Content
2007
Loss-of-Function of Human PINK1 Results in Mitochondrial Pathology and Can Be Rescued by parkin
07.11.2007
Degeneration of dopaminergic neurons in the substantia nigra is characteristic for Parkinson’s disease (PD), the second most common neurodegenerative disorder. Mitochondrial dysfunction is believed to contribute to the etiology of PD. Although most cases are sporadic, recent evidence points to anumberof genes involved in familial variants of PD.Amongthem, a loss-of-function of phosphatase and tensin homolog-induced kinase 1 (PINK1; PARK6) is associated with rare cases of autosomal recessive parkinsonism. In HeLa cells, RNA interference-mediated downregulation of PINK1 results in abnormal mitochondrial morphology and altered membrane potential. Morphological changes of mitochondria can be rescued by expression of wild-type PINK1 but not by PD-associated PINK1 mutants. Moreover, primary cells derived from patients with two different PINK1 mutants showed a similar defect in mitochondrial morphology. Human parkin but not PD-associated mutants could rescue mitochondrial pathology in human cells like wild-type PINK1. Our results may therefore suggest that PINK1 deficiency in humans results in mitochondrial abnormalities associated with cellular stress, a pathological phenotype, which can be ameliorated by enhanced expression of parkin.
FTLD-U linked missense mutations in the progranulin gene reduce progranulin production and secretion
05.11.2007
J. Biol. Chem.,
2007,
doi/10.1074/jbc.M705115200,
1-13
published on 05.11.2007
http://www.jbc.org, online article
http://www.jbc.org, online article
Loss of function mutations in progranulin
cause tau-negative frontotemporal lobar
degeneration with ubiquitin positive inclusions.
A major protein component of these inclusions
is TDP-43, which becomes
hyperphosphorylated, ubiquitinated and
cleaved to generate C-terminal fragments,
which apparently translocate from nuclei to the
cytoplasm. Most progranulin mutations are
nonsense mutations resulting in nonsensemediated
mRNA decay and consequently
reduced progranulin protein levels. However,
some missense mutations are described, which
occur within the signal sequence and mature
progranulin. We now demonstrate that a
progranulin mutation located within the signal
sequence (PGRN A9D) results in cytoplasmic
missorting with extremely low expression. In
contrast, two other progranulin mutations
(PGRN P248L and R432C) are expressed as
immature proteins, but are inefficiently
transported through and partially degraded
within the secretory pathway resulting in a
significantly reduced secretion. Thus apparently
all progranulin mutations cause reduced protein
expression or secretion, although by different
cellular mechanisms. To investigate a putative
relationship between reduced expression of
progranulin and TDP-43 relocalization and
deposition, we downregulated progranulin in
human cell lines and in zebrafish. Upon
reduction of progranulin, neither a major
redistribution of TDP-43 nor proteolytic
processing to disease characterizing C-terminal
fragments could be observed.
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Regulated Intramembrane Proteolysis of Bri2 (Itm2b) by ADAM10 and SPPL2a/b
25.10.2007
J. Biol. Chem.,
2007,
doi/10.1074/jbc.M706661200,
published on 25.10.2007
http://www.jbc.org, online article
http://www.jbc.org, online article
Presenilin, the catalytic component of the gamma-secretase complex, type IV prepilin peptidases
and signal peptide peptidase (SPP) are the
founding members of the family of
intramembrane cleaving GxGD aspartyl
proteases. SPP-like (SPPL) proteases, such as
SPPL2a, b, and c and SPPL3 also belong to
the GxGD family. In contrast to gamma-secretase,
where numerous substrates have been
identified, very few in vivo substrates are
known for SPP and SPPLs. Here we
demonstrate that Bri2 (Itm2b), a type-II
oriented transmembrane protein associated
with familial British and Danish dementia
undergoes regulated intramembrane
proteolysis (RIP). In addition to the
previously described ectodomain processing
by furin and related proteases, we now
describe that the Bri2 protein, similar to gamma-
secretase substrates, undergoes an additional
cleavage by ADAM10 in its ectodomain. This
cleavage releases a soluble variant of Bri2, the
BRICHOS domain, which is secreted into the
extracellular space. Upon this shedding event
a membrane bound N-terminal Bri2 fragment
(NTF) remains, which undergoes
intramembrane proteolysis to produce an
intracellular domain (ICD) as well as a
secreted low molecular weight C-terminal
peptide. By expressing all known SPP/SPPL
family members as well as their loss of
function variants, we demonstrate that
selectively SPPL2a and SPPL2b mediate the
intramembrane cleavage, while neither SPP
nor SPPL3 are capable of processing the Bri2
NTF.
