Daniel R. Dries

TDP-43 is directed to stress granules by sorbitol, a novel physiological osmotic and oxidative stressor

ABSTRACT TDP-43, or TAR DNA-binding protein 43, is a pathological marker of a spectrum of neurodegenerative disorders, including amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. TDP-43 is an RNA/DNA-binding protein implicated in transcriptional and posttranscriptional regulation. Recent work also suggests that TDP-43 associates with cytoplasmic stress granules, which are transient structures that form in response to stress. In this study, we establish sorbitol as a novel physiological stressor that directs TDP-43 to stress granules in Hek293T cells and primary cultured glia. We quantify the association of TDP-43 with stress granules over time and show that stress granule association and size are dependent on the glycine-rich region of TDP-43, which harbors the majority of pathogenic mutations. Moreover, we establish that cells harboring wild-type and mutant TDP-43 have distinct stress responses: mutant TDP-43 forms significantly larger stress granules, and is incorporated into stress granules earlier, than wild-type TDP-43; in striking contrast, wild-type TDP-43 forms more stress granules over time, but the granule size remains relatively unchanged. We propose that mutant TDP-43 alters stress granule dynamics, which may contribute to the progression of TDP-43 proteinopathies.

A Single Residue in the C1 Domain Sensitizes Novel Protein Kinase C Isoforms to Cellular Diacylglycerol Production

The C1 domain mediates the diacylglycerol (DAG)-dependent translocation of conventional and novel protein kinase C (PKC) isoforms. In novel PKC isoforms (nPKCs), this domain binds membranes with sufficiently high affinity to recruit nPKCs to membranes in the absence of any other targeting mechanism. In conventional PKC (cPKC) isoforms, however, the affinity of the C1 domain for DAG is two orders of magnitude lower, necessitating the coordinated binding of the C1 domain and a Ca2+-regulated C2 domain for translocation and activation. Here we identify a single residue that tunes the affinity of the C1b domain for DAG- (but not phorbol ester-) containing membranes. This residue is invariant as Tyr in the C1b domain of cPKCs and invariant as Trp in all other PKC C1 domains. Binding studies using model membranes, as well as live cell imaging studies of yellow fluorescent protein-tagged C1 domains, reveal that Trp versus Tyr toggles the C1 domain between a species with sufficiently high affinity to respond to agonist-produced DAG to one that is unable to respond to physiological levels of DAG. In addition, we show that while Tyr at this switch position causes cytosolic localization of the C1 domain under unstimulated conditions, Trp targets these domains to the Golgi, likely due to basal levels of DAG at this region. Thus, Trp versus Tyr at this key position in the C1 domain controls both the membrane affinity and localization of PKC. The finding that a single residue controls the affinity of the C1 domain for DAG-containing membranes provides a molecular explanation for why 1) DAG alone is sufficient to activate nPKCs but not cPKCs and 2) nPKCs target to the Golgi.

Suberoylanilide Hydroxamic Acid (Vorinostat) Up-regulates Progranulin Transcription RATIONAL THERAPEUTIC APPROACH TO FRONTOTEMPORAL DEMENTIA

Progranulin (GRN) haploinsufficiency is a frequent cause of familial frontotemporal dementia, a currently untreatable progressive neurodegenerative disease. By chemical library screening, we identified suberoylanilide hydroxamic acid (SAHA), a Food and Drug Administration-approved histone deacetylase inhibitor, as an enhancer of GRN expression. SAHA dose-dependently increased GRN mRNA and protein levels in cultured cells and restored near-normal GRN expression in haploinsufficient cells from human subjects. Although elevation of secreted progranulin levels through a post-transcriptional mechanism has recently been reported, this is, to the best of our knowledge, the first report of a small molecule enhancer of progranulin transcription. SAHA has demonstrated therapeutic potential in other neurodegenerative diseases and thus holds promise as a first generation drug for the prevention and treatment of frontotemporal dementia.

Assembly, maturation, and trafficking of the gamma-secretase complex in Alzheimer's disease.

