2010). Meth use is associated with high-risk sexual behaviors and drug injection, both of which are proposed causal factors of HIV transmission (Degenhardt et al. 2010). Thus, HIV infection is prevalent among Meth abuse population. (Buchacz et al. 2005; Colfax and

Shoptaw 2005). Both HIV infection and long-term Meth abuse can result in neurotoxicity, and Meth abuse accelerated the pathological process of HIV-associated dementia and more severe encephalitis (Bell et al. 1998; Bouwman et al. 1998; Langford et al. 2003;

Nath et al. 2001). Although the individual mechanism of Meth- or HIV-induced neurotoxicity has been well investigated, the underlying mechanisms of the synergistic effects of Meth and HIV are still largely unknown. Persistent neuroinflammation featured by reactive microgliosis exists both in HIV and Meth-associated neurotoxicity. However, the pathways dysregulated in microglia under the intersecting influence of both HIV and

Meth are less clear. Thus, this study aims to investigate intracellular signaling pathways potentiated by Meth in HIV-protein treated microglia.

The contribution of microglia to neurodegeneration in HAND is well established.

Residual inflammation persists even with well-controlled viremia by HAART. Certain studies even suggest microglial activation could be an indicator of further neurocognitive decline (Saylor et al. 2016). Similar microglial activation patterns were also observed in long-term Meth abusers and experimental animals. Blocking microglial activation has proven to be neuroprotective in Meth-induced neurotoxicity. Thus,

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microglia-associated inflammation is considered to be a central pathological change of both HAND and Meth. Although rapid disease progression of HAND with Meth abuse suggests a synergistic effect on sustained neuroinflammation, very few studies have investigated the common downstream pathways that are affected by both HIV protein and Meth. We hypothesize that Meth could potentiate HIV protein gp120-induced inflammasome activation by amplification of upstream danger signals. The synergistic effects of Meth and gp120 induce sustained inflammasome activation and thus enhance neuroinflammation-induced neurotoxicity.

To this day, most experiments related to Meth-induced microglial activation were performed in vivo, where it is extremely hard to identify the key factor contributing to the sustained microglial activation. To better investigate the direct cellular mechanism of

Meth in microglia, an extended study on isolated microglia is clearly needed. Taking advantage of our cultured microglial system, we investigated whether Meth combined with gp120 will activate the inflammasome, which enhances the proinflammatory signaling leading to neuronal death. The outcome of this study may identify an intersecting site for Meth potentiation of and gp120-associated inflammasome activation and promote the development of therapeutic strategies for HAND.

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2. Materials and methods

Information on microglial culture and total protein extraction are same as described in Chapters 2 and 3 Materials section. The primary purpose of this section is to illustrate the difference of treatment of gp120 combined with Meth.

2.1. Western blotting

The microglia were treated with HIV-1 protein gp120 for 48 h before washing off with pre-warmed DMEM. The different concentrations of Meth were then added into the microglia for another 12 h. It is worth noting that gp120 primed the microglia, but did not co-incubate with Meth.

2.2. ELISA

After priming with gp120 for the first 48 h, different concentrations of Meth were added into the microglial culture and incubated for 24 h. The supernatants of each well were collected and was subjected to the detection of IL-1β ELISA Kit.

The experiments performed following manufacturer’s instructions. Briefly, the plates coated with capture antibody overnight at room temperature. BSA (0.1%) filtered through a 0.2-µm strainer used as a blocking reagent. Supernatants incubated 2h followed by incubation with detection antibody for another 2h. Then the Streptavidin-

HRP working solution incubated for another 20 minutes before substrate solution added to each well.

2.3. Immunofluorescent staining

Immunofluorescent staining was conducted to observe the co-localization of NLRP3 and caspase-1 as a readout of inflammasome activation. Microglia were seeded on coverslips in a 12-well plate at a density of 0.5 × 106 cells per well. Cells were directly primed with gp120 for 48 h and stimulated with Meth 50 µM. The cells were fixed with 4%

PFA for 20 min. The microglia blocked with 3% filtered BSA and permeabilized by 0.1%

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Triton X-100 for 10 min. The primary antibodies against NLRP3 and caspase-1 were all purchased from San ta Cruz (Dallas, TX). Both primary antibodies were in a 1:50 dilution.

2.4. Fluorescent dye loading and imaging

H2DCFDA was used to specifically detect the total ROS production. After microglia primed with gp120 for 48 h, the Meth further incubated for another 12 h. Then, the medium was removed and cells were washed three times with pre-warmed PBS.

H2DCFDA were made into 5 µM solution with DMEM and incubated with microglia for

30 min. The fluorescent intensities were quantified by a microplate reader. Meanwhile, the 20X picture of each well was taken as a representative image.