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Generation of Aß38 and Aß42 is independendently and differentially affected by FAD-associated presenilin mutations and gamma-secretase modulation
24.10.2007
Alzheimer's disease (AD) amyloid beta-peptide (Aß) is generated via proteolytic processing of the ß-amyloid precursor protein (APP) by ß- and gamma-secretase. gamma-Secretase can be blocked by selective inhibitors but can also be modulated by a subset of non-steroidal anti-inflammatory drugs (NSAIDs) including sulindac sulfide. These drugs selectively reduce the generation of the aggregation-prone 42 amino acid Aß42 and concomitantly increase the levels of the rather benign Aß38. Here we show that Aß42 and Aß38 generation occurs independently from each other. The amount of Aß42 produced by cells expressing ten different familial AD (FAD)-associated mutations in presenilin (PS) 1, the catalytic subunit of gamma-secretase, appeared to correlate with the respective age of onset in patients. However, Aß38 levels did not show a negative correlation with the age of onset.
Modulation of gamma-secretase activity by sulindac sulfide reduced Aß42 in the case of wt PS1 and two FAD-associated PS1 mutations (M146L and A285V). The remaining eight PS1 FAD mutants showed either no reduction of Aß42 or only rather subtle effects. Strikingly, even the mutations, which showed no effect on Aß42 levels allowed a robust increase of Aß38 upon treatment with sulindac sulfide. Similar observations were made for fenofibrate, a compound known to increase Aß42 and to decrease Aß38. For mutants that predominantly produce Aß42, the ability of fenofibrate to further increase Aß42 levels became diminished, whereas Aß38 levels were altered to varying extents for all mutants analyzed. Thus, we conclude that Aß38 and Aß42 production do not depend on each other. Using an independent NSAID derivative, we obtained similar results for PS1 as well as for PS2. These in vitro results were confirmed by in vivo experiments in transgenic mice expressing the PS2 N141I FAD mutant. Our findings therefore have strong implications on the selection of transgenic mouse models used for screening of the Aß42 lowering capacity of gamma-secretase modulators. Furthermore, human patients with certain PS mutations may not respond to gamma-secretase modulators.
Active gamma-secretase complexes contain only one of each component
10.08.2007
J. Biol. Chem.,
2007,
doi:10.1074/jbc.M705248200,
published on 02.10.2007
The Journal of Biological Chemistry, online article
The Journal of Biological Chemistry, online article
gamma-Secretase is an intramembrane aspartyl protease complex that cleaves type I integral membrane proteins, including the amyloid beta-protein precursor (APP) and the Notch receptor, and is composed of presenilin, Pen-2, nicastrin and Aph-1. Although all four of these membrane proteins are essential for assembly and activity, the stoichiometry of the complex is unknown, with the number of presenilin molecules present being especially controversial. Here we analyze functional gamma-secretase complexes, isolated by immunoprecipitation from solubilized membrane fractions and able to produce amyloid beta-peptides and APP intracellular domain. We show that the active isolated protease contains only one presenilin per complex, which excludes certain models of the active site that require aspartate dyads formed between two presenilin molecules. We also quantified components in the isolated complexes by western blot using protein standards, and found that the amounts of Pen-2 and nicastrin were the same as that of presenilin. Moreover, we found that one Aph-1 was not co-immunoprecipitated with another in active complexes, evidence that Aph-1 is likewise present as a monomer. Taken together, these results demonstrate that the stoichiometry of gamma-components presenilin : Pen-2 : nicastrin : Aph-1 is 1 : 1 : 1 : 1.
Function and Dysfunction of CNG Channels: Insights from Channelopathies and Mouse Models
18.09.2007
Channels directly gated by cyclic nucleotides (CNG channels) are important cellular switches that mediate influx of Na+ and Ca2+ in response to increases in the intracellular concentration of cAMP and cGMP. In photoreceptors and olfactory receptor neurons, these channels serve as final targets for cGMP and cAMP signaling pathways that are initiated by the absorption of photons and the binding of odorants, respectively. CNG channels have been also found in other types of neurons and in non-excitable cells. However, in most of these cells, the physiological role of CNG channels has yet to be determined. CNG channels have a complex heteromeric structure. The properties of individual subunits that assemble in specific stoichiometries to the native channels have been extensively investigated in heterologous expression systems. Recently, mutations in human CNG channel genes leading to inherited diseases (so-called channelopathies) have been functionally characterized. Moreover, mouse knockout models were generated to define the role of CNG channel proteins in vivo. In this review, we will summarize recent insights into the physiological and patho- physiological role of CNG channel proteins that have emerged from genetic studies in mice and humans.
Contribution of the receptor guanylyl cyclase GC-D to chemosensory function in the olfactory epithelium
04.09.2007
The mammalian main olfactory epithelium (MOE) recognizes and transduces olfactory cues through a G protein-coupled, cAMPdependent signaling cascade. Additional chemosensory transduction mechanisms have been suggested but remain controversial. We show that a subset of MOE neurons expressing the orphan receptor guanylyl cyclase GC-D and the cyclic nucleotide-gated channel subunit CNGA3 employ an excitatory cGMP-dependent transduction mechanism for chemodetection. By combining gene targeting of Gucy2d, which encodes GC-D, with patch clamp recording and confocal Ca2+ imaging from single dendritic knobs in situ, we find that GC-D cells recognize the peptide hormones uroguanylin and guanylin as well as natural urine stimuli. These molecules stimulate an excitatory, cGMP-dependent signaling cascade that increases intracellular Ca2+ and action potential firing. Responses are eliminated in both Gucy2d- and Cnga3-null mice, demonstrating the essential role of GC-D and CNGA3 in the transduction of these molecules. The sensitive and selective detection of two important natriuretic peptides by the GC-D neurons suggests the possibility that these cells contribute to the maintenance of salt and water homeostasis or the detection of cues related to hunger, satiety, or thirst.
Endoplasmic reticulum retetion of the gamma-secretase complex component Pen2 by Rer1
06.07.2007
EMBO reports,
2007,
1-6
(pdf)
published on 06.07.2007
Gamma-Secretase is involved in the production of amyloid beta-peptide, which is the principle component of amyloid plaques in the brains of patients with Alzheimer disease. Gamma-Secretase is a complex composed of presenilin (PS), nicastrin, anterior pharynx-defective phenotype 1 (Aph1) and PS enhancer 2 (Pen2). We previously proposed a mechanism of complex assembly by which unassembled subunits are retained in the endoplasmic reticulum (ER) and only the fully assembled complex is exported from the ER. We have now identified Retention in endoplasmic reticulum 1 (Rer1) as a protein that is involved in the retention/retrievel of unassembled Pen2 to the ER. Direct binding of unassembled Pen2 to Rer1 is mediated by the first transmembrane domain of Pen2, and a conversed asparagine in this domain is required. Downregualtion of Rer1 leads to increased surface localization of Pen2, whereas overexpression of Rer1 stabilizes unassembled Pen2. To our knowledge, Per1 is the first identified interaction partner of mammalian transmembrane-based retention/retrieval signals.
The Nicastrin-like Protein Nicalin Regulates Assembly and Stability of the Nicalin-Nodal Modulator (NOMO) Membrane Protein Complex
29.01.2007
The assembly of the gamma-secretase complex, an Alzheimer disease-related protease required for ß-amyloid generation, is tightly regulated and predominantly limited by the stoichiometrical availability of its components. We have identified a novel endoplasmic reticulum-located protein complex that is regulatedin a similar fashion. It contains the recently identified Nodal signaling antagonists Nicalin (a distant homolog of the gamma-secretase component Nicastrin) and NOMO (Nodal modulator). Using an RNA interference approach, we found that Nicalin and NOMO became unstable in the absence of the respective binding partner, suggesting that complex formation has a stabilizing effect. Overexpression of Nicalin resulted in an increase in NOMO, whereas endogenous Nicalin was reduced below the detection limit. Both effects were shown to occur at a post-transcriptional level. Thus, NOMO is most likely produced in excess amounts and either stabilized by Nicalin or rapidly degraded. Incontrast, Nicalin levels are limited independently of NOMO. We, therefore, propose that Nicalin controls the assembly andstability of the Nicalin-NOMO complex.