In this review, we discuss the biology of gamma-secretase, an enigmatic enzyme complex that is responsible for the generation of the amyloid-beta peptide that constitutes the amyloid plaques of Alzheimers disease. We begin with a brief review on the processing of the amyloid precursor protein and a brief discussion on the family of enzymes involved in regulated intramembrane proteolysis, of which gamma-secretase is a member. We then identify the four major components of the gamma-secretase complex - presenilin, nicastrin, Aph-1, and Pen-2 - with a focus on the identification of each and the role that each plays in the maturation and activity of the complex. We also discuss two new proteins that may play a role in modulating the assembly and activity of the gamma-secretase complex. Next, we summarize the known subcellular locations of each gamma-secretase component and the sites of gamma-secretase activity, as defined by the production of Abeta. Finally, we close by synthesizing all of the included topics into an overarching model for the assembly and trafficking of the gamma-secretase complex, which serves as a launching point for further questions into the biology and function of gamma-secretase in Alzheimers disease.

Glu-333 of Nicastrin Directly Participates in γ-Secretase Activity

γ-Secretase is a proteolytic membrane complex that processes a variety of substrates including the amyloid precursor protein and the Notch receptor. Earlier we showed that one of the components of this complex, nicastrin (NCT), functions as a receptor for γ-secretase substrates. A recent report challenged this, arguing instead that the Glu-333 residue of NCT predicted to participate in substrate recognition only participates in γ-secretase complex maturation and not in activity per se. Here, we present evidence that Glu-333 directly participates in γ-secretase activity. By normalizing to the active pool of γ-secretase with two separate methods, we establish that γ-secretase complexes containing NCT-E333A are indeed deficient in intrinsic activity. We also demonstrate that the NCT-E333A mutant is deficient in its binding to substrates. Moreover, we find that the cleavage of substrates by γ-secretase activity requires a free N-terminal amine but no minimal length of the extracellular N-terminal stub. Taken together, these studies provide further evidence supporting the role of NCT in substrate recognition. Finally, because γ-secretase cleaves itself during its maturation and because NCT-E333A also shows defects in γ-secretase complex maturation, we present a model whereby Glu-333 can serve a dual role via similar mechanisms in the recruitment of both Type 1 membrane proteins for activity and the presenilin intracellular loop during complex maturation.

Expression, localization, and biochemical characterization of nicotinamide mononucleotide adenylyltransferase 2.

Nicotinamide mononucleotide (NMN) adenylyltransferase 2 (Nmnat2) catalyzes the synthesis of NAD from NMN and ATP. The Nmnat2 transcript is expressed predominately in the brain; we report here that Nmnat2 is a low abundance protein expressed in neurons. Previous studies indicate that Nmnat2 localizes to Golgi. As Nmnat2 is not predicted to contain a signal sequence, lipid-binding domain, or transmembrane domain, we investigated the nature of this interaction. These experiments reveal that Nmnat2 is palmitoylated in vitro, and this modification is required for membrane association. Surprisingly, exogenous Nmnat2 is toxic to neurons, indicating that protein levels must be tightly regulated. To analyze Nmnat2 localization in neurons (previous experiments relied on exogenous expression in HeLa cells), mouse brains were fractionated, showing that Nmnat2 is enriched in numerous membrane compartments including synaptic terminals. In HeLa cells, in addition to Golgi, Nmnat2 localizes to Rab7-containing late endosomes. These studies show that Nmnat2 is a neuronal protein peripherally attached to membranes via palmitoylation and suggest that Nmnat2 is transported to synaptic terminals via an endosomal pathway.

Kinetic Analysis of the Interaction of the C1 Domain of Protein Kinase C with Lipid Membranes by Stopped-flow Spectroscopy

