Correction: The Impact of Juvenile Coxsackievirus Infection on Cardiac Progenitor Cells and Postnatal Heart Development.

[This corrects the article DOI: 10.1371/journal.ppat.1004249.].

Leucine-rich repeat kinase 2 modulates neuroinflammation and neurotoxicity in models of human immunodeficiency virus 1-associated neurocognitive disorders.

Leucine-rich repeat kinase 2 (LRRK2) is the single most common genetic cause of both familial and sporadic Parkinson's disease (PD), both of which share pathogenetic and neurologic similarities with human immunodeficiency virus 1 (HIV-1)-associated neurocognitive disorders (HAND). Pathologic LRRK2 activity may also contribute to neuroinflammation, because microglia lacking LRRK2 exposed to proinflammatory stimuli have attenuated responses. Because microglial activation is a hallmark of HIV-1 neuropathology, we have investigated the role of LRRK2 activation using in vitro and in vivo models of HAND. We hypothesize that LRRK2 is a key modulator of microglial inflammatory responses, which play a pathogenic role in both HAND and PD, and that these responses may cause or exacerbate neuronal damage in these diseases. The HIV-1 Tat protein is a potent neurotoxin produced during HAND that induces activation of primary microglia in culture and long-lasting neuroinflammation and neurotoxicity when injected into the CNS of mice. We found that LRRK2 inhibition attenuates Tat-induced pS935-LRRK2 expression, proinflammatory cytokine and chemokine expression, and phosphorylated p38 and Jun N-terminal kinase signaling in primary microglia. In our murine model, cortical Tat injection in LRRK2 knock-out (KO) mice results in significantly diminished neuronal damage, as assessed by microtubule-associated protein 2 (MAP2), class III ß-tubulin TUJ1, synapsin-1, VGluT, and cleaved caspase-3 immunostaining. Furthermore, Tat-injected LRRK2 KO animals have decreased infiltration of peripheral neutrophils, and the morphology of microglia from these mice were similar to that of vehicle-injected controls. We conclude that pathologic activation of LRRK2 regulates a significant component of the neuroinflammation associated with HAND.

The impact of juvenile coxsackievirus infection on cardiac progenitor cells and postnatal heart development.

Coxsackievirus B (CVB) is an enterovirus that most commonly causes a self-limited febrile illness in infants, but cases of severe infection can manifest in acute myocarditis. Chronic consequences of mild CVB infection are unknown, though there is an epidemiologic association between early subclinical infections and late heart failure, raising the possibility of subtle damage leading to late-onset dysfunction, or chronic ongoing injury due to inflammatory reactions during latent infection. Here we describe a mouse model of juvenile infection with a subclinical dose of coxsackievirus B3 (CVB3) which showed no evident symptoms, either immediately following infection or in adult mice. However following physiological or pharmacologically-induced cardiac stress, juvenile-infected adult mice underwent cardiac hypertrophy and dilation indicative of progression to heart failure. Evaluation of the vasculature in the hearts of adult mice subjected to cardiac stress showed a compensatory increase in CD31+ blood vessel formation, although this effect was suppressed in juvenile-infected mice. Moreover, CVB3 efficiently infected juvenile c-kit+ cells, and cardiac progenitor cell numbers were reduced in the hearts of juvenile-infected adult mice. These results suggest that the exhausted cardiac progenitor cell pool following juvenile CVB3 infection may impair the heart's ability to increase capillary density to adapt to increased load.

Distinct neural stem cell tropism, early immune activation, and choroid plexus pathology following coxsackievirus infection in the neonatal central nervous system.

Coxsackievirus B3 (CVB3) and lymphocytic choriomeningitis virus (LCMV) are both neurotropic RNA viruses, which can establish a persistent infection and cause meningitis and encephalitis in the neonatal host. Utilizing our neonatal mouse model of infection, we evaluated the consequences of early viral infection upon the host central nervous system (CNS) by comparing CVB3 and LCMV infection. Both viruses expressed high levels of viral protein in the choroid plexus and subventricular zone (SVZ), a region of neurogenesis. LCMV infected a greater number of cells in the SVZ and targeted both nestin(+) (neural progenitor cell marker) and olig2(+) (glial progenitor marker) cells at a relatively equal proportion. In contrast, CVB3 preferentially infected nestin(+) cells within the SVZ. Microarray analysis revealed differential kinetics and unique host gene expression changes for each infection. MHC class I gene expression, several developmental-related Hox genes, and transthyretin (TTR), a protein secreted in the cerebrospinal fluid by the choroid plexus, were specifically downregulated following CVB3 infection. Also, we identified severe pathology in the choroid plexus of CVB3-infected animals at 48 h post infection accompanied by a decrease in the level of TTR and carbonic anhydrase II. These results demonstrate broader neural progenitor and stem cell (NPSC) tropism for LCMV in the neonatal CNS, whereas CVB3 targeted a more specific subset of NPSCs, stimulated a distinct early immune response, and induced significant acute damage in the choroid plexus.

The new small-molecule mixed-lineage kinase 3 inhibitor URMC-099 is neuroprotective and anti-inflammatory in models of human immunodeficiency virus-associated neurocognitive disorders.

