Multiple system atrophy is a parkinsonian neurodegenerative disorder. It is cytopathologically characterized by accumulation of the protein p25α in cell bodies of oligodendrocytes followed by accumulation of aggregated α-synuclein in so-called glial cytoplasmic inclusions. p25α is a stimulator of α-synuclein aggregation, and coexpression of α-synuclein and p25α in the oligodendroglial OLN-t40-AS cell line causes α-synuclein aggregate-dependent toxicity. In this study, we investigated whether the FAS system is involved in α-synuclein aggregate dependent degeneration in oligodendrocytes and may play a role in multiple system atrophy. Using rat oligodendroglial OLN-t40-AS cells we demonstrate that the cytotoxicity caused by coexpressing α-synuclein and p25α relies on stimulation of the death domain receptor FAS and caspase-8 activation. Using primary oligodendrocytes derived from PLP-α-synuclein transgenic mice we demonstrate that they exist in a sensitized state expressing pro-apoptotic FAS receptor, which makes them sensitive to FAS ligand-mediated apoptosis. Immunoblot analysis shows an increase in FAS in brain extracts from multiple system atrophy cases. Immunohistochemical analysis demonstrated enhanced FAS expression in multiple system atrophy brains notably in oligodendrocytes harboring the earliest stages of glial cytoplasmic inclusion formation. Oligodendroglial FAS expression is an early hallmark of oligodendroglial pathology in multiple system atrophy that mechanistically may be coupled to α-synuclein dependent degeneration and thus represent a potential target for protective intervention.

The identification of causative mutations in the (pro)granulin gene (GRN) has been a major breakthrough in the research on frontotemporal dementia (FTD). So far, all FTD-associated GRN mutations are leading to neurodegeneration through a “loss-of-function” mechanism, encouraging researchers to develop a growing number of cellular and animal models for GRN deficiency. GRN is a multifunctional secreted growth factor, and loss of its function can affect different cellular processes. Besides loss-of-function (i.e., mostly premature termination codons) mutations, which cause GRN haploinsufficiency through reduction of GRN expression, FTD-associated GRN missense mutations have also been identified. Several of these missense mutations are predicted to increase the risk of developing neurodegenerative diseases through altering various key biological properties of GRN-like protein secretion, proteolytic processing, and neurite outgrowth. With the use of cellular and animal models for GRN deficiency, the portfolio of GRN functions has recently been extended to include functions in important biological processes like energy and protein homeostasis, inflammation as well as neuronal survival, neurite outgrowth, and branching. Furthermore, GRN-deficient animal models have been established and they are believed to be promising disease models as they show accelerated aging and recapitulate at least some neuropathological features of FTD. In this review, we summarize the current knowledge on the molecular mechanisms leading to GRN deficiency and the lessons we learned from the established cellular and animal models. Furthermore, we discuss how these insights might help in developing therapeutic strategies for GRN-associated FTD.