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3. Results

3.1. HIV-1 gp120 works as the priming signal for Meth-induced inflammasome

activation

The priming of gp120 elevated the expressional level of pro-IL-1β. The upregulation of IL-1β by gp120 application was in a dose-dependent manner. At the higher concentration (500 and 1000 pM), gp120 alone also promoted the maturation of IL-1β.

However, due to the decreased cellular viability (data not shown), 1000 pM of gp120 was not appropriate to serve as priming signal for Meth. For this reason, we selected

250 and 500 pM as priming concentrations of gp120. As shown in the Fig. 27A, the

Meth obviously increased the processing of IL-1β in the group of microglia primed with

500 pM of gp120. In the experiment of Fig. 27B, all groups of microglia were primed with 500 pM of gp120 and test the effect of different concentrations of Meth as the second activation signal. In consistent with the results in LPS, the processing of IL-1β was maximal after stimulation with 50 µM of Meth. Similarly, the release of IL-1β in supernatants further confirmed that 500 pM of gp120 and 50 µM of Meth were the best combination.

Our previous experiments in LPS-primed microglia demonstrated that Meth promoted the assembling of NLRP3 inflammasome. For this reason, we observed the co-localization of caspase-1 and NLRP3 with gp120-Meth combination. In the group of

Ctrl and gp120 alone, very few co-localized sites were found within the cell while both two proteins evenly distributed all over the cells. The combinational stimulation promoted the overlapping of two signals, and the number of aggregated NLRP3 increased.

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Figure 27. Meth promoted the HIV-1 gp120-induced IL-1β processing. In panel A, microglia were primed with different concentrations of gp120. After washing off the gp120 with PBS, Meth 50 µM was added for another 12 h. The expression levels of pro-

IL-1β and IL-1β were quantified in the bar graph below. Panel B was represented by the dose response of Meth in gp120-primed microglia. The processing of IL-1β was measured and quantified.

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Figure 28. Meth potentiated the HIV-1 gp120-induced IL-1β release. The secreted IL-1β was detected by ELISA. Microglia were primed with gp120 (0.5 nM) for 48 h and treated with different concentrations of Meth. *** p < .001 vs gp120.

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Figure 29. Meth induced the co-localization of NLRP3 and caspase-1 in gp120-primed microglia. Co-localization of NLRP3 and caspse-1 was observed by immunofluorescent staining with two primary antibodies, separately. All pictures were taken at 60X magnification.

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3.2. Activation of ROS contributes to the Meth-induced inflammasome activation

in gp120-primed microglia

Recent study demonstrated that cocaine abuse and HIV infection could activate the

NLRP3 inflammasome in a synergistic manner (Atluri et al. 2016). The ROS activation as signaling pathway converging from HIV-infection and cocaine abuse that was associated with the potentiated inflammasome activation. Our previous results indicated that gp120 could prime the microglia for Meth-induced inflammasome as the second activation signal. Thus, we also investigated the activation of total ROS after combinational stimulation of gp120 and Meth. The intensity of fluorescent signal of total

ROS was comparable to positive control in the group of microglia treated with gp120 and Meth. The gp120 alone also elevated the level of total ROS compared to the group of controls. However, the increase of gp120 group did not reach the significant level.

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Figure 30. Meth activated the total ROS synergistically with HIV-1 gp120. Total ROS was measure after receiving the combined stimulation of gp120 and Meth. The quantification of results was displayed as a bar graph. ** p < .01 vs Ctrl. # p < .05, # p

< .05 vs gp120. All images were taken at 40X original magnification.

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4. Discussion

Despite successes with highly active antiretroviral therapy (HAART), 50% of the

HIV-infected population still suffers from HIV-associated neurological disorder (HAND)

(Chen et al. 2014). The persistent neuroinflammation was found in HAND patients, even in the HAART era (Eden et al. 2007; Everall et al. 2009; Harezlak et al. 2011;

Kumar et al. 2007). A neuroimaging study found that the degree of neuroinflammation marked by activated microglia inversely correlated with executive function even in the patient receiving effective antiretroviral therapy (Garvey et al. 2014). Because the neuron is refractory to the HIV infection, the activated microglia are primary contributors to neuronal damage and cognitive dysfunction due to their releasing of neurotoxic agents (Gonzalez-Scarano and Martin-Garcia 2005). The neurotoxic HIV proteins released from infected microglia, in turn, sustained the microglial over-activation and the release of other potent neurotoxins. It is well-accepted that the most important inducers of persistent neuroinflammation are HIV proteins shed off from viruses or released from infected microglia or astrocytes, ultimately leading to neuronal damage

(Bagashev and Sawaya 2013). However, how these proinflammatory signals are sensed and maintained by microglia is still not understood.

We want to investigate Meth-induced inflammasome activation in a pre-existing inflammatory condition like HIV brain infection. To test this hypothesis, we first determined whether the gp120 could prime the microglia for subsequent Meth-induced inflammasome activation. Because the primary goal of inflammasome is to promote maturation and release of IL-1β, the secretion level of IL-1β was characterized by

ELISA. The optimized dose and time of gp120 and Meth treatment were studied by

Western blot analysis of maturation of IL-1β; we determine to prime the microglia with gp120 500 pM for 48h and then stimulate microglia with Meth 50 μM for 12 h. The

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supernatants of treated microglia were collected for detection of matured form of IL-1β that converting from pro-IL-1β by caspase-1. Finally, we examined the co-localization of

NLRP3 and caspase-1 as an indicator of NLRP3 inflammasome activation. After priming with gp120, Meth promoted the assembling of inflammasome evidenced by the increased overlapping of two signals.

In next step, we need to dissect further the pathways that directly mediated the

Meth-potentiated inflammasome activation in gp120-primed microglia. Because abnormalities of mitochondrial, efflux of potassium (K+) and cathepsin B release were all reported associated with HIV infection, it is still unclear whether gp120 synergistically impairs mitochondrial and lysosomal function with Meth or potentiates inflammasome activation by activation of an additional pathway (K+ efflux). We will pre-treat microglia with inhibitors including Ac-YVAD-CMK (caspase-1), CA-074Me (Cathepsin B), Mito-

TEMPO (Mitochondrial ROS) and high extracellular concentration of KCl (K+ efflux, 5 –

45 mM) before stimulation of Meth. Supernatants will be collected, and maturation and release of IL-1β will be measured by ELISA and WB. Subsequently, the activity of caspase-1 and aggregation of ASC will be studied after pre-treatment of pathway- specific inhibitors.

Because intensive activation of inflammasome initiates the pyroptosis pathway, microglial apoptosis should be examined after combined stimulation. The overall goals of this study are to determine and characterize whether the inflammasome is the intersecting site orchestrating Meth/120-induced microglial neurotoxic activity and to identify the potential target(s) for the development of therapeutic strategies for HAND.

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Chapter 5

General discussion

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In the highly effective anti-retroviral era, the plasma viral loads in HIV-infected patient have been well controlled by the deployment of combination anti-retroviral therapy (cART). Accordingly, the proportion of HIV-1 infected patient with HIV-1- associated dementia has been dramatically declined (Chan and Brew 2014). However, the milder form of HIV-1-associated neurocognitive disorder (HAND) remains common in HIV-1 infected patient (Heaton et al. 2010; Heaton et al. 2011). The abuse of psychoactive drugs, particularly the Meth, further exacerbates the deleterious effects of

HIV-1 infection on neurocognition (Weber et al. 2013). Because Meth abuse is associated with high-risk sexual behaviors and the injection sharing, HIV infection is prevalent among Meth abusers (Degenhardt et al. 2010). The HIV-1 patient with Meth abuse history shows a more severe neurocognitive deficits and neuropathology

(Langford et al. 2003). However, the exact molecular mechanisms underlying the potentiation effect of Meth in HAND is still largely unknown. Emerging evidence indicates that Meth abuse accelerates the progression of the pathogenesis of HAND through enhanced neuroinflammation (Chana et al. 2006; Soontornniyomkij et al. 2016;

Wires et al. 2012). Because the CNS inflammation is sustained in HIV patient even treated with cART (Saylor et al. 2016), the Meth-induced microglial activation might result in an overabundant inflammatory response that leads to an potentiated neuronal damage. We hypothesized that there is a common signaling pathway involved both in

HIV- and Meth-induced neurotoxicity and thus mediated their concerted effect on neuroinflammation.

Although the neuron is refractory to HIV infection, neuronal damage and apoptosis were still found in HAND patient (Kovalevich and Langford 2012). One proposed model of HIV-associated neurotoxicity suggested that viral proteins released from infected glial cell could directly induced neuronal death. While the another model believed that the

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death of neurons is caused by soluble neurotoxins derived from proinflammatory response to HIV infection (Lindl et al. 2010). In fact, the viral proteins will not only directly induce the neuronal damage, but also activate the glial cells to sustain the proinflammatory response (Liu et al. 2012a; Liu et al. 2013). Thus, it is reasonable to investigate whether Meth administration could potentiate the HIV protein-induced microglial activation.

