Modulation of the endoplasmic reticulum-mitochondria interface in Alzheimer's disease and related models.

It is well-established that subcompartments of endoplasmic reticulum (ER) are in physical contact with the mitochondria. These lipid raft-like regions of ER are referred to as mitochondria-associated ER membranes (MAMs), and they play an important role in, for example, lipid synthesis, calcium homeostasis, and apoptotic signaling. Perturbation of MAM function has previously been suggested in Alzheimer's disease (AD) as shown in fibroblasts from AD patients and a neuroblastoma cell line containing familial presenilin-2 AD mutation. The effect of AD pathogenesis on the ER-mitochondria interplay in the brain has so far remained unknown. Here, we studied ER-mitochondria contacts in human AD brain and related AD mouse and neuronal cell models. We found uniform distribution of MAM in neurons. Phosphofurin acidic cluster sorting protein-2 and σ1 receptor, two MAM-associated proteins, were shown to be essential for neuronal survival, because siRNA knockdown resulted in degeneration. Up-regulated MAM-associated proteins were found in the AD brain and amyloid precursor protein (APP)Swe/Lon mouse model, in which up-regulation was observed before the appearance of plaques. By studying an ER-mitochondria bridging complex, inositol-1,4,5-triphosphate receptor-voltage-dependent anion channel, we revealed that nanomolar concentrations of amyloid ß-peptide increased inositol-1,4,5-triphosphate receptor and voltage-dependent anion channel protein expression and elevated the number of ER-mitochondria contact points and mitochondrial calcium concentrations. Our data suggest an important role of ER-mitochondria contacts and cross-talk in AD pathology.

The pathogenic aß43 is enriched in familial and sporadic Alzheimer disease.

The amyloid-cascade hypothesis posits that the role of amyloid ß-peptide (Aß) in Alzheimer disease (AD) involves polymerization into structures that eventually are deposited as amyloid plaques. During this process, neurotoxic oligomers are formed that induce synaptic loss and neuronal death. Several different isoforms of Aß are produced, of which the 40 and 42 residue variants (Aß40 and Aß42) are the most common. Aß42 has a strong tendency to form neurotoxic aggregates and is involved in AD pathogenesis. Longer Aß isoforms, like the less studied Aß43, are gaining attention for their higher propensity to aggregate into neurotoxic oligomers. To further investigate Aß43 in AD, we conducted a quantitative study on Aß43 levels in human brain. We homogenized human brain tissue and prepared fractions of various solubility; tris buffered saline (TBS), sodium dodecyl sulfate (SDS) and formic acid (FA). Levels of Aß43, as well as Aß40 and Aß42, were quantified using ELISA. We compared quantitative data showing Aß levels in occipital and frontal cortex from sporadic (SAD) and familial (FAD) AD cases, as well as non-demented (ND) controls. Results showed Aß43 present in each fraction from the SAD and FAD cases, while its level was lower than the detection limit in the majority of the ND-cases. Aß42 and Aß43 were enriched in the less soluble fractions (SDS and FA) of SAD and FAD cases in both occipital and frontal cortex. Thus, although the total levels of Aß43 in human brain are low compared to Aß40 and Aß42, we suggest that Aß43 could initiate the formation of oligomers and amyloid plaques and thereby be crucial to AD pathogenesis.

Thioredoxin-80 is a product of alpha-secretase cleavage that inhibits amyloid-beta aggregation and is decreased in Alzheimer's disease brain.

Thioredoxin-1 (Trx1) is an endogenous dithiol reductant and antioxidant that was shown to be decreased in Alzheimer's disease (AD) neurons. A truncated form of Trx1, thioredoxin 80 (Trx80), was reported to be secreted from monocytes having cytokine activity. Here, we show that Trx80 is present in human brain in an aggregated form. Trx80 localizes mainly to neurons and is dramatically decreased in AD brains. Trx80 levels in cerebrospinal fluid (CSF) correlate with those of the classical AD biomarkers amyloid-ß (Aß) 1-42 and total tau. Moreover, Trx80 measurements in CSF discriminate between patients with stable mild cognitive impairment, prodomal AD and mild AD. We report that ADAM10 and 17, two α-secretases processing the Aß precursor protein, are responsible for Trx80 generation. In contrast to the periphery, Trx80 has no pro-inflammatory effects in glia, either by itself or in combination with Aß or apolipoprotein E. Instead, Trx80 inhibits Aß(1-42) aggregation and protects against its toxicity. Thus, a reduction in Trx80 production would result in increased Aß polymerization and enhanced neuronal vulnerability. Our data suggest that a deficit in Trx80 could participate in AD pathogenesis.

