Ubiquitination of a new form of alpha-synuclein by parkin from human brain: implications for Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the progressive accumulation in selected neurons of protein inclusions containing alpha-synuclein and ubiquitin. Rare inherited forms of PD are caused by autosomal dominant mutations in alpha-synuclein or by autosomal recessive mutations in parkin, an E3 ubiquitin ligase. We hypothesized that these two gene products interact functionally, namely, that parkin ubiquitinates alpha-synuclein normally and that this process is altered in autosomal recessive PD. We have now identified a protein complex in normal human brain that includes parkin as the E3 ubiquitin ligase, UbcH7 as its associated E2 ubiquitin conjugating enzyme, and a new 22-kilodalton glycosylated form of alpha-synuclein (alphaSp22) as its substrate. In contrast to normal parkin, mutant parkin associated with autosomal recessive PD failed to bind alphaSp22. In an in vitro ubiquitination assay, alphaSp22 was modified by normal but not mutant parkin into polyubiquitinated, high molecular weight species. Accordingly, alphaSp22 accumulated in a non-ubiquitinated form in parkin-deficient PD brains. We conclude that alphaSp22 is a substrate for parkin's ubiquitin ligase activity in normal human brain and that loss of parkin function causes pathological alphaSp22 accumulation. These findings demonstrate a critical biochemical reaction between the two PD-linked gene products and suggest that this reaction underlies the accumulation of ubiquitinated alpha-synuclein in conventional PD.

K45A mutation of RIPK1 results in poor necroptosis and cytokine signaling in macrophages, which impacts inflammatory responses in vivo.

Receptor interacting protein kinase 1 (RIPK1) participates in several cell signaling complexes that promote cell activation and cell death. Stimulation of RIPK1 in the absence of caspase signaling induces regulated necrosis (necroptosis), which promotes an inflammatory response. Understanding of the mechanisms through which RIPK1 promotes inflammation has been unclear. Herein we have evaluated the impact of a K45A mutation of RIPK1 on necroptosis of macrophages and the activation of inflammatory response. We show that K45A mutation of RIPK1 results in attenuated necroptosis of macrophages in response to stimulation with LPS, TNFα and IFNß in the absence of caspase signaling. Impairment in necroptosis correlated with poor phosphorylation of RIPK1, RIPK3 and reduced trimerization of MLKL. Furthermore, K45A mutation of RIPK1 resulted in poor STAT1 phosphorylation (at S727) and expression of RANTES and MIP-1α following TNF-R engagement in the absence of caspase activation. Our results further indicate that in the absence of stimulation by pathogen-associated molecular patterns (PAMPs), cellular inhibitors of apoptotic proteins (cIAPs) prevent the K45-dependent auto-phosphorylation of RIPK1, leading to resistance against necroptosis. Finally, RIPK1(K45A) mice displayed attenuated inflammatory response in vivo as they were significantly resistant against endotoxin shock, but highly susceptible against a challenge with Salmonella typhimurium. This correlated with reduced expression of IL-1ß and ROS, and poor processing of caspase 8 by RIPK1(K45A) macrophages. Overall, these results indicate that K45 mediated kinase activity of RIPK1 is not only important for necroptosis but it also has a key role in promoting cytokine signaling and host response to inflammatory stimuli.

Detection of distinct isoform patterns of the beta-amyloid precursor protein in human platelets and lymphocytes.

Cerebral deposition of the amyloid beta-protein (A beta P), approximately 40 residue fragment of the integral membrane protein, beta-amyloid precursor protein (beta APP), has been implicated as the probable cause of some cases of familial Alzheimer's disease (AD). The parallels between A beta P deposition in AD and the deposition of certain plasma proteins in systemic amyloid diseases has heightened interest in the analysis of beta APP in circulating cells and plasma. Here, we describe distinct isoform patterns of beta APP in peripheral platelets and lymphocytes. PCR-mediated amplification of mRNA from purified platelets demonstrated the expression of all three major beta APP transcripts (beta APP770,751,695). The full-length, approximately 140 kDa form of beta APP751,770 was detected in membranes of resting and activated platelets but very little immature, approximately 122 kDa beta APP751,770 was found, suggesting a different processing of beta APP in platelets than that described in a variety of cultured cells and tissues. Platelets stimulated with thrombin, calcium ionophore, or collagen released the soluble, carboxyl-truncated form of beta APP (protease nexin-II), but no evidence for the shedding of full-length beta APP associated with platelet microparticles was found, in contrast to previous reports. As a positive control marker for microparticles, the fibrinogen receptor subunit, GPIIIa, was readily detected in platelet releasates. Resting and activated platelets contained similar amounts of the approximately 10 kDa carboxyl terminal beta APP fragment that is retained in platelet membranes following the constitutive cleavage of protease nexin-II. Nonstimulated peripheral B and T lymphocytes contained small amounts of membrane-associated mature and immature beta APP751,770. The potentially amyloidogenic full-length beta APP molecules present in circulating platelets and lymphocytes but not in microparticles could serve as a source of the microvascular A beta P deposited during aging and particularly in AD.

Normal cellular processing of the beta-amyloid precursor protein results in the secretion of the amyloid beta peptide and related molecules.

