Delaying clinical disease onset would greatly reduce neurodegenerative disease burden, but the mechanisms influencing early preclinical progression are poorly understood. Here, we show that in mouse models of familial motoneuron (MN) disease, SOD1 mutants specifically render vulnerable MNs dependent on endogenous neuroprotection signaling involving excitability and mammalian target of rapamycin (mTOR). The most vulnerable low-excitability FF MNs already exhibited evidence of pathology and endogenous neuroprotection recruitment early postnatally. Enhancing MN excitability promoted MN neuroprotection and reversed misfolded SOD1 (misfSOD1) accumulation and MN pathology, whereas reducing MN excitability augmented misfSOD1 accumulation and accelerated disease. Inhibiting metabotropic cholinergic signaling onto MNs reduced ER stress, but enhanced misfSOD1 accumulation and prevented mTOR activation in alpha-MNs. Modulating excitability and/or alpha-MN mTOR activity had comparable effects on the progression rates of motor dysfunction, denervation, and death. Therefore, excitability and mTOR are key endogenous neuroprotection mechanisms in motoneurons to counteract clinically important disease progression in ALS.

Members of the BTB-kelch superfamily play important roles during fundamental cellular processes, such as the regulation of cell morphology, migration, and gene expression. The BTB-kelch protein LZTR-1 is deleted in the majority of DiGeorge syndrome patients and is believed to act as a transcriptional regulator. However, functional and expression profiling studies of LZTR-1 have not been performed thus far. Therefore, we examined the subcellular localization and function of LZTR-1 to gain insights into its biological role. Analysis of the primary structure of the protein revealed six N-terminal kelch motifs and two BTB/POZ domains at the C terminus within LZTR-1. Confocal analysis of the subcellular distribution of LZTR-1 using the Golgi markers GM130, Golgin-97, and TGN46 identified a localization of LZTR-1 exclusively on the cytoplasmic surface of the Golgi network that is mediated by its second BTB/POZ domain. In contrast to most other BTB-kelch proteins, LZTR-1 did not co-localize with actin. Treatment with brefeldin A did not lead to redistribution of LZTR-1 to the endoplasmic reticulum but caused its relocalization in dispersed, punctuated structures that were also positive for GM130. These data demonstrate that LZTR-1 is a Golgi matrix-associated protein. Upon induction of apoptosis, LZTR-1 was phosphorylated on tyrosine residues and subsequently degraded; that could be rescued partially by the addition of the caspase inhibitor Z-VAD-fmk and the proteasome inhibitors lactacystin and MG132. Taken together, our experiments identify LZTR-1 as the first BTB-kelch protein that exclusively localizes to the Golgi network, and the binding of LZTR-1 to the Golgi complex is mediated by its second BTB/POZ domain.

Abstract-Sprouting and invasive migration of endothelial cells are important steps of the angiogenic cascade. Vascular endothelial growth factor (VEGF) induces angiogenesis by activating intracellular signal transduction cascades, which regulate endothelial cell morphology and function. BTB-kelch proteins are intracellular proteins that control cellular architecture and cellular functions. The BTB-kelch protein KLEIP has been characterized as an actin-binding protein that interacts with the nucleotide exchange factor ECT2. We report that KLEIP is preferentially expressed in endothelial cells, suggesting that it may play a critical role in controlling the functions of migrating, proliferating, and invading endothelial cells during angiogenesis. KLEIP mRNA level in endothelial cells is strongly regulated by hypoxia which is controlled by hypoxia-inducible factor-1␣. Functional analysis of KLEIP in endothelial cells revealed that it acts as an essential downstream regulator of VEGF-and basic fibroblast growth factor-induced migration and in-gel sprouting angiogenesis. Yet, it is not involved in controlling VEGF-or basic fibroblast growth factor-mediated proliferative responses. The depletion of KLEIP in endothelial cells blunted the VEGF-induced activation of the monomeric GTPase RhoA but did not alter the VEGF-stimulated activation of extracellular signal-regulated kinase 1/2. Moreover, VEGF induced a physical association of KLEIP with the guanine nucleotide-exchange factor ECT2, the depletion of which also blunted VEGF-induced sprouting. We conclude that the BTB-kelch protein KLEIP is a novel regulator of endothelial function during angiogenesis that controls the VEGF-induced activation of Rho GTPases. (Circ Res. 2007;100:1155-1163.)Key Words: BTB-kelch protein KLEIP Ⅲ angiogenesis Ⅲ migration Ⅲ hypoxia Ⅲ G proteins A ngiogenesis, the formation of blood vessels from preexisting ones, is a crucial process during embryonic development and in several pathological conditions. Vascular endothelial growth factor (VEGF) and its receptors are major regulators of endothelial cell (EC) function and angiogenesis. 1 VEGF is upregulated under hypoxic conditions and induces several angiogenesis-related functions in ECs, such as proliferation, migration, and NO release, which are mediated by cascades of intracellular signaling pathways. [2][3][4] Genetic experiments suggest that VEGF receptor Flk-1/KDR signaling is required for proper positioning of hemangioblastic cells from the posterior primitive streak in the yolk sac. 5 Furthermore, cells lacking Flk1/KDR are unable to coalesce to form blood islands, suggesting that Flk1/KDR signaling controls the migration of ECs. 5 The signaling pathways regulating EC migration involve the phosphorylation of p38 mitogen-activated protein kinase and focal adhesion kinase, 6 activation of phosphatidylinositol 3-kinase, 7 as well as the phosphorylation of protein kinase Akt/protein kinase B, 8,9 with subsequent formation of NO by endothelial NO synthase (eNOS). 10 Yet, mechanistically, V...