Proteomic identification of synaptic caspase substrates.

The dismantling and elimination of excess neurons and their connections (pruning) is essential for brain development and may be aberrantly reactivated in some neurodegenerative diseases. Growing evidence implicates caspase-mediated apoptotic and nonapoptotic cascades in the dysfunction and death of neurons in neurodegenerative disorders such as Alzheimer's, Parkinson, and Huntington's diseases. It is the cleaved caspase substrates that are the effectors of synapse elimination. However, their identities, specific cleavage sites, and functional consequences of cleavage are largely unknown. An important gap in our knowledge is a comprehensive catalog of synapse-specific or synapse-enriched caspase targets. Traditional biochemical approaches have revealed only a small number of neuronal caspase targets. Instead, we utilized a gel-based proteomics approach to enable the first global analysis of caspase-mediated cleavage events in mammalian brain synapses, employing both an in vitro system with recombinant activated caspases and an in vivo model of ethanol-induced neuronal apoptosis. Of the more than 70 putative cleavage substrates that were identified, 22 were previously known caspase substrates. Among the novel targets identified and validated by Western blot were the proton pump ATPase subunit ATP6V1B2 and the N-ethylmaleimide-sensitive fusion protein (NSF). Our work represents the first comprehensive, proteome-wide screen for proteolytic targets of caspases in neuronal synapses. Our discoveries will have significance for both furthering basic understanding of roles of caspases in synaptic plasticity and synaptic loss in neurodegeneration, and on a more immediately practical level, may provide candidate biomarkers for measuring synapse loss in human disease states.

Fractalkine is a "find-me" signal released by neurons undergoing ethanol-induced apoptosis.

Apoptotic neurons generated during normal brain development or secondary to pathologic insults are efficiently cleared from the central nervous system. Several soluble factors, including nucleotides, cytokines, and chemokines are released from injured neurons, signaling microglia to find and clear debris. One such chemokine that serves as a neuronal-microglial communication factor is fractalkine, with roles demonstrated in several models of adult neurological disorders. Lacking, however, are studies investigating roles for fractalkine in perinatal brain injury, an important clinical problem with no effective therapies. We used a well-characterized mouse model of ethanol-induced apoptosis to assess the role of fractalkine in neuronal-microglial signaling. Quantification of apoptotic debris in fractalkine-knockout (KO) and CX3CR1-KO mice following ethanol treatment revealed increased apoptotic bodies compared to wild type mice. Ethanol-induced injury led to release of soluble, extracellular fractalkine. The extracellular media harvested from apoptotic brains induces microglial migration in a fractalkine-dependent manner that is prevented by neutralization of fractalkine with a blocking antibody or by deficiency in the receptor, CX3CR1. This suggests fractalkine acts as a "find-me" signal, recruiting microglial processes toward apoptotic cells to promote their clearance. Next, we aimed to determine whether there are downstream alterations in cytokine gene expression due to fractalkine signaling. We examined mRNA expression in fractalkine-KO and CX3CR1-KO mice after alcohol-induced apoptosis and found differences in cytokine production in the brains of these KOs by 6 h after ethanol treatment. Collectively, this suggests that fractalkine acts as a "find me" signal released by apoptotic neurons, and subsequently plays a critical role in modulating both clearance and inflammatory cytokine gene expression after ethanol-induced apoptosis.

Caspase-mediated cleavage of actin and tubulin is a common feature and sensitive marker of axonal degeneration in neural development and injury.

BACKGROUND: Axon degeneration is a characteristic feature of multiple neuropathologic states and is also a mechanism of physiological neurodevelopmental pruning. The vast majority of in vivo studies looking at axon degeneration have relied on the use of classical silver degeneration stains, which have many limitations including lack of molecular specificity and incompatibility with immunolabeling methods. Because Wallerian degeneration is well known to involve cytoskeletal disassembly and because caspases are recently implicated in aspects of this process, we asked whether antibodies directed at caspase-generated neoepitopes of beta-actin and alpha-tubulin would be useful immunohistochemical markers of pathological and developmental axon degeneration. RESULTS: Here we demonstrate that several forms of axon degeneration involve caspase-mediated cleavage of these cytoskeletal elements and are well-visualized using this approach. We demonstrate the generation of caspase-induced neoepitopes in a) an in vitro neuronal culture model using nerve growth factor-deprivation-induced degeneration and b) an in vivo model using ethanol-induced neuronal apoptosis, and c) during normal developmental pruning and physiological turnover of neurons. CONCLUSIONS: Our findings support recent experimental data that suggests caspase-3 and caspase-6 have specific non-redundant roles in developmental pruning. Finally, these findings may have clinical utility, as these markers highlight degenerating neurites in human hypoxic-ischemic injury. Our work not only confirms a common downstream mechanism involved in axon degeneration, but also illuminates the potential utility of caspase-cleavage-neoepitope antibodies as markers of neurodegeneration.

Brain-specific angiogenesis inhibitor-1 expression in astrocytes and neurons: implications for its dual function as an apoptotic engulfment receptor.