The diacylglycerol (DG)/phorbol ester-dependent translocation of conventional protein kinase C (PKC) isozymes is mediated by the C1 domain, a membrane-targeting module that also selectively binds phosphatidylserine (PS). Using stopped-flow spectroscopy, we dissect the contribution of DG/phorbol esters (C1 ligand) and PS in driving the association and dissociation of the C1 domain from membranes. Specifically, we examine the binding to membranes of the C1B domain of PKCβ with a substituted Trp (Y123W) whose fluorescence is quenched upon binding to membranes. Binding of this construct (C1Bβ-Y123W) to phospholipid vesicles is cooperative with respect to PS content and dependent on C1 ligand, as previously characterized. Stopped-flow analysis reveals that the apparent association rate (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(k_{\mathrm{on}}^{\mathrm{app}}\) \end{document}), but not the apparent dissociation rate (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(k_{\mathrm{off}}^{\mathrm{app}}\) \end{document}), is highly sensitive to PS content: the 60-fold increase in membrane affinity for vesicles containing no PS compared with 40 mol % PS results primarily from a robust (30-fold) increase in \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(k_{\mathrm{on}}^{\mathrm{app}}\) \end{document} with little effect (2-fold) on \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(k_{\mathrm{off}}^{\mathrm{app}}\) \end{document}. Membrane affinity is also controlled by the content and structure of the C1 ligand. In contrast to PS, these ligands markedly alter \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(k_{\mathrm{off}}^{\mathrm{app}}\) \end{document} with smaller effects on \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(k_{\mathrm{on}}^{\mathrm{app}}\) \end{document}. We also show that the affinity for phorbol ester-containing membranes is 2 orders of magnitude higher than that for DG-containing membranes primarily resulting from differences in \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(k_{\mathrm{off}}^{\mathrm{app}}\) \end{document}. Our data are consistent with a model in which the C1 domain is recruited to the membrane via an initial weak electrostatic interaction with PS, followed by a rapid two-dimensional search for ligand, the binding of which retains the domain at the membrane. Thus, PS drives the initial encounter, and DG/phorbol esters retain the domain on membranes. The decreased effectiveness of DG compared with phorbol esters in retaining the C1 domain on membranes contributes to the molecular dichotomy of the rapid, transient nature of DG-dependent PKC signaling versus the chronic hyperactivity of phorbol ester-activated PKC.

γ-Secretase-regulated Proteolysis of the Notch Receptor by Mitochondrial Intermediate Peptidase

Notch is a transmembrane receptor that controls a diverse array of cellular processes including cell proliferation, differentiation, survival, and migration. The cellular outcome of Notch signaling is dependent on extracellular and intracellular signals, but the complexities of its regulation are not well understood. Canonical Notch signaling involves ligand association that triggers sequential and regulated proteolysis of Notch at several sites. Ligand-dependent proteolysis at the S2 site removes the bulk of the extracellular domain of Notch. Subsequent γ-secretase-mediated intramembrane proteolysis of the remaining membrane-tethered Notch fragment at the S3 site produces a nuclear-destined Notch intracellular domain (NICD). Here we show that following γ-secretase cleavage, Notch is proteolyzed at a novel S5 site. We have identified this S5 site to be eight amino acids downstream of the S3 site. Biochemical fractionation and purification resulted in the identification of the S5 site protease as the mitochondrial intermediate peptidase (MIPEP). Expression of the MIPEP-cleaved NICD (ΔNICD) results in a decrease in cell viability and mitochondria membrane potential. The sequential and regulated proteolysis by γ-secretase and MIPEP suggests a new means by which Notch function can be modulated.

Extracting β-amyloid from Alzheimer's disease

The amyloid hypothesis of Alzheimers disease (AD) posits that extracellular plaques comprised of the β-amyloid (Aβ) peptide are a root cause of neuronal loss in AD (1). To date, three therapeutic strategies targeting Aβ have been used: (i) inhibiting the production of Aβ, (ii) inhibiting the oligomerization of Aβ, and (iii) promoting the clearance and/or degradation of Aβ. None have led to a therapeutic or preventive medication for AD. As an alternative approach, natural compounds have demonstrated remarkable promise in diseases ranging from cancer to diabetes. In PNAS, Sehgal et al. (2) describe how an extract from the root of Withania somnifera (WS; also known as Ashwagandha or Indian ginseng) reverses AD pathology via the peripheral clearance of Aβ.

Rip exposed: how ectodomain shedding regulates the proteolytic processing of transmembrane substrates.

Regulated intramembrane proteolysis (Rip) controls a wide variety of cellular mechanisms such as cholesterol homeostasis, immune surveillance, cellular signaling, and β-amyloid formation in Alzheimers disease (1). Rip of substrates is mediated by several families of intramembranously cleaving proteases (I-CLiPs), all of which perform the unique chemistry of hydrolysis within the hydrophobic lipid bilayer (2). There are four known families of I-CLiPs, each denoted by the protease that typifies each group: site-2 protease (S2P) metalloproteases, the γ-secretase and signal peptide peptidase (SPP) aspartyl proteases, and the rhomboid serine proteases. Rip cleavage of transmembrane substrates by S2P, SPP, and γ-secretase is preceded and regulated by an initial distinct cleavage in a process termed “ectodomain shedding” (Fig. 1). The reason ectodomain shedding is necessary in most Rip cases is not understood. An article in this issue of PNAS (3) has shed new light on this important question.