Tripeptidyl peptidase II coordinates the homeostasis of calcium and lipids in the central nervous system and its depletion causes presenile dementia in female mice through calcium/lipid dyshomeostasis-induced autophagic degradation of CYP19A1.

Rationale: Tripeptidyl peptidase II (TPP2) has been proven to be related to human immune and neurological diseases. It is generally considered as a cytosolic protein which forms the largest known protease complex in eukaryotic cells to operate mostly downstream of proteasomes for degradation of longer peptides. However, this canonical function of TPP2 cannot explain its role in a wide variety of biological and pathogenic processes. The mechanistic interrelationships and hierarchical order of these processes have yet to be clarified. Methods: Animals, cells, plasmids, and viruses established and/or used in this study include: TPP2 knockout mouse line, TPP2 conditional knockout mouse lines (different neural cell type oriented), TRE-TPP2 knockin mouse line on the C57BL/6 background; 293T cells with depletion of TPP2, ATF6, IRE1, PERK, SYVN1, UCHL1, ATG5, CEPT1, or CCTα, respectively; 293T cells stably expressing TPP2, TPP2 S449A, TPP2 S449T, or CCTα-KDEL proteins on the TPP2-depleted background; Plasmids for eukaryotic transient expression of rat CYP19A1-Flag, CYP19A1 S118A-Flag, CYP19A1 S118D-Flag, Sac I ML GFP Strand 11 Long, OMMGFP 1-10, G-CEPIA1er, GCAMP2, CEPIA3mt, ACC-GFP, or SERCA1-GFP; AAV2 carrying the expression cassette of mouse CYP19A1-3 X Flag-T2A-ZsGreen. Techniques used in this study include: Flow cytometry, Immunofluorescence (IF) staining, Immunohistochemical (IHC) staining, Luxol fast blue (LFB) staining, ß-galactosidase staining, Lipid droplet (LD) staining, Calcium (Ca2+) staining, Stimulated emission depletion (STED) imaging, Transmission electron microscopic imaging, Two-photon imaging, Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end Labeling (TUNEL) assay, Bromodeoxyuridine (BrdU) assay, Enzymatic activity assay, Proximity ligation assay (PLA), In vivo electrophysiological recording, Long-term potentiation (LTP) recording, Split-GFP-based mitochondria-associated membrane (MAM) detection, Immunoprecipitation (IP), Cellular fractionation, In situ hybridization, Semi-quantitative RT-PCR, Immunoblot, Mass spectrometry-based lipidomics, metabolomics, proteomics, Primary hippocampal neuron culture and Morris water maze (MWM) test. Results: We found that TPP2, independent of its enzymatic activity, plays a crucial role in maintaining the homeostasis of intracellular Ca2+ and phosphatidylcholine (PC) in the central nervous system (CNS) of mice. In consistence with the critical importance of Ca2+ and PC in the CNS, TPP2 gene ablation causes presenile dementia in female mice, which is closely associated with Ca2+/PC dysregulation-induced endoplasmic reticulum (ER) stress, abnormal autophagic degradation of CYP19A1 (aromatase), and estrogen depletion. This work therefore uncovers a new role of TPP2 in lipogenesis and neurosteroidogenesis which is tightly related to cognitive function of adult female mice. Conclusion: Our study reveals a crucial role of TPP2 in controlling homeostasis of Ca2+ and lipids in CNS, and its deficiency causes sexual dimorphism in dementia. Thus, this study is not only of great significance for elucidating the pathogenesis of dementia and its futural treatment, but also for interpreting the role of TPP2 in other systems and their related disorders.

Role of primary aging hallmarks in Alzheimer´s disease.

Alzheimer's disease (AD) is the most common neurodegenerative disease, which severely threatens the health of the elderly and causes significant economic and social burdens. The causes of AD are complex and include heritable but mostly aging-related factors. The primary aging hallmarks include genomic instability, telomere wear, epigenetic changes, and loss of protein stability, which play a dominant role in the aging process. Although AD is closely associated with the aging process, the underlying mechanisms involved in AD pathogenesis have not been well characterized. This review summarizes the available literature about primary aging hallmarks and their roles in AD pathogenesis. By analyzing published literature, we attempted to uncover the possible mechanisms of aberrant epigenetic markers with related enzymes, transcription factors, and loss of proteostasis in AD. In particular, the importance of oxidative stress-induced DNA methylation and DNA methylation-directed histone modifications and proteostasis are highlighted. A molecular network of gene regulatory elements that undergoes a dynamic change with age may underlie age-dependent AD pathogenesis, and can be used as a new drug target to treat AD.

