Henri J. Huttunen

Heparin-binding proteins HB-GAM (pleiotrophin) and amphoterin in the regulation of cell motility ☆

Fractionation of proteins from perinatal rat brain was monitored using a neurite outgrowth assay. Two neurite-promoting proteins, HB-GAM (heparin-binding growth-associated molecule; also known as pleiotrophin) and amphoterin, were isolated, cloned and produced by baculovirus expression for structural and functional studies. HB-GAM is highly expressed in embryonic and early post-natal fiber pathways of the nervous system, and it enhances axonal growth/guidance by binding to N-syndecan (syndecan-3) at the neuron surface. N-syndecan in turn communicates with the cytoskeleton through the cortactin/src-kinase pathway to enhance neurite extension. In addition to N-syndecan, the chondroitin sulfate proteoglycan RPTP beta/zeta (receptor-type tyrosine phosphatase beta/zeta) is implicated in the receptor mechanism of HB-GAM. HB-GAM is also prominently expressed in developing and regenerating bone as a matrix-bound cue for migration of osteoblasts/osteoblast precursors to the site of bone deposition. HB-GAM is suggested to regulate motility in osteoblasts through a similar mechanism as in neurons. Structural studies using heteronuclear NMR reveal two similar protein domains in HB-GAM, both consisting of three anti-parallel beta-strands. Search of sequence databases shows that the beta structures of HB-GAM and of the similar domains of MK (midkine) correspond to the thrombospondin type I (TSR) sequence motif. We suggest that the TSR sequence motif, found in several neurite outgrowth-promoting and other cell surface and matrix-binding proteins, defines a beta structure similar to those found in HB-GAM and MK. In general, amphoterin is highly expressed in immature and transformed cells. We suggest a model, according to which amphoterin is an autocrine/paracrine regulator of invasive migration. Amphoterin binds to RAGE (receptor of advanced glycation end products), an immunoglubulin superfamily member related to N-CAM (neural cell adhesion molecule), that communicates with the GTPases Cdc42 and Rac to regulate cell motility. In addition, ligation of RAGE by amphoterin activates NF-kappaB to regulate transcription.

N-Glycans on the receptor for advanced glycation end products influence amphoterin binding and neurite outgrowth

In this study we show that embryonic neurite growth‐promoting protein amphoterin binds to carboxylated N‐glycans previously identified on mammalian endothelial cells. Since amphoterin is a ligand for the receptor for advanced glycation end products (RAGE), and the ligand‐binding V‐domain of the receptor contains two potential N‐glycosylation sites, we hypothesized that N‐glycans on RAGE may mediate its interactions with amphoterin. In support of this, anti‐carboxylate antibody mAbGB3.1 immunoprecipitates bovine RAGE, and PNGase F treatment reduces its molecular mass by 4.5 kDa, suggesting that the native receptor is a glycoprotein. The binding potential of amphoterin to RAGE decreases significantly in presence of soluble carboxylated glycans or when the receptor is deglycosylated. Oligosaccharide analysis shows that RAGE contains complex type anionic N‐glycans with non‐sialic acid carboxylate groups, but not the HNK‐1 (3‐sulfoglucuronyl β1–3 galactoside) epitope. Consistent with the functional localization of RAGE and amphoterin at the leading edges of developing neurons, mAbGB3.1 stains axons and growth cones of mouse embryonic cortical neurons, and inhibits neurite outgrowth on amphoterin matrix. The carboxylated glycans themselves promote neurite outgrowth in embryonic neurons and RAGE‐transfected neuroblastoma cells. This outgrowth requires full‐length, signalling‐competent RAGE, as cells expressing cytoplasmic domain‐deleted RAGE are unresponsive. These results indicate that carboxylated N‐glycans on RAGE play an important functional role in amphoterin‐RAGE‐mediated signalling.

Microglial activation induces cell death, inhibits neurite outgrowth and causes neurite retraction of differentiated neuroblastoma cells

Abstract.Activation of glial cells has been proposed to contribute to neuronal dysfunction and neuronal cell death in Alzheimers disease. In this study, we attempt to determine some of the effects of secreted factors from activated murine N-11 microglia on viability and morphology of neurons using the differentiated neuroblastoma cell line Neuro2a. Microglia were activated either by lipopolysaccharide (LPS), bacterial cell wall proteoglycans, or advanced glycation endproducts (AGEs), protein-bound sugar oxidation products. At high LPS or AGE concentrations, conditioned medium from microglia caused neuronal cell death in a dose-dependent manner. At sublethal LPS or AGE concentrations, conditioned media inhibited retinoic acid-induced neurite outgrowth and stimulated retraction of already extended neurites. Among the many possible secreted factors, the contribution of NO or NO metabolites in the cytotoxicity of conditioned medium was investigated. Cell death and changes in neurite morphology were partly reduced when NO production was inhibited by nitric oxide synthase inhibitors. The results suggest that even in the absence of significant cell death, inflammatory processes, which are partly transmitted via NO metabolites, may affect intrinsic functions of neurons such as neurite extension that are essential components of neuronal morphology and thus may contribute to degenerative changes in Alzheimers disease.