Alzheimer's disease is characterized by the extracellular deposition in the brain and its blood vessels of insoluble aggregates of the amyloid beta peptide (A beta). This peptide is derived from a large integral membrane protein, the beta-amyloid precursor protein (beta APP), by proteolytic processing. The A beta has previously been found only in the brains of patients with Alzheimer's disease or advanced aging. We describe here the finding that A beta is produced continuously by normal processing in tissue culture cells. A beta and closely related peptides were identified in the media of cells transfected with cDNAs coding for beta APP in a variety of cell lines and primary tissue cultured cells. The identity of these peptides was confirmed by epitope mapping and radiosequencing. Peptides of a molecular weight of approximately 3 and approximately 4 kDa are described. The 4 kDa range contains mostly the A beta and two related peptides starting N-terminal to the beginning of A beta. In the 3 kDa range, the majority of peptides start at the secretase site; in addition, two longer peptides were found starting at amino acid F(4) and E(11) of the A beta sequence. To identify the processing pathways which lead to the secretion of these peptides, we used a variety of drugs known to interfere with certain cell biological pathways. We conclude that lysosomes may not play a predominant role in the formation of 3 and 4 kDa peptides. We show that an acidic environment is necessary to create the N-terminus of the A beta and postulate that alternative secretory cleavage might result in the formation of the N-terminus of A beta and related peptides. This cleavage takes place either in the late Golgi, at the cell-surface or in early endosomes, but not in lysosomes. The N-terminus of most of the 3 kDa peptides is created by secretory cleavage on the cell surface or within late Golgi.

The life of Eugene Braunwald: a remarkable journey that began in Vienna.

The life of Dr. Eugene Braunwald, who is without doubt the preeminent cardiologist of our time, represents a remarkable journey that began in Vienna, Austria. It took an early and decisive turn in July 1938 when his family was forced into exile by Austrian Nazism, ultimately settling in the United States. During his career Dr. Braunwald has provided unprecedented contributions to the theory and practice of medicine, foremost in cardiovascular research and clinical cardiology. Professionally, his pursuit of excellence continues in the roles of physician-scientist, academic teacher, editor and administrator. Personally, Dr. Braunwald's voyage came full circle in the summer of 1995, some fifty seven years after his emigration, when the University of Vienna awarded him an honorary Medical Doctorate. This article reviews Dr. Eugene Braunwald's remarkable journey from his childhood in pre-World War II Vienna to the current titles of Distinguished Hersey Professor of Medicine and Faculty Dean for Academic Programs at Harvard Medical School and Vice President of Partner's HealthCare Systems in Boston, Massachusetts. This essay is also intended to raise awareness of, and foster academic investigation into, the long-lasting implications of Austrian Nazism on the "Viennese School of Medicine".

beta-Amyloid peptide and a 3-kDa fragment are derived by distinct cellular mechanisms.

We have analyzed the cellular processing pathways which produce the 4-kDa amyloid beta-peptide (A beta) and a 3-kDa derivative (p3) of the beta-amyloid precursor protein (beta APP) found in conditioned media of tissue culture cells and in cerebrospinal fluid. Pulse-chase experiments reveal that both peptides are secreted in parallel with soluble beta APP (APPs); no precursor-product relation between A beta and p3 was found. The protease inhibitor leupeptin did not influence the production of either peptide. In contrast, the weak base ammonium chloride (NH4Cl) showed a dose-dependent inhibition of A beta production with less decrease in p3. A similar effect was observed using the monovalent ionophore monensin. Brefeldin A completely inhibited the generation of both peptides, indicating that proteases located in the endoplasmic reticulum or early Golgi are not sufficient for the production of the small peptides. Deletion of the beta APP cytoplasmic domain, which removes a consensus sequence that probably mediates reinternalization, caused an increase in secretion of both APPs and p3 and did not abolish A beta production. These observations suggest that completely mature beta APP within the late Golgi and/or at the cell surface is a prerequisite for A beta production but processing within the lysosome might not be directly required. p3 appears to derive from the 10-kDa C-terminal stub of beta APP following secretion of APPs.

Detection of soluble forms of the beta-amyloid precursor protein in human plasma.

A approximately 40-residue fragment of the beta-amyloid precursor protein (APP) is progressively deposited in the extracellular spaces of brain and blood vessels in Alzheimer's disease (AD), Down's syndrome and aged normal subjects. Soluble, truncated forms of APP lacking the carboxyl terminus are normally secreted from cultured cells expressing this protein and are found in cerebrospinal fluid. Here, we report the detection of a similar soluble APP isoform in human plasma. This approximately 125 kDa protein, which was isolated from plasma by Affi-Gel Blue chromatography or dialysis-induced precipitation, comigrates with the larger of the two major soluble APP forms present in spinal fluid and contains the Kunitz protease inhibitor insert. It thus derives from the APP751 and APP770 precursors; a soluble form of APP695 has not yet been detected in plasma. The approximately 125 kDa plasma form lacks the C-terminal region and is unlikely to serve as a precursor for the beta-protein that forms the amyloid in AD.

Amyloid beta-peptide is produced by cultured cells during normal metabolism.

Alzheimer's disease is characterized by the extracellular deposition in the brain and its blood vessels of insoluble aggregates of the amyloid beta-peptide (A beta), a fragment, of about 40 amino acids in length, of the integral membrane protein beta-amyloid precursor protein (beta-APP). The mechanism of extracellular accumulation of A beta in brain is unknown and no simple in vitro or in vivo model systems that produce extracellular A beta have been described. We report here the unexpected identification of the 4K (M(r) 4,000) A beta and a truncated form of A beta (approximately 3K) in media from cultures of primary cells and untransfected and beta-APP-transfected cell lines grown under normal conditions. These peptides were immunoprecipitated readily from culture medium by A beta-specific antibodies and their identities confirmed by sequencing. The concept that pathological processes are responsible for the production of A beta must not be reassessed in light of the observation that A beta is produced in soluble form in vitro and in vivo during normal cellular metabolism. Further, these findings provide the basis for using simple cell culture systems to identify drugs that block the formation or release of A beta, the primary protein constituent of the senile plaques of Alzheimer's disease.