Identification of two novel synaptic γ-secretase associated proteins that affect amyloid ß-peptide levels without altering Notch processing.

Synaptic degeneration is one of the earliest hallmarks of Alzheimer disease (AD) and results in loss of cognitive function. One of the causative agents for the synaptic degeneration is the amyloid ß-peptide (Aß), which is formed from its precursor protein by two sequential cleavages mediated by ß- and γ-secretase. We have earlier shown that γ-secretase activity is enriched in synaptic compartments, suggesting that the synaptotoxic Aß is produced locally. Proteins that interact with γ-secretase at the synapse and regulate the production of Aß can therefore be potential therapeutic targets. We used a recently developed affinity purification approach to identify γ-secretase associated proteins (GSAPs) in synaptic membranes and synaptic vesicles prepared from rat brain. Liquid chromatography-tandem mass spectrometry analysis of the affinity purified samples revealed the known γ-secretase components presenilin-1, nicastrin and Aph-1b along with a number of novel potential GSAPs. To investigate the effect of these GSAPs on APP processing, we performed siRNA experiments to knock down the expression of the GSAPs and measured the Aß levels. Silencing of NADH dehydrogenase [ubiquinone] iron-sulfur protein 7 (NDUFS7) resulted in a decrease in Aß levels whereas silencing of tubulin polymerization promoting protein (TPPP) resulted in an increase in Aß levels. Treatment with γ-secretase inhibitors often results in Notch-related side effects and therefore we also studied the effect of the siRNAs on Notch processing. Interestingly, silencing of TPPP or NDUFS7 did not affect cleavage of Notch. We also studied the expression of TPPP and NDUFS7 in control and AD brain and found NDUFS7 to be highly expressed in vulnerable neurons such as pyramidal neurons in the hippocampus, whereas TPPP was found to accumulate in intraneuronal granules and fibrous structures in hippocampus from AD cases. In summary, we here report on two proteins, TPPP and NDUFS7, which interact with γ-secretase and alter the Aß levels without affecting Notch cleavage.

DJ-1 acts in parallel to the PINK1/parkin pathway to control mitochondrial function and autophagy.

Mutations in DJ-1, PINK1 (PTEN-induced putative kinase 1) and parkin all cause recessive parkinsonism in humans, but the relationships between these genes are not clearly defined. One event associated with loss of any of these genes is altered mitochondrial function. Recent evidence suggests that turnover of damaged mitochondria by autophagy might be central to the process of recessive parkinsonism. Here, we show that loss of DJ-1 leads to loss of mitochondrial polarization, fragmentation of mitochondria and accumulation of markers of autophagy (LC3 punctae and lipidation) around mitochondria in human dopaminergic cells. These effects are due to endogenous oxidative stress, as antioxidants will reverse all of them. Similar to PINK1 and parkin, DJ-1 also limits mitochondrial fragmentation in response to the mitochondrial toxin rotenone. Furthermore, overexpressed parkin will protect against loss of DJ-1 and, although DJ-1 does not alter PINK1 mitochondrial phenotypes, DJ-1 is still active against rotenone-induced damage in the absence of PINK1. None of the three proteins complex together using size exclusion chromatography. These data suggest that DJ-1 works in parallel to the PINK1/parkin pathway to maintain mitochondrial function in the presence of an oxidative environment.

Parkin deficiency disrupts calcium homeostasis by modulating phospholipase C signalling.