S100B Interaction With the Receptor for Advanced Glycation End Products (RAGE): A Novel Receptor-Mediated Mechanism for Myocyte Apoptosis Postinfarction

Rationale: Post–myocardial infarction ventricular remodeling is associated with the expression of a variety of factors including S100B that can potentially modulate myocyte apoptosis. Objective: This study was undertaken to investigate the expression and function of S100B and its receptor, the receptor for advanced glycation end products (RAGE) in both postinfarction myocardium and in a rat neonatal myocyte culture model. Methods and Results: In a rat model of myocardial infarction following coronary artery ligation, we demonstrate in periinfarct myocytes, upregulation of RAGE, induction of S100B, and release into plasma with consequent myocyte apoptosis. Using a coimmunoprecipitation strategy, we demonstrate a direct interaction between S100B and RAGE. In rat neonatal cardiac myocyte cultures, S100B at concentrations ≥50 nmol/L induced myocyte apoptosis, as evidenced by increased terminal DNA fragmentation, TUNEL, cytochrome c release from mitochondria to cytoplasm, phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and p53, increased expression and activity of proapoptotic caspase-3, and decreased expression of antiapoptotic Bcl-2. Transfection of a full-length cDNA of RAGE or a dominant-negative mutant of RAGE resulted in increased or attenuated S100B-induced myocyte apoptosis, respectively. Inhibition of ERK1/2 by U0126/PD-98059 or overexpression of a dominant negative p53 comparably inhibited S100B-induced myocyte apoptosis. Conclusions: These results suggest that interaction of RAGE and its ligand S100B after myocardial infarction may play a role in myocyte apoptosis by activating ERK1/2 and p53 signaling. This receptor-mediated mechanism is uniquely amenable to therapeutic intervention.

HtrA2 Regulates β-Amyloid Precursor Protein (APP) Metabolism through Endoplasmic Reticulum-associated Degradation

Alzheimer disease-associated β-amyloid peptide is generated from its precursor protein APP. By using the yeast two-hybrid assay, here we identified HtrA2/Omi, a stress-responsive chaperone-protease as a protein binding to the N-terminal cysteinerich region of APP. HtrA2 coimmunoprecipitates exclusively with immature APP from cell lysates as well as mouse brain extracts and degrades APP in vitro. A subpopulation of HtrA2 localizes to the cytosolic side of the endoplasmic reticulum (ER) membrane where it contributes to ER-associated degradation of APP together with the proteasome. Inhibition of the proteasome results in accumulation of retrotranslocated forms of APP and increased association of APP with HtrA2 and Derlin-1 in microsomal membranes. In cells lacking HtrA2, APP holoprotein is stabilized and accumulates in the early secretory pathway correlating with elevated levels of APP C-terminal fragments and increased Aβ secretion. Inhibition of ER-associated degradation (either HtrA2 or proteasome) promotes binding of APP to the COPII protein Sec23 suggesting enhanced trafficking of APP out of the ER. Based on these results we suggest a novel function for HtrA2 as a regulator of APP metabolism through ER-associated degradation.

PCSK9 regulates neuronal apoptosis by adjusting ApoER2 levels and signaling

The secreted protease proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to low-density lipid (LDL) receptor family members LDLR, very low density lipoprotein receptor (VLDLR) and apolipoprotein receptor 2 (ApoER2), and promotes their degradation in intracellular acidic compartments. In the liver, LDLR is a major controller of blood LDL levels, whereas VLDLR and ApoER2 in the brain mediate Reelin signaling, a critical pathway for proper development of the nervous system. Expression level of PCSK9 in the brain is highest in the cerebellum during perinatal development, but is also increased in the adult brain after ischemia. The mechanism of PCSK9 function and its involvement in neuronal apoptosis is poorly understood. We show here that RNAi-mediated knockdown of PCSK9 significantly reduced the death of potassium-deprived cerebellar granule neurons (CGN), as shown by reduced levels of nuclear phosphorylated c-Jun and activated caspase-3, as well as condensed apoptotic nuclei. ApoER2 protein levels were increased in PCSK9 RNAi cells. Knockdown of ApoER2 but not of VLDLR was sufficient to reverse the protection provided by PCSK9 RNAi, suggesting that proapoptotic signaling of PCSK9 is mediated by altered ApoER2 function. Pharmacological inhibition of signaling pathways associated with lipoprotein receptors suggested that PCSK9 regulates neuronal apoptosis independently of NMDA receptor function but in concert with ERK and JNK signaling pathways. PCSK9 RNAi also reduced staurosporine-induced CGN apoptosis and axonal degeneration in the nerve growth factor-deprived dorsal root ganglion neurons. We conclude that PCSK9 potentiates neuronal apoptosis via modulation of ApoER2 levels and related anti-apoptotic signaling pathways.

Knockdown of ACAT-1 reduces amyloidogenic processing of APP

Previous studies have shown that acyl‐coenzyme A:cholesterol acyl transferase (ACAT), an enzyme that controls cellular equilibrium between free cholesterol and cholesteryl esters, modulates proteolytic processing of APP in cell‐based and animal models of Alzheimers disease. Here we report that ACAT‐1 RNAi reduced cellular ACAT‐1 protein by ∼50% and cholesteryl ester levels by 22% while causing a slight increase in the free cholesterol content of ER membranes. This correlated with reduced proteolytic processing of APP and 40% decrease in Aβ secretion. These data show that even a modest decrease in ACAT activity can have robust suppressive effects on Aβ generation.

Inhibition of acyl-coenzyme A: cholesterol acyl transferase modulates amyloid precursor protein trafficking in the early secretory pathway

Amyloid β‐peptide (Aβ) has a central role in the pathogenesis of Alzheimers disease (AD). Cellular cholesterol homeostasis regulates endoproteo‐ lytic generation of Aβ from the amyloid precursor protein (APP). Previous studies have identified acyl‐ coenzyme A: cholesterol acyltransferase (ACAT)’ an enzyme that regulates subcellular cholesterol distribu‐tion, as a potential therapeutic target for AD. Inhibition of ACAT activity decreases Aβ generation in cell‐ and animal‐based models of AD through an unknown mechanism. Here we show that ACAT inhibition retains a fraction of APP molecules in the early secretory pathway, limiting the availability of APP for secretase‐ mediated proteolytic processing. ACAT inhibitors delayed the trafficking of immature APP molecules from the endoplasmic reticulum (ER) as shown by metabolic labeling and live‐cell imaging. This resulted in partial ER retention of APP and enhanced ER‐associated deg‐radation of APP by the proteasome, without activation of the unfolded protein response pathway. The ratio of mature APP to immature APP was reduced in brains of mice treated with ACAT inhibitors’ and strongly correlated with reduced brain APP‐C99 and cerebrospinal fluid Aβ levels in individual animals. Our results identify a novel ACAT‐dependent mechanism that regulates secretory trafficking of APP, likely contributing to decreased Aβ generation in vivo.—Huttunen, H. J., Peach, C., Bhattacharyya, R., Barren, C., Pettingell, W., Hutter‐Paier, B., Windisch, M., Berezovska, O., Kovacs, D. M. Inhibition of acyl‐coenzyme A: cholesterol acyl transferase modulates amyloid precursor protein trafficking in the early secretory pathway. FASEB J. 23, 3819‐3828 (2009). www.fasebj.org

Thyroxin regulates BDNF expression to promote survival of injured neurons.

A growing amount of evidence indicates that neuronal trauma can induce a recapitulation of developmental-like mechanisms for neuronal survival and regeneration. Concurrently, ontogenic dependency of central neurons for brain-derived neurotrophic factor (BDNF) is lost during maturation but is re-acquired after injury. Here we show in organotypic hippocampal slices that thyroxin, the thyroid hormone essential for normal CNS development, induces up-regulation of BDNF upon injury. This change in the effect of thyroxin is crucial to promote survival and regeneration of damaged central neurons. In addition, the effect of thyroxin on the expression of the K-Cl cotransporter (KCC2), a marker of neuronal maturation, is changed from down to up-regulation. Notably, previous results in humans have shown that during the first few days after traumatic brain injury or spinal cord injury, thyroid hormone levels are often diminished. Our data suggest that maintaining normal levels of thyroxin during the early post-traumatic phase of CNS injury could have a therapeutically positive effect.

The Acyl-Coenzyme A:Cholesterol Acyltransferase Inhibitor CI-1011 Reverses Diffuse Brain Amyloid Pathology in Aged Amyloid Precursor Protein Transgenic Mice

Cerebral accumulation of amyloid-&bgr; (A&bgr;) is characteristic of Alzheimer disease and of amyloid precursor protein (APP) transgenic mice. Here, we assessed the efficacy of CI-1011, an inhibitor of acyl-coenzyme A:cholesterol acyltransferase, which is suitable for clinical use, in reducing amyloid pathology in both young (6.5 months old) and aged (16 months old) human APP transgenic mice. Treatment of young animals with CI-1011 decreased amyloid plaque load in the cortex and hippocampus and reduced the levels of insoluble A&bgr;40 and A&bgr;42 and C-terminal fragments of APP in brain extracts. In aged mice, CI-1011 specifically reduced diffuse amyloid plaques with a minor effect on thioflavin S-positive dense-core plaques. Reduced diffusible amyloid was accompanied by suppression of astrogliosis and enhanced microglial activation. Collectively, these data suggest that CI-1011 treatment reduces amyloid burden in human APP mice by limiting generation and increasing clearance of diffusible A&bgr;.