Human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND) is a significant source of disability in the HIV-infected population. Even with stringent adherence to anti-retroviral therapy, >50% of patients living with HIV-1 will develop HAND (Heaton et al., 2010). Because suppression of viral replication alone is not enough to stop HAND progression, there is a need for an adjunctive neuroprotective therapy in this population. To this end, we have developed a small-molecule brain-penetrant inhibitor with activity against mixed-lineage kinase 3 (MLK3), named URMC-099. MLK3 activation is associated with many of the pathologic hallmarks of HAND (Bodner et al., 2002, 2004; Sui et al., 2006) and therefore represents a prime target for adjunctive therapy based on small-molecule kinase inhibition. Here we demonstrate the anti-inflammatory and neuroprotective effects of URMC-099 in multiple murine and rodent models of HAND. In vitro, URMC-099 treatment reduced inflammatory cytokine production by HIV-1 Tat-exposed microglia and prevented destruction and phagocytosis of cultured neuronal axons by these cells. In vivo, URMC-099 treatment reduced inflammatory cytokine production, protected neuronal architecture, and altered the morphologic and ultrastructural response of microglia to HIV-1 Tat exposure. In conclusion, these data provide compelling in vitro and in vivo evidence to investigate the utility of URMC-099 in other models of HAND with the goal of advancement to an adjunctive therapeutic agent.

Ultrastructure of microglia-synapse interactions in the HIV-1 Tat-injected murine central nervous system.

The destruction of normal synaptic architecture is the main pathogenetic substrate in HIV-associated neurocognitive disorder (HAND), but the sequence of cellular events underlying this outcome is not completely understood. Our recent work in a mouse model of HAND using a single intraparenchymal injection of the HIV-1 regulatory protein trans-activator of transcription revealed increased microglial phagocytosis that was accompanied by an increased release of pro-inflammatory cytokines and elimination of dendritic spines in vivo, thus suggesting that microglia-synapse interactions could be dysregulated in HAND. Here, we further examine the relationships between microglia and synaptic structures in our mouse model, at high spatial resolution using immunocytochemical electron microscopy. Our ultrastructural analysis reveals the prevalence of putative microglial filopodial protrusions, which are targeting excitatory and inhibitory synapses, some of which contain phagocytic inclusions at various distances from their distal extremities to the microglial cell bodies. These observations thus suggest that cell-to-cell contacts mediated by microglial filopodia might be a crucial preliminary step in the elimination of synaptic structures in a neuroinflammatory milieu that occurs in HAND.

LRRK2 kinase inhibition prevents pathological microglial phagocytosis in response to HIV-1 Tat protein.

BACKGROUND: Human Immunodeficiency Virus-1 (HIV-1) associated neurocognitive disorders (HANDs) are accompanied by significant morbidity, which persists despite the use of combined antiretroviral therapy (cART). While activated microglia play a role in pathogenesis, changes in their immune effector functions, including phagocytosis and proinflammatory signaling pathways, are not well understood. We have identified leucine-rich repeat kinase 2 (LRRK2) as a novel regulator of microglial phagocytosis and activation in an in vitro model of HANDs, and hypothesize that LRRK2 kinase inhibition will attenuate microglial activation during HANDs. METHODS: We treated BV-2 immortalized mouse microglia cells with the HIV-1 trans activator of transcription (Tat) protein in the absence or presence of LRRK2 kinase inhibitor (LRRK2i). We used Western blot, qRT-PCR, immunocytochemistry and latex bead engulfment assays to analyze LRRK2 protein levels, proinflammatory cytokine and phagocytosis receptor expression, LRRK2 cellular distribution and phagocytosis, respectively. Finally, we utilized ex vivo microfluidic chambers containing primary hippocampal neurons and BV-2 microglia cells to investigate microglial phagocytosis of neuronal axons. RESULTS: We found that Tat-treatment of BV-2 cells induced kinase activity associated phosphorylation of serine 935 on LRRK2 and caused the formation of cytoplasmic LRRK2 inclusions. LRRK2i decreased Tat-induced phosphorylation of serine 935 on LRRK2 and inhibited the formation of Tat-induced cytoplasmic LRRK2 inclusions. LRRK2i also decreased Tat-induced process extension in BV-2 cells. Furthermore, LRRK2i attenuated Tat-induced cytokine expression and latex bead engulfment. We examined relevant cellular targets in microfluidic chambers and found that Tat-treated BV-2 microglia cells cleared axonal arbor and engulfed neuronal elements, whereas saline treated controls did not. LRRK2i was found to protect axons in the presence of Tat-activated microglia, as well as AnnexinV, a phosphatidylserine-binding protein. In addition, LRRK2i decreased brain-specific angiogenesis inhibitor 1 (BAI1) receptor expression on BV-2 cells after Tat-treatment, a key receptor in phosphatidylserine-mediated phagocytosis. CONCLUSION: Taken together, these results implicate LRRK2 as a key player in microglial inflammation and, in particular, in the phagocytosis of neuronal elements. These studies show that LRRK2 kinase inhibition may prove an effective therapeutic strategy for HANDs, as well as other neuroinflammatory conditions.