Brain-specific angiogenesis inhibitor-1 (BAI1) is a transmembrane protein highly expressed in normal brain that has been ascribed two apparently distinct functions: inhibition of angiogenesis and recognition and engulfment of apoptotic cells by phagocytes. A previous localization study reported BAI1 expression only in neurons. Because a phagocytic function of BAI1 could be important for neuroglial antigen processing and presentation, we performed immunolocalization studies in adult mouse brain and cultured neural cells, using a pair of antibodies directed against N- and C-terminal epitopes. BAI1 immunoreactivity is enriched in gray matter structures and largely excluded from myelinated axon tracts. Neuronal BAI1 expression was readily detectable in the cerebellar molecular layer as well as in primary hippocampal cultures. In some brain regions, especially olfactory bulb glomeruli, BAI1 was expressed by GFAP-positive astrocytes. Cultured cortical astrocytes show small (∼0.4µm(2)) BAI1 immunoreactive membrane puncta as well as prominent focal adhesion localization in a subset of cells. In mixed neuronal-glial cultures, BAI1-expressing astrocytes frequently contained engulfed apoptotic debris. Cultured astrocytes engulfed apoptotic targets, and BAI1 showed accumulation within the phagocytic cup. We hypothesize that glial BAI1 may subserve an engulfment function in adult brain regions such as olfactory bulb with ongoing apoptotic turnover, whereas neuronal-derived BAI1 may serve primarily as an anti-angiogenic factor in the mature neuropil.

Serotonergic dystrophy induced by excess serotonin.

Administration of certain serotonin-releasing amphetamine derivatives (fenfluramine and/or 3,4-methylenedioxymethamphetamine, MDMA, 'ecstasy') results in dystrophic serotonergic morphology in the mammalian brain. In addition to drug administration, dystrophic serotonergic neurites are also associated with neurodegenerative disorders. We demonstrate here that endogenously elevated serotonin in the Drosophila CNS induces aberrant enlarged varicosities, or spheroids, that are morphologically similar to dystrophic mammalian serotonergic fibers. In Drosophila these spheroids are specific to serotonergic neurons, distinct from typical varicosities, and form only after prolonged increases in cytoplasmic serotonin. Our results also suggest that serotonin levels during early development determine later sensitivity of spheroid formation to manipulations of the serotonin transporter (SERT). Elevated serotonin also interacts with canonical protein aggregation and autophagic pathways to form spheroids. The data presented here support a model in which excess cytoplasmic neurotransmitter triggers a cell-specific pathway inducing aberrant morphology in fly serotonergic neurons that may be shared in certain mammalian pathologies.

Brain-specific deletion of extracellular signal-regulated kinase 2 mitogen-activated protein kinase leads to aberrant cortical collagen deposition.

The mitogen-activated protein kinases extracellular signal-regulated kinase (ERK)1 and 2 are essential intracellular mediators of numerous transmembrane signals. To investigate neural-specific functions of ERK2 in the brain, we used a Cre/lox strategy using Nestin:Cre to drive recombination in neural precursor cells. Nestin:Cre;ERK2(fl/fl) conditional knockout (cKO) mice have architecturally normal brains and no gross behavioral deficits. However, all cKO mice developed early-onset (postnatal day 35 to 40) frontal cortical astrogliosis, without evidence of neuronal degeneration. Frontoparietal cortical gray matter, but not underlying white matter, was found to contain abundant pericapillary and parenchymal reticulin fibrils, which were shown by immunohistochemistry to contain fibrillar collagens, including type I collagen. ERK1 general KO mice showed neither fibrils nor astrogliosis, indicating a specific role for ERK2 in the regulation of brain collagen. Collagen fibrils were also observed to a lesser extent in GFAP:Cre;ERK2(fl/fl) mice but not in CamKII-Cre;ERK2(fl/fl) mice (pyramidal neuron specific), consistent with a possible astroglial origin. Primary astroglial cultures from cKO mice expressed elevated fibrillar collagen levels, providing further evidence that the phenotype may be cell autonomous for astroglia. Unlike most other tissues, brain and spinal cord parenchyma do not normally contain fibrillar collagens, except in disease states. Determining mechanisms of ERK2-mediated collagen regulation may enable targeted suppression of glial scar formation in diverse neurological disorders.

Opposing roles of ERK and p38 MAP kinases in FGF2-induced astroglial process extension.

The stellate processes of astroglial cells undergo extensive remodeling in response to neural injury. Little is known about intracellular signaling mechanisms controlling process extension. We tested roles for the ERK and p38 MAP kinase pathways in a simplified culture model. FGF2-induced process extension was preceded by a strong and transient phosphorylation of ERK, and a modest activation of p38 MAP kinase, which exhibited significant basal activity. Phosphorylated ERK was found predominantly in the cytoplasm, whereas activated p38 MAP kinase was nuclear. Process extension was completely blocked by the specific MEK inhibitor U0126. Conversely, inhibition of the p38 MAP kinase pathway with SB202190 stimulated spontaneous process growth and greatly potentiated FGF2-induced process extension. The p38 inhibitor effect was reproduced with an adenovirus expressing dominant-negative p38 MAP kinase. Selective pharmacological blockade of MAP kinase pathways may enable modulation of the astroglial response to injury so as to promote neural regeneration.