Accumulation of the DNA/RNA binding protein fused in sarcoma as cytoplasmic inclusions in neurons and glial cells is the pathological hallmark of all patients with amyotrophic lateral sclerosis with mutations in FUS as well as in several subtypes of frontotemporal lobar degeneration, which are not associated with FUS mutations. The mechanisms leading to inclusion formation and fused in sarcoma-associated neurodegeneration are only poorly understood. Because fused in sarcoma belongs to a family of proteins known as FET, which also includes Ewing’s sarcoma and TATA-binding protein-associated factor 15, we investigated the potential involvement of these other FET protein family members in the pathogenesis of fused in sarcoma proteinopathies. Immunohistochemical analysis of FET proteins revealed a striking difference among the various conditions, with pathology in amyotrophic lateral sclerosis with FUS mutations being labelled exclusively for fused in sarcoma, whereas fused in sarcoma-positive inclusions in subtypes of frontotemporal lobar degeneration also consistently immunostained for TATA-binding protein-associated factor 15 and variably for Ewing’s sarcoma. Immunoblot analysis of proteins extracted from post-mortem tissue of frontotemporal lobar degeneration with fused in sarcoma pathology demonstrated a relative shift of all FET proteins towards insoluble protein fractions, while genetic analysis of the TATA-binding protein-associated factor 15 and Ewing’s sarcoma gene did not identify any pathogenic variants. Cell culture experiments replicated the findings of amyotrophic lateral sclerosis with FUS mutations by confirming the absence of TATA-binding protein-associated factor 15 and Ewing’s sarcoma alterations upon expression of mutant fused in sarcoma. In contrast, all endogenous FET proteins were recruited into cytoplasmic stress granules upon general inhibition of Transportin-mediated nuclear import, mimicking the findings in frontotemporal lobar degeneration with fused in sarcoma pathology. These results allow a separation of fused in sarcoma proteinopathies caused by FUS mutations from those without a known genetic cause based on neuropathological features. More importantly, our data imply different pathological processes underlying inclusion formation and cell death between both conditions; the pathogenesis in amyotrophic lateral sclerosis with FUS mutations appears to be more restricted to dysfunction of fused in sarcoma, while a more global and complex dysregulation of all FET proteins is involved in the subtypes of frontotemporal lobar degeneration with fused in sarcoma pathology.

Accumulations of insoluble deposits of amyloid β-peptide are major pathological hallmarks of Alzheimer disease. Amyloid β-peptide is derived by sequential proteolytic processing from a large type I trans-membrane protein, the β-amyloid precursor protein. The proteolytic enzymes involved in its processing are named secretases. β- and γ-secretase liberate by sequential cleavage the neurotoxic amyloid β-peptide, whereas α-secretase prevents its generation by cleaving within the middle of the amyloid domain. In this chapter we describe the cell biological and biochemical characteristics of the three secretase activities involved in the proteolytic processing of the precursor protein. In addition we outline how the precursor protein maturates and traffics through the secretory pathway to reach the subcellular locations where the individual secretases are preferentially active. Furthermore, we illuminate how neuronal activity and mutations which cause familial Alzheimer disease affect amyloid β-peptide generation and therefore disease onset and progression.

The neurotoxic amyloid β-peptide protein in Alzheimer disease is produced when γ- and β-secretase cleave the β-amyloid precursor protein (APP). α-Secretase prevents its generation.

Background

Mutations in the gene encoding the E3 ubiquitin ligase parkin (PARK2) are responsible for the majority of autosomal recessive parkinsonism. Similarly to other knockout mouse models of PD-associated genes, parkin knockout mice do not show a substantial neuropathological or behavioral phenotype, while loss of parkin in Drosophila melanogaster leads to a severe phenotype, including reduced lifespan, apoptotic flight muscle degeneration and male sterility. In order to study the function of parkin in more detail and to address possible differences in its role in different species, we chose Danio rerio as a different vertebrate model system.

Methodology/Principal Findings

We first cloned zebrafish parkin to compare its biochemical and functional aspects with that of human parkin. By using an antisense knockdown strategy we generated a zebrafish model of parkin deficiency (knockdown efficiency between 50% and 60%) and found that the transient knockdown of parkin does not cause morphological or behavioral alterations. Specifically, we did not observe a loss of dopaminergic neurons in parkin-deficient zebrafish. In addition, we established transgenic zebrafish lines stably expressing parkin by using a Gal4/UAS-based bidirectional expression system. While parkin-deficient zebrafish are more vulnerable to proteotoxicity, increased parkin expression protected transgenic zebrafish from cell death induced by proteotoxic stress.

Conclusions/Significance

Similarly to human parkin, zebrafish parkin is a stress-responsive protein which protects cells from stress-induced cell death. Our transgenic zebrafish model is a novel tool to characterize the protective capacity of parkin in vivo.

Summary

Beta-site APP cleaving enzyme-1 (BACE1), the rate-limiting enzyme for β-amyloid (Aβ) production, is elevated in Alzheimer’s disease (AD). Here, we show that energy deprivation induces phosphorylation of the translation initiation factor eIF2α eIF2α-P), which increases the translation of BACE1. Salubrinal, an inhibitor of eIF2α-P phosphatase PP1c, directly increases BACE1 and elevates Aβ production in primary neurons. Preventing eIF2α phosphorylation by transfection with constitutively active PP1c regulatory subunit, dominant negative eIF2α kinase PERK, or PERK inhibitor P58IPK blocks the energy deprivation-induced BACE1 increase. Furthermore, chronic treatment of aged Tg2576 mice with energy inhibitors increases levels of eIF2α-P, BACE1, Aβ, and amyloid plaques. Importantly, eIF2α-P and BACE1 are elevated in aggressive plaque-forming 5XFAD transgenic mice, and BACE1, eIF2α-P, and amyloid load are all correlated in humans with AD. These results strongly suggest that eIF2α phosphorylation increases BACE1 levels and causes Aβ overproduction, which could be an early, initiating molecular mechanism in sporadic AD.

In synucleinopathies, including Parkinson's disease, partially ubiquitylated α-synuclein species phosphorylated on serine 129 (PS129-α-synuclein) accumulate abnormally. Parkin, an ubiquitin-protein ligase that is dysfunctional in autosomal recessive parkinsonism, protects against α-synuclein-mediated toxicity in various models.

We analyzed the effects of Parkin deficiency in a mouse model of synucleinopathy to explore the possibility that Parkin and α-synuclein act in the same biochemical pathway. Whether or not Parkin was present, these mice developed an age-dependent neurodegenerative disorder preceded by a progressive decline in performance in tasks predictive of sensorimotor dysfunction. The symptoms were accompanied by the deposition of PS129-α-synuclein but not PS87-α-synuclein in neuronal cell bodies and neuritic processes throughout the brainstem and the spinal cord; activation of caspase 9 was observed in 5% of the PS129-α-synuclein-positive neurons. As in Lewy bodies, ubiquitin-immunoreactivity, albeit less abundant, was invariably co-localized with PS129-α-synuclein. During late disease stages, the disease-specific neuropathological features revealed by ubiquitin- and PS129-α-synuclein-specific antibodies were similar in mice with or without Parkin. However, the proportion of PS129-α-synuclein-immunoreactive neuronal cell bodies and neurites co-stained for ubiquitin was lower in the absence than in the presence of Parkin, suggesting less advanced synucleinopathy. Moreover, sensorimotor impairment and manifestation of the neurodegenerative phenotype due to overproduction of human α-synuclein were significantly delayed in Parkin-deficient mice.

These findings raise the possibility that effective compensatory mechanisms modulate the phenotypic expression of disease in parkin-related parkinsonism.

Surrogate markers for the Alzheimer disease (AD)-associated 42-amino acid form of amyloid-β (Aβ42) have been sought because they may aid in the diagnosis of AD and for clarification of disease pathogenesis. Here, we demonstrate that human cerebrospinal fluid (CSF) contains three APLP1-derived Aβ-like peptides (APL1β) that are generated by β- and γ-cleavages at a concentration of ∼4.5 nM. These novel peptides, APL1β25, APL1β27 and APL1β28, were not deposited in AD brains. Interestingly, most γ-secretase modulators (GSMs) and familial AD-associated presenilin1 mutants that up-regulate the relative production of Aβ42 cause a parallel increase in the production of APL1β28 in cultured cells. Moreover, in CSF from patients with pathological mutations in presenilin1 gene, the relative APL1β28 levels are higher than in non-AD controls, while the relative Aβ42 levels are unchanged or lower. Most strikingly, the relative APL1β28 levels are higher in CSF from sporadic AD patients (regardless of whether they are at mild cognitive impairment or AD stage), than those of non-AD controls. Based on these results, we propose the relative level of APL1β28 in the CSF as a candidate surrogate marker for the relative level of Aβ42 production in the brain.

Our aging society is confronted with a dramatic increase of patients suffering from tauopathies, which include Alzheimer disease and certain frontotemporal dementias. These disorders are characterized by typical neuropathological lesions including hyperphosphorylation and subsequent aggregation of TAU protein and neuronal cell death. Currently, no mechanism-based cures are available. We generated fluorescently labeled TAU transgenic zebrafish, which rapidly recapitulated key pathological features of tauopathies, including phosphorylation and conformational changes of human TAU protein, tangle formation, neuronal and behavioral disturbances, and cell death. Due to their optical transparency and small size, zebrafish larvae are well suited for both in vivo imaging and drug development. TAU-induced neuronal cell death was imaged by time-lapse microscopy in vivo. Furthermore, we used this zebrafish model to identify compounds targeting the TAU kinase glycogen synthase kinase 3β (GSK3β). We identified a newly developed highly active GSK3β inhibitor, AR-534, by rational drug design. AR-534 reduced TAU phosphorylation in TAU transgenic zebrafish. This transgenic zebrafish model may become a valuable tool for further studies of the neuropathology of dementia.

Several receptor protein tyrosine phosphatases (RPTPs) are cell adhesion molecules involved in homophilic interactions, suggesting that RPTP outside-in signaling is coupled to cell contact formation. However, little is known about the mechanisms by which cell density regulates RPTP function. We show that the MAM family prototype RPTPκ is cleaved by three proteases: furin, ADAM 10, and γ-secretase. Cell density promotes ADAM 10-mediated cleavage and shedding of RPTPκ. This is followed by γ-secretase-dependent intramembrane proteolysis of the remaining transmembrane part to release the phosphatase intracellular portion (PIC) from the membrane, thereby allowing its translocation to the nucleus. When cells were treated with leptomycin B, a nuclear export inhibitor, PIC accumulated in nuclear bodies. PIC is an active protein tyrosine phosphatase that binds to and dephosphorylates β-catenin, an RPTPκ substrate. The expression of RPTPκ suppresses β-catenin's transcriptional activity, whereas the expression of PIC increases it. Notably, this increase required the phosphatase activity of PIC. Thus, both isoforms have acquired opposing roles in the regulation of β-catenin signaling. We also found that RPTPμ, another MAM family member, undergoes γ-secretase-dependent processing. Our results identify intramembrane proteolysis as a regulatory switch in RPTPκ signaling and implicate PIC in the activation of β-catenin-mediated transcription.

Millions of patients suffer from Alzheimer's disease, and intensive efforts to find a cure for this devastating disorder center on the proteases, which release the deadly amyloid β-peptide from its precursor. The cutting procedure is thought to be cholesterol dependent and strategies to lower cholesterol as therapeutic treatment are under intensive investigation. Recent findings suggest that the complete proteolytic machinery required for amyloid β-peptide generation is located within lipid rafts. Data by Dotti and colleagues (Abad-Rodriguez et al., 2004), in this issue, suggest that rafts isolate the cutting machinery away from its deadly substrate. These findings describe a novel mechanism for controlling proteolytic activity by building a lipid boundary between proteases and their substrates.

The amyloid β-peptide (Aβ peptide) is assumed to play a crucial and early role in the pathogenesis of Alzheimer disease. Thus, strategies for a pharmacotherapy aim at reducing Aβ peptide generation, which proteolytically derives from the amyloid precursor protein (APP). The main targets so far have been β- and γ-secretase, the two proteases that cleave APP at the N- and C-terminus of the Aβ peptide and are thus directly responsible for Aβ peptide generation. A different strategy, namely the activation of α-secretase, has barely been investigated for its therapeutic potential. α-Secretase cleaves within the Aβ peptide domain and thus precludes Aβ peptide generation. Now, new results demonstrate that activation of α-secretase indeed reduces Aβ peptide generation and toxicity in vivo.