Mutations in the E3 ubiquitin ligase parkin cause early-onset, autosomal-recessive juvenile parkinsonism (AJRP), presumably as a result of a lack of function that alters the level, activity, aggregation or localization of its substrates. Recently, we have reported that phospholipase Cgamma1 is a substrate for parkin. In this article, we show that parkin mutants and siRNA parkin knockdown cells possess enhanced levels of phospholipase Cgamma1 phosphorylation, basal phosphoinositide hydrolysis and intracellular Ca2+ concentration. The protein levels of Ca2+-regulated protein kinase Calpha were decreased in AJRP parkin mutant cells. Neomycin and dantrolene both decreased the intracellular Ca2+ levels in parkin mutants in comparison with those seen in wild-type parkin cells, suggesting that the differences were a consequence of altered phospholipase C activity. The protection of wild-type parkin against 6-hydroxydopamine (6OHDA) toxicity was also established in ARJP mutants on pretreatment with dantrolene, implying that a balancing Ca2+ release from ryanodine-sensitive stores decreases the toxic effects of 6OHDA. Our findings suggest that parkin is an important factor for maintaining Ca2+ homeostasis and that parkin deficiency leads to a phospholipase C-dependent increase in intracellular Ca2+ levels, which make cells more vulnerable to neurotoxins, such as 6OHDA.

Mitochondrial alterations in PINK1 deficient cells are influenced by calcineurin-dependent dephosphorylation of dynamin-related protein 1.

PTEN-induced novel kinase 1 (PINK1) mutations are associated with autosomal recessive parkinsonism. Previous studies have shown that PINK1 influences both mitochondrial function and morphology although it is not clearly established which of these are primary events and which are secondary. Here, we describe a novel mechanism linking mitochondrial dysfunction and alterations in mitochondrial morphology related to PINK1. Cell lines were generated by stably transducing human dopaminergic M17 cells with lentiviral constructs that increased or knocked down PINK1. As in previous studies, PINK1 deficient cells have lower mitochondrial membrane potential and are more sensitive to the toxic effects of mitochondrial complex I inhibitors. We also show that wild-type PINK1, but not recessive mutant or kinase dead versions, protects against rotenone-induced mitochondrial fragmentation whereas PINK1 deficient cells show lower mitochondrial connectivity. Expression of dynamin-related protein 1 (Drp1) exaggerates PINK1 deficiency phenotypes and Drp1 RNAi rescues them. We also show that Drp1 is dephosphorylated in PINK1 deficient cells due to activation of the calcium-dependent phosphatase calcineurin. Accordingly, the calcineurin inhibitor FK506 blocks both Drp1 dephosphorylation and loss of mitochondrial integrity in PINK1 deficient cells but does not fully rescue mitochondrial membrane potential. We propose that alterations in mitochondrial connectivity in this system are secondary to functional effects on mitochondrial membrane potential.

Parkin-mediated ubiquitination regulates phospholipase C-gamma1.

Mutations in parkin cause autosomal recessive forms of Parkinson's disease (PD), with an early age of onset and similar pathological phenotype to the idiopathic disease. Parkin has been identified as an E3 ubiquitin ligase that mediates different types of ubiquitination, which has made the search for substrates an intriguing possibility to identify pathological mechanisms linked to PD. In this study, we present PLCgamma1 as a novel substrate for parkin. This association was found in non-transfected human neuroblastoma SH-SY5Y cells as well as in stable cell lines expressing parkin WT and familial mutants R42P and G328E. Analysis of cortical, striatal and nigral human brain homogenates revealed that the interaction between parkin and PLCgamma1 is consistent throughout these regions, suggesting that the interaction is likely to have a physiological relevance for humans. Unlike many of the previously identified substrates, we could also show that the steady-state levels of PLCgamma1 is significantly higher in parkin KO mice and lower in parkin WT human neuroblastoma cells, suggesting that parkin ubiquitination of PLCgamma1 is required for proteasomal degradation. In line with this idea, we show that the ability to ubiquitinate PLCgamma1 in vitro differs significantly between WT and familial mutant parkin. In this study, we demonstrate that parkin interacts with PLCgamma1, affecting PLCgamma1 steady state protein levels in human and murine models with manipulated parkin function and expression levels. This finding could be of relevance for finding novel pathogenic mechanisms leading to PD.