Research ReportAttenuation of oxidative stress, inflammation and apoptosis by minocycline prevents retrovirus-induced neurodegeneration in mice
Introduction
ts1, a neuropathogenic mutant of Moloney murine leukemia virus (MoMuLV), induces a progressive neurodegenerative disease in susceptible strains of mice (Stoica et al., 1993, Wong et al., 1991, Wong, 1990). This neurodegenerative disease is morphologically manifested as spongiform encephalomyelopathy, and clinically manifested as hindlimb paralysis and wasting, eventually leading to death of infected animals (Wong et al., 1992). The ts1 virus has a mutation that results in a substitution of isoleucine for valine at position 25 in the viral envelope precursor protein gPr80env (Szurek et al., 1990). This alteration makes the virus cytopathic to astrocytes, due to abnormal accumulation of uncleaved gPr80env. In turn, the infected astrocytes may induce motor neuron loss in infected mice (Stoica et al., 2000, Wong and Yuen, 1994, Wong P.K.Y. and Y.P., 1992).
Although neurodegeneration is the end result of central nervous system (CNS) damage after ts1 infection, the virus replicates in astrocytes, microglia, oligodentrocytes and endothelial cells, but not in neurons (Stoica et al., 1993, Wong and Yuen, 1994). In this respect, the ts1 induced neurodegenerative syndrome is to some extent similar to the neuropathology of HIV-1-associated dementia (HAD) (Clark et al., 2001, Gonzalez-Scarano et al., 1995, Mollace et al., 2001, Stoica et al., 1993). ts1- and HIV-1-induced neurodegeneration also share pathological features with other nonviral neurological diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). These conditions resemble the ts1 syndrome in that they all involve oxidative stress, inflammation and apoptosis (Infante-Duarte et al., 2008, Liu et al., 2004, Qiang et al., 2004, Reynolds et al., 2007).
Minocycline is a semisynthetic second-generation tetracycline compound that effectively crosses the blood–brain barrier (Yong et al., 2004). This drug has been shown to have neuroprotective activity in primates infected with Simian immunodeficiency virus (SIV) (Zink et al., 2005). It also protects the CNS in ischemia–perfusion injury (Yrjanheikki et al., 1999), Huntington's disease (Chen et al., 2000), PD (Du et al., 2001), AD(Noble et al., 2009) and ALS (Zhu et al., 2002). The mechanisms involved in neuroprotection by minocycline appear to be distinct from its antimicrobial activities (Tikka et al., 2001).
Recent work has shown that minocycline is a potent antioxidant, with radical scavenging activities (Kraus et al., 2005, Morimoto et al., 2005). Both laboratory and clinical studies have also shown that minocycline has broad-spectrum anti-inflammatory properties (Sapadin and Fleischmajer, 2006) that are manifested via inhibition of cyclooxygenase-2 (COX-2) (Yrjanheikki et al., 1999), nuclear factor-κB (NF-κB) (Nikodemova et al., 2006, Si et al., 2004) and activation of microglia (Dheen et al., 2007, Tikka et al., 2001, Zink et al., 2005). Minocycline also exerts anti-apoptotic effects (Choi et al., 2007, Stirling et al., 2004), via both caspase-dependent and caspase-independent pathways (Stirling et al., 2005).
In this study, we first demonstrated that minocycline significantly delays ts1-induced neurodegeneration in infected mice. We then identified mechanisms underlying the neuroprotective effects of minocycline on cultured astrocytes, and on primary neurons that were either cocultured with ts1-infected astrocytes, or exposed to spent medium from these astrocyte cultures. We conclude that neuroprotection by minocycline involves both direct radical scavenging and upregulation of Nrf2-mediated antioxidant defenses. Its neuroprotection also involves anti-inflammatory and anti-apoptotic mechanisms.
Section snippets
Minocycline not only delays paralysis and death, but also attenuates astrogliosis lesion in ts1-infected FVB/N mice
The paralysis/survival curves in Fig. 1A show that untreated ts1-infected (ts1-only) mice became paralyzed at around 35 dpi, while ts1-infected minocycline-treated (ts1-mino) mice lived longer, and became paralyzed much later (p < 0.001). Brainstem and spinal cord tissues were prepared from uninfected, ts1-only and ts1-mino mice, and stained with hematoxylin and eosin (HE). The sections from uninfected mice showed normal neuropil in the brainstem, while those from ts1-only mice contained many
Discussion
Minocycline has been shown to reduce the incidence and severity of encephalitis in SIV-infected rhesus macaques (Zink et al., 2005), and to slow other nonviral neurodegenerative diseases (Chen et al., 2000, Du et al., 2001, Yrjanheikki et al., 1999). At present, clinical trials of minocycline in HIV-associated dementia patients are underway, but more work is needed in nonhuman animal models for HAD regarding the mechanism of minocycline's protection in retrovirus-induced neurodegeneration. Such
Animals and infection
FVB/N mouse pups were injected intraperitoneally (i.p.) with 0.1 ml/pup ts1 virus suspension containing 1 × 107 infectious units (IU)/ml of ts1, at 3 days after birth. The infected animals were then separated into groups. ts1-infected minocycline-treated mice (ts1-mino, n = 8) were treated with minocycline (Sigma), delivered intraperitoneally at 10 mg/kg/day at 5 days after birth (2 days post-infection, or dpi) continuously, until the infected untreated mice become paralyzed just prior to death.
Acknowledgments
This work was supported in part by NIH Grants RO1 MH071583 RO1 NS043984 (to P.K.Wong), and by NIEHS center grant P30 ES007784 the National Cancer Institute (MD. Anderson Core Grant P30 CA016672) and by funds from The Longevity Foundation in Austin, Texas. We thank Christine Brown and Rebecca Deen for their assistance in preparing the manuscript. We are also most grateful to Ms. Lifang Zhang for technical assistance.
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