Formation of senile plaques containing the β-amyloid peptide (Aβ) derived from the amyloid precursor protein (APP) is an invariant feature of Alzheimer's disease (AD). APP is cleaved either by β-secretase or by α-secretase to initiate amyloidogenic (release of Aβ) or nonamyloidogenic processing of APP, respectively. A key to understanding AD is to unravel how access of these enzymes to APP is regulated. Here, we demonstrate that lipid rafts are critically involved in regulating Aβ generation. Reducing cholesterol levels in N2a cells decreased Aβ production. APP and the β-site APP cleavage enzyme (BACE1) could be induced to copatch at the plasma membrane upon cross-linking with antibodies and to segregate away from nonraft markers. Antibody cross-linking dramatically increased production of Aβ in a cholesterol-dependent manner. Aβ generation was dependent on endocytosis and was reduced after expression of the dynamin mutant K44A and the Rab5 GTPase-activating protein, RN-tre. This inhibition could be overcome by antibody cross-linking. These observations suggest the existence of two APP pools. Although APP inside raft clusters seems to be cleaved by β-secretase, APP outside rafts undergoes cleavage by α-secretase. Thus, access of α- and β-secretase to APP, and therefore Aβ generation, may be determined by dynamic interactions of APP with lipid rafts.

Amyloid β-peptide (Aβ) is generated by the consecutive cleavages of β- and γ-secretase. The intramembraneous γ-secretase cleavage critically depends on the activity of presenilins (PS1 and PS2). Although there is evidence that PSs are aspartyl proteases with γ-secretase activity, it remains controversial whether their subcellular localization overlaps with the cellular sites of Aβ production. We now demonstrate that biologically active GFP-tagged PS1 as well as endogenous PS1 are targeted to the plasma membrane (PM) of living cells. On the way to the PM, PS1 binds to nicastrin (Nct), an essential component of the γ-secretase complex. This complex is targeted through the secretory pathway where PS1-bound Nct becomes endoglycosidase H resistant. Moreover, surface-biotinylated Nct can be coimmunoprecipitated with PS1 antibodies, demonstrating that this complex is located to cellular sites with γ-secretase activity. Inactivating PS1 or PS2 function by mutagenesis of one of the critical aspartate residues or by γ-secretase inhibitors results in delayed reinternalization of the β-amyloid precursor protein and its accumulation at the cell surface. Our data suggest that PS is targeted as a biologically active complex with Nct through the secretory pathway to the cell surface and suggest a dual function of PS in γ-secretase processing and in trafficking.

The pathological modifications of α-synuclein (αS) in Parkinson disease and related diseases are poorly understood. We have detected misfolded αS in situ based on the proteinase K resistance (PK resistance) of αS fibrils, and using specific antibodies against S129-phosphorylated αS as well as oxidized αS. Unexpectedly massive neuritic pathology was found in affected human brain regions, in addition to classical αS pathology. PK resistance and abnormal phosphorylation of αS developed with increasing age in (Thy1)-h[A30P] αS transgenic mice, concomitant with formation of argyrophilic, thioflavin S-positive, and electron-dense inclusions that were occasionally ubiquitinated. αS pathology in the transgenic mice was predominantly in the brainstem and spinal cord. Astrogliosis was found in these heavily affected tissues. Homozygous mice showed the same pathology approximately one year earlier. The transgenic mice showed a progressive deterioration of locomotor function.

Myelin sheath thickness is precisely adjusted to axon caliber, and in the peripheral nervous system, neuregulin 1 (NRG1) type III is a key regulator of this process. It has been proposed that the protease BACE1 activates NRG1 dependent myelination. Here, we characterize the predicted product of BACE1-mediated NRG1 type III processing in transgenic mice. Neuronal overexpression of a NRG1 type III-variant, designed to mimic prior cleavage in the juxtamembrane stalk region, induces hypermyelination in vivo and is sufficient to restore myelination of NRG1 type III-deficient neurons. This observation implies that the NRG1 cytoplasmic domain is dispensable and that processed NRG1 type III is sufficient for all steps of myelination. Surprisingly, transgenic neuronal overexpression of full-length NRG1 type III promotes hypermyelination also in BACE1 null mutant mice. Moreover, NRG1 processing is impaired but not abolished in BACE1 null mutants. Thus, BACE1 is not essential for the activation of NRG1 type III to promote myelination. Taken together, these findings suggest that multiple neuronal proteases collectively regulate NRG1 processing. © 2011 Wiley Periodicals, Inc.