Lipid Dyshomeostasis and Inherited Cerebellar Ataxia.

Cerebellar ataxia is a form of ataxia that originates from dysfunction of the cerebellum, but may involve additional neurological tissues. Its clinical symptoms are mainly characterized by the absence of voluntary muscle coordination and loss of control of movement with varying manifestations due to differences in severity, in the site of cerebellar damage and in the involvement of extracerebellar tissues. Cerebellar ataxia may be sporadic, acquired, and hereditary. Hereditary ataxia accounts for the majority of cases. Hereditary ataxia has been tentatively divided into several subtypes by scientists in the field, and nearly all of them remain incurable. This is mainly because the detailed mechanisms of these cerebellar disorders are incompletely understood. To precisely diagnose and treat these diseases, studies on their molecular mechanisms have been conducted extensively in the past. Accumulating evidence has demonstrated that some common pathogenic mechanisms exist within each subtype of inherited ataxia. However, no reports have indicated whether there is a common mechanism among the different subtypes of inherited cerebellar ataxia. In this review, we summarize the available references and databases on neurological disorders characterized by cerebellar ataxia and show that a subset of genes involved in lipid homeostasis form a new group that may cause ataxic disorders through a common mechanism. This common signaling pathway can provide a valuable reference for future diagnosis and treatment of ataxic disorders.

Structural Properties and Interaction Partners of Familial ALS-Associated SOD1 Mutants.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron degenerative disease in adults and has also been proven to be a type of conformational disease associated with protein misfolding and dysfunction. To date, more than 150 distinct genes have been found to be associated with ALS, among which Superoxide Dismutase 1 (SOD1) is the first and the most extensively studied gene. It has been well-established that SOD1 mutants-mediated toxicity is caused by a gain-of-function rather than the loss of the detoxifying activity of SOD1. Compared with the clear autosomal dominant inheritance of SOD1 mutants in ALS, the potential toxic mechanisms of SOD1 mutants in motor neurons remain incompletely understood. A large body of evidence has shown that SOD1 mutants may adopt a complex profile of conformations and interact with a wide range of client proteins. Here, in this review, we summarize the fundamental conformational properties and the gained interaction partners of the soluble forms of the SOD1 mutants which have been published in the past decades. Our goal is to find clues to the possible internal links between structural and functional anomalies of SOD1 mutants, as well as the relationships between their exposed epitopes and interaction partners, in order to help reveal and determine potential diagnostic and therapeutic targets.

Precise Somatotopic Thalamocortical Axon Guidance Depends on LPA-Mediated PRG-2/Radixin Signaling.

Precise connection of thalamic barreloids with their corresponding cortical barrels is critical for processing of vibrissal sensory information. Here, we show that PRG-2, a phospholipid-interacting molecule, is important for thalamocortical axon guidance. Developing thalamocortical fibers both in PRG-2 full knockout (KO) and in thalamus-specific KO mice prematurely entered the cortical plate, eventually innervating non-corresponding barrels. This misrouting relied on lost axonal sensitivity toward lysophosphatidic acid (LPA), which failed to repel PRG-2-deficient thalamocortical fibers. PRG-2 electroporation in the PRG-2-/- thalamus restored the aberrant cortical innervation. We identified radixin as a PRG-2 interaction partner and showed that radixin accumulation in growth cones and its LPA-dependent phosphorylation depend on its binding to specific regions within the C-terminal region of PRG-2. In vivo recordings and whisker-specific behavioral tests demonstrated sensory discrimination deficits in PRG-2-/- animals. Our data show that bioactive phospholipids and PRG-2 are critical for guiding thalamic axons to their proper cortical targets.

PRG-1 Regulates Synaptic Plasticity via Intracellular PP2A/ß1-Integrin Signaling.

Alterations in dendritic spine numbers are linked to deficits in learning and memory. While we previously revealed that postsynaptic plasticity-related gene 1 (PRG-1) controls lysophosphatidic acid (LPA) signaling at glutamatergic synapses via presynaptic LPA receptors, we now show that PRG-1 also affects spine density and synaptic plasticity in a cell-autonomous fashion via protein phosphatase 2A (PP2A)/ß1-integrin activation. PRG-1 deficiency reduces spine numbers and ß1-integrin activation, alters long-term potentiation (LTP), and impairs spatial memory. The intracellular PRG-1 C terminus interacts in an LPA-dependent fashion with PP2A, thus modulating its phosphatase activity at the postsynaptic density. This results in recruitment of adhesome components src, paxillin, and talin to lipid rafts and ultimately in activation of ß1-integrins. Consistent with these findings, activation of PP2A with FTY720 rescues defects in spine density and LTP of PRG-1-deficient animals. These results disclose a mechanism by which bioactive lipid signaling via PRG-1 could affect synaptic plasticity and memory formation.

