Recruitment of actin modifiers to TrkA endosomes governs retrograde NGF signaling and survival.

The neurotrophins NGF and NT3 collaborate to support development of sympathetic neurons. Although both promote axonal extension via the TrkA receptor, only NGF activates retrograde transport of TrkA endosomes to support neuronal survival. Here, we report that actin depolymerization is essential for initiation of NGF/TrkA endosome trafficking and that a Rac1-cofilin signaling module associated with TrkA early endosomes supports their maturation to retrograde transport-competent endosomes. These actin-regulatory endosomal components are absent from NT3/TrkA endosomes, explaining the failure of NT3 to support retrograde TrkA transport and survival. The inability of NT3 to activate Rac1-GTP-cofilin signaling is likely due to the labile nature of NT3/TrkA complexes within the acidic environment of TrkA early endosomes. Thus, TrkA endosomes associate with actin-modulatory proteins to promote F-actin disassembly, enabling their maturation into transport-competent signaling endosomes. Differential control of this process explains how NGF but not NT3 supports retrograde survival of sympathetic neurons.

Long-distance control of synapse assembly by target-derived NGF.

We report a role for long-distance retrograde neurotrophin signaling in the establishment of synapses in the sympathetic nervous system. Target-derived NGF is both necessary and sufficient for formation of postsynaptic specializations on dendrites of sympathetic neurons. This, in turn, is a prerequisite for formation of presynaptic specializations, but not preganglionic axonal ingrowth from the spinal cord into sympathetic ganglia. We also find that NGF-TrkA signaling endosomes travel from distal axons to cell bodies and dendrites where they promote PSD clustering. Furthermore, the p75 neurotrophin receptor restricts PSD formation, suggesting an important role for antagonistic NGF-TrkA and p75 signaling pathways during retrograde control of synapse establishment. Thus, in addition to defining the appropriate number of sympathetic neurons that survive the period of developmental cell death, target-derived NGF also exerts control over the degree of connectivity between the spinal cord and sympathetic ganglia through retrograde control of synapse assembly.

LIG family receptor tyrosine kinase-associated proteins modulate growth factor signals during neural development.

Genome-wide screens were performed to identify transmembrane proteins that mediate axonal growth, guidance and target field innervation of somatosensory neurons. One gene, Linx (alias Islr2), encoding a leucine-rich repeat and immunoglobulin (LIG) family protein, is expressed in a subset of developing sensory and motor neurons. Domain and genomic structures of Linx and other LIG family members suggest that they are evolutionarily related to Trk receptor tyrosine kinases (RTKs). Several LIGs, including Linx, are expressed in subsets of somatosensory and motor neurons, and select members interact with TrkA and Ret RTKs. Moreover, axonal projection defects in mice harboring a null mutation in Linx resemble those in mice lacking Ngf, TrkA, and Ret. In addition, Linx modulates NGF-TrkA- and GDNF-GFRalpha1/Ret-mediated axonal extension in cultured sensory and motor neurons, respectively. These findings show that LIGs physically interact with RTKs and modulate their activities to control axonal extension, guidance and branching.

Aquaporin 4 is increased in association with human immunodeficiency virus dementia: implications for disease pathogenesis.

Changes in astrocyte shape and function are known to occur in association with human immunodeficiency virus (HIV) dementia (HIVD). However, the causes and consequences of such changes are not completely understood. In vitro data suggest that changes in the expression of aquaporin 4 (AQP4), the aquaporin subtype expressed by astrocytes, can significantly influence cell shape and physiology. In the present study, the authors therefore investigated the possibility that AQP4 levels may be altered in HIVD. Using Western blot, the authors show that immunoreactivity for AQP4 is elevated in brain homogenates from the mid frontal gyrus of patients who died with HIVD (P < .005 HIV seronegative versus HIVD). Of interest, a significant increase was also observed in homogenates from HIV-infected individuals without dementia (P < .05 HIV seronegative versus neurologically normal HIV seropositive). In the present study the authors also examined the stimulated expression of AQP4 in cultured cells. Previous in vitro studies have shown that AQP4 expression may be increased by stimuli that induce cytoskeletal changes and/or the activation of p38 mitogen-activated protein (MAP) kinase. The authors therefore focused on tumor necrosis factor (TNF)-alpha, which has been linked to p38 MAP kinase activation, and thrombin, which may also induce changes in the actin cytoskeleton. Both may be elevated with HIVD. Again using Western blot, the authors show an increase in both AQP4 and phosphorylated p38 MAP kinase in homogenates from TNF-alpha- and thrombin-stimulated organotypic cerebellar and spinal cord cultures. Together, these studies suggest that AQP4 expression may be altered in HIVD and/or in response to exogenous proteinases. Additional studies may be warranted to determine whether altered AQP4 expression represents a protective and/or maladaptive response to central nervous system (CNS) inflammation.

Attenuated central nervous system infection in advanced HIV/AIDS with combination antiretroviral therapy.

BACKGROUND: Before the introduction of combination antiretroviral therapy (CART), neurological disease correlated with cerebrospinal fluid (CSF) levels of human immunodeficiency virus (HIV) RNA. OBJECTIVE: To investigate the relationships among HIV RNA levels, immune activation markers, and neurological status in patients receiving CART. DESIGN: Multicenter cohort study. SETTING: Academic neurology departments. PATIENTS: A total of 371 patients unselected for neurological complaints and with CD4 cell counts less than 200/microL or with cognitive symptoms and CD4 cell counts less than 300/microL were enrolled into the Northeastern AIDS Dementia cohort in 1998-2002. Diagnoses of HIV-associated dementia (HIV-D) and minor cognitive-motor disorder (MCMD) were obtained with a computerized algorithm. Plasma and CSF levels of HIV RNA, monocyte chemotactic protein 1, macrophage colony-stimulating factor, and tumor necrosis factor alpha were quantified. RESULTS: The mean +/- SD age was 41.5 +/- 7.2 years, and the mean +/- SD educational level was 12.3 +/- 2.2 years. Seventy percent of the cohort was black, and 30% were women. The mean +/- SD CD4 cell count was 136.8 +/- 87.9/microL, and CART was used in 71%. Twenty-nine percent of the patients were unimpaired (n = 106), 36% had MCMD (n = 133), and 35% had HIV-D (n = 128). Mean log(10) CSF HIV RNA copies per milliliter was 2.6 +/- 0.8, with no differences among the neurological groups, even after adjustments for baseline CD4 cell counts and antiretroviral therapy. Cerebrospinal fluid HIV RNA was undetectable in 47% of unimpaired, 46% of MCMD, and 43% of HIV-D patients (P = .91). Plasma levels of monocyte chemotactic protein type 1 and tumor necrosis factor alpha correlated weakly with HIV RNA levels but did not distinguish those with neurological deficits. CONCLUSIONS: In contrast to observations in individuals not treated with CART, we found no relationship between CSF markers and neurological status in this CART-using cohort with advanced HIV/AIDS. This was not explicable by demographic differences or plasma virological control. CART may substantially attenuate the degree of central nervous system HIV infection and immune activation, and in CART users, CSF HIV RNA and immune activation markers may fail to discriminate milder degrees of HIV-D and MCMD.

PGE2 receptors rescue motor neurons in a model of amyotrophic lateral sclerosis.

Recent studies suggest that the inducible isoform of cyclooxygenase, COX-2, promotes motor neuron loss in rodent models of ALS. We investigated the effects of PGE2, a principal downstream prostaglandin product of COX-2 activity, on motor neuron survival in an organotypic culture model of ALS. We find that PGE2 paradoxically protects motor neurons at physiological concentrations in this model. PGE2 exerts its downstream effects by signaling through a class of four distinct G-protein-coupled E-prostanoid receptors (EP1-EP4) that have divergent effects on cAMP. EP2 and EP3 are dominantly expressed in ventral spinal cord in neurons and astrocytes, and activation of these receptor subtypes individually or in combination also rescued motor neurons. The EP2 receptor is positively coupled to cAMP, and its neuroprotection was mimicked by application of forskolin and blocked by inhibition of PKA, suggesting that its protective effect is mediated by downstream effects of cAMP. Conversely, the EP3 receptor is negatively coupled to cAMP, and its neuroprotective effect was blocked by pertussis toxin, suggesting that its protective effect is dependent on Gi-coupled heterotrimeric signaling. Taken together, these data demonstrate an unexpected neuroprotective effect mediated by PGE2, in which activation of its EP2 and EP3 receptors protected motor neurons from chronic glutamate toxicity.

Antiretroviral treatment alters relationship between MCP-1 and neurometabolites in HIV patients.

OBJECTIVE: The relationships between neurometabolites and macrophage chemoattractant protein (MCP-1) in serum and cerebrospinal fluid (CSF) were evaluated in HIV patients before and after antiretroviral treatment. DESIGN: Prior studies found higher CSF MCP-1 levels in patients with HIV-associated dementia compared to those in neuroasymptomatic. We hypothesized that CSF MCP-1 levels would correlate inversely to neuronal metabolites [including N-acetyl compounds, glutamate+glutamine, as assessed by principal component analyses (PCA)] and positively to glial metabolites (including myo-inositol and choline compounds). METHODS: Thirty-nine antiretroviral-naive HIV patients were evaluated prospectively with proton magnetic resonance spectroscopy (1H MRS), and serum and CSF MCP-1 measurements prior to highly active antiretroviral therapy (HAART); 31 of these patients completed follow-up studies after 3 months of HAART but only 24 had follow-up CSF studies. RESULTS: After HAART, brain metabolites and clinical signs showed no change despite improvements in systemic (CD4 counts, plasma viral load, MCP-1) and CSF (viral load and MCP-1) variables. CSF, but not serum, MCP-1 levels correlated inversely with the neuronal component (from PCA) prior to treatment (r=-0.59, P=0.0008). Conversely, after 3 months of HAART, the glial component (from PCA) correlated positively with CSF MCP-1 levels (r=0.70, P=0.0002; ANCOVA interaction for treatment status, P=0.003). CONCLUSIONS: These findings suggest that higher CSF MCP-1 levels are associated with neuronal dysfunction in untreated patients. After 3 months of HAART, the decreased systemic factors (viral burden, systemically derived MCP-1) no longer associate with neuronal dysfunction, but subjects with the strongest glial response in the brain continue to produce the highest levels of MCP-1.

Human immunodeficiency virus type 1 Tat and methamphetamine affect the release and activation of matrix-degrading proteinases.

Human immunodeficiency virus (HIV) dementia (HIVD) is associated with an increase in the number of activated monocytes within the central nervous system (CNS), a pathological feature that may be more remarkable in the setting of superimposed substance abuse. Monocytes may transport HIV to the brain, and, moreover, activated and/or infected monocytes have been shown to release a number of potent neurotoxins. Although the mechanisms responsible for the increase in the CNS ingress of monocytes are multiple, blood-brain barrier (BBB)-degrading matrix metalloproteinases (MMPs) are likely to play an important role. The current study investigates the effects of the HIV-1-encoded protein Tat, and the drug of abuse methamphetamine, on MMP release from brain derived cells. The release of urokinase plasminogen activator (uPA), an activator of MMPs, was also investigated. Mixed human neuron/astrocyte cultures were stimulated with Tat or methamphetamine, and supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA) and/or gelatin substrate zymography. Results showed that Tat and methamphetamine increased the release of MMP-1 from these cultures. Tat also increased supernatant levels of active MMP-2. In addition, both Tat and methamphetamine stimulated the release of the MMP activator uPA, and in a manner that was sensitive to inhibition with pertussis toxin. Together, these results suggest that in HIVD, Tat and methamphetamine may contribute to CNS inflammation by stimulating increased release and/or activation of matrix-degrading proteinases through mechanisms that include Gi/Go-coupled signaling. These results also suggest a potential mechanism for acceleration of HIVD with methamphetamine use.

Matrix metalloproteinase 1 interacts with neuronal integrins and stimulates dephosphorylation of Akt.

Several studies have demonstrated that matrix metalloproteinases (MMPs) are cytotoxic. The responsible mechanisms, however, are not well understood. MMPs may promote cytotoxicity through their ability to disrupt or degrade matrix proteins that support cell survival, and MMPs may also cleave substrates to generate molecules that stimulate cell death. In addition, MMPs may themselves act on cell surface receptors that affect cell survival. Among such receptors is the alpha(2)beta(1) integrin, a complex that has previously been linked to leukocyte death. In the present study we show that human neurons express alpha(2)beta(1) and that pro-MMP-1 interacts with this integrin complex. We also show that stimulation of neuronal cultures with MMP-1 is associated with a rapid reduction in the phosphorylation of Akt, a kinase that can influence caspase activity and cell survival. Moreover, MMP-1-associated dephosphorylation of Akt is inhibited by a blocking antibody to the alpha(2) integrin, but not by batimastat, an inhibitor of MMP-1 enzymatic activity. Such dephosphorylation is also stimulated by a catalytic mutant of pro-MMP-1. Additional studies show that MMP-1 causes neuronal death, which is significantly diminished by both a general caspase inhibitor and anti-alpha(2) but not by batimastat. Together, these results suggest that MMP-1 can stimulate dephosphorylation of Akt and neuronal death through a non-proteolytic mechanism that involves changes in integrin signaling.

Matrix metalloproteinase-1 activates a pertussis toxin-sensitive signaling pathway that stimulates the release of matrix metalloproteinase-9.

The matrix metalloproteinases (MMPs) are a family of structurally related metalloendopeptidases so named due to their propensity to target extracellular matrix (ECM) proteins. Accumulating evidence, however, suggests that these proteases cleave numerous non-ECM substrates including enzymes and cell surface receptors. MMPs may also bind to cell surface receptors, though such binding has typically been thought to mediate internalization and degradation of the bound protease. More recently, it has been shown that MMP-1 coimmunoprecipitates with the alpha2beta1 integrin, a receptor for collagen. This association may serve to localize the enzymatic activity of MMP-1 so that collagen is cleaved and cell migration is facilitated. In other studies, however, it has been shown that integrin engagement may be linked to the activation of signaling cascades including those mediated by Gialpha containing heterotrimers. As an example, alpha2beta1 can form a complex with CD47 that may associate with Gialpha. In the present study we have therefore investigated the possibility that MMP-1 may affect intracellular changes that are linked to the activation of a Gi protein-coupled receptor. We show that treatment of neural cells with MMP-1 is followed by a rapid reduction in cytosolic levels of cAMP. Moreover, MMP-1 potentiates proteinase activated receptor-1 (PAR-1) agonist-linked increases in intracellular calcium, an effect which is often observed when an agonist of a Gi protein-coupled receptor is administered in association with an agonist of a Gq coupled receptor. In addition, MMP-1 stimulates pertussis toxin sensitive release ofMMP-9 both from cultured neural cells and monocyte/macrophages. Together, these results suggest that MMP-1 signals through a pertussis toxin-sensitive G protein-coupled receptor.