Meth-induced neurotoxicity in the human abuser was first evidenced by the reduction of dopamine transporter with PET imaging (Volkow et al. 2001b). They first identified the human abuser and proved the recovery took more than one year, which excluded the possibility of transient downregulation (Volkow et al. 2001a). Later studies confirmed that long-term abuse of Meth is associated with degeneration of monoamine- containing terminals. The induction of local inflammatory response was well-accepted as the cellular mechanism of Meth-induced neurotoxicity (Thomas and Kuhn 2005a;

Thomas and Kuhn 2005c; Thomas et al. 2004b). However, the molecular mechanism underlying the Meth-induced microglial activation is still largely unknown. Currently, neuroinflammation has been recognized as a promising mechanism for Meth-induced dopaminergic terminal damage (Beardsley et al. 2010; Worley et al. 2016). Better understanding the molecular pathways involved in Meth-induced microglia activation is imperative.

Recently, certain polymorphisms of NLRP3 have been linked with increased susceptibility to HIV infection (Pontillo et al. 2010; Pontillo et al. 2012). The investigation of inflammasome-related components revealed that IL-1β and caspase-1 were induced in the HIV-infected brain, chiefly in microglia (Walsh et al. 2014b). Moreover, both Meth treatment and HIV infection are associated with upstream signaling pathways of inflammasome including ROS activation, mitochondrial damage, and lysosomal

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damage (Potula et al. 2010; Rodriguez-Franco et al. 2012; Talloczy et al. 2008;

Turchan-Cholewo et al. 2009; Villeneuve et al. 2016). Based on these evidence, we hypothesized that Meth could potentiate the HIV proteins-induced inflammasome activaiton and thus enhanced the HIV-associated neurotoxicity.

Because there are only limited studies of Meth effects in vitro, we first examined whether Meth alone could stimulate the proinflammatory response on cultured microglia.

Surprisingly, Meth treatment alone failed to replicate the inflammatory response in vivo.

Then, we hypothesized that Meth could potentiate pre-existing neuroinflammation stimulation (HIV infection), and thus shift the microglia from neuroprotective to neurotoxic phenotype.

The ideal pathorelevant pre-existing inflammatory stimulation should be HIV proteins. However, the role of these viral proteins in inflammasome activation is still not fully understood. To better investigate the Meth-induced inflammasome activation, we used LPS to mimic the HIV protein-induced pre-existing neuroinflammation. Both LPS and HIV protein gp120 can activate the NF-κB signal pathway and induce the expression of the pro-IL-1β (Heese et al. 1998; Shah and Kumar 2010; Walsh et al.

2014b). However, while LPS is well-accepted first priming signal, the role of gp120 in inflammasome activation is less clear. For this reason, we used LPS as priming signal to investigate whether Meth could activate the inflammasome, and thus potentiate the neuroinflammation-induced neurotoxicity.

We found that the LPS-induced release of IL-1β was enhanced while the transcriptional induction was not altered, which demonstrated that Meth regulation of IL-

1β is primarily at the post-translational level. After priming with LPS, Meth treatment activated the inflammasome, and thus promoted the processing and release of IL-1β.

Specific inhibition of inflammasome activation could block Meth-potentiated

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neurotoxicity. We also identified that Meth-induced mitochondrial ROS and lysosomal cathepsin B were primary molecular pathways mediating the Meth-induced inflammasome activation. Inhibition of mitochondrial ROS and cathepsin B could not only block the Meth-induced release of IL-1β, but also reverse the Meth-potentiated neurotoxicity.

After the role of Meth as second inflammasome activation signal was demonstrated, we further investigated whether gp120 could serve as the priming signal for Meth- induced inflammasome activation. Our results demonstrated that gp120 combined with

Meth could promote assembly of inflammasome evident by co-localization of NLRP3 and caspase-1, resulting in the processing and release of IL-1β.

Our studies identify the NLRP3 inflammasome as the intersecting molecular target of Meth and HIV-1 gp120. By specific inhibition of Meth-induced inflammasome activation, the Meth-potentiated neurotoxicity could be reversed in vitro. In future, it will be interesting to investigate whether specific inhibition of inflammasome in vivo could attenuate Meth-potentiated neurotoxicity. Taking advantage of the transgenic HIV rat model, we could assess whether specific inhibition of inflammasome could block HIV- and Meth-associated cognitive deficits. Further studies could even be performed on humanized mice. Chronic HIV infection in this model could investigate whether inflammasome inhibition could prevent the HIV neuroinvasion by protecting Blood Brain

Barrier (BBB) from Meth-induced leakage. It has been suggested that even with low viral burdens, HAND still persisted in chronic HIV-infected NSG-hCD34+ mice due to the residual microglial activation (Boska et al. 2014; Gorantla et al. 2010). Except the cognitive test, Meth- and HIV-associated behavior changes can also be examined

(Boska et al. 2014; Gorantla et al. 2010). Overall, these rodent models of neuro-HIV

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hold promise for future tests on novel therapeutic approaches based on specific inflammasome inhibition.

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