Molecular cause and functional impact of altered synaptic lipid signaling due to a prg-1 gene SNP.

Loss of plasticity-related gene 1 (PRG-1), which regulates synaptic phospholipid signaling, leads to hyperexcitability via increased glutamate release altering excitation/inhibition (E/I) balance in cortical networks. A recently reported SNP in prg-1 (R345T/mutPRG-1) affects ~5 million European and US citizens in a monoallelic variant. Our studies show that this mutation leads to a loss-of-PRG-1 function at the synapse due to its inability to control lysophosphatidic acid (LPA) levels via a cellular uptake mechanism which appears to depend on proper glycosylation altered by this SNP. PRG-1(+/-) mice, which are animal correlates of human PRG-1(+/mut) carriers, showed an altered cortical network function and stress-related behavioral changes indicating altered resilience against psychiatric disorders. These could be reversed by modulation of phospholipid signaling via pharmacological inhibition of the LPA-synthesizing molecule autotaxin. In line, EEG recordings in a human population-based cohort revealed an E/I balance shift in monoallelic mutPRG-1 carriers and an impaired sensory gating, which is regarded as an endophenotype of stress-related mental disorders. Intervention into bioactive lipid signaling is thus a promising strategy to interfere with glutamate-dependent symptoms in psychiatric diseases.

TNFα-induced lysosomal membrane permeability is downstream of MOMP and triggered by caspase-mediated NDUFS1 cleavage and ROS formation.

When NF-κB activation or protein synthesis is inhibited, tumor necrosis factor alpha (TNFα) can induce apoptosis through Bax- and Bak-mediated mitochondrial outer membrane permeabilization (MOMP) leading to caspase-3 activation. Additionally, previous studies have implicated lysosomal membrane permeability (LMP) and formation of reactive oxygen species (ROS) as early steps of TNFα-induced apoptosis. However, how these two events connect to MOMP and caspase-3 activation has been largely debated. Here, we present the novel finding that LMP induced by the addition of TNFα plus cycloheximide (CHX), the release of lysosomal cathepsins and ROS formation do not occur upstream but downstream of MOMP and require the caspase-3-mediated cleavage of the p75 NDUFS1 subunit of respiratory complex I. Both a caspase non-cleavable p75 mutant and the mitochondrially localized antioxidant MitoQ prevent LMP mediated by TNFα plus CHX and partially interfere with apoptosis induction. Moreover, LMP is completely blocked in cells deficient in both Bax and Bak, Apaf-1, caspase-9 or both caspase-3 and -7. Thus, after MOMP, active caspase-3 exerts a feedback action on complex I to produce ROS. ROS then provoke LMP, cathepsin release and further caspase activation to amplify TNFα apoptosis signaling.

Role of caspases and non-caspase proteases in cell death.

Undoubtedly, caspases are the major driving force for apoptosis execution and mechanisms of their activation and inhibition have been largely unveiled. Recent progress has been made with regard to the exact intracellular ordering of caspases, monitoring their activities in vivo and unveiling their substrate degradomes. Moreover, non-caspase proteases seem to assist caspases in the completion of the death execution program. Here we will consider some very recent data dealing with these aspects. We will also provide novel insights into the mechanisms that dictate apoptotic variability within a cell population.

Non-caspase proteases: triggers or amplifiers of apoptosis?

Caspases are the most important effectors of apoptosis, the major form of programmed cell death (PCD) in multicellular organisms. This is best reflected by the appearance of serious development defects in mice deficient for caspase-8, -9, and -3. Meanwhile, caspase-independent PCD, mediated by other proteases or signaling components has been described in numerous publications. Although we do not doubt that such cell death exists, we propose that it has evolved later during evolution and is most likely not designed to execute, but to amplify and speed-up caspase-dependent cell death. This review shall provide evidence for such a concept.

Viability and DNA damage responses of TPPII-deficient Myc- and Ras-transformed fibroblasts.

Tripeptidyl peptidase II (TPPII) is a giant cytosolic protease. Previous protease inhibitor, overexpression and siRNA studies suggested that TPPII is important for viability and proliferation of tumor cells, and for their ionizing radiation-induced DNA damage response. The possibility that TPPII could be targeted for tumor therapy prompted us to study its role in transformed cells following genetic TPPII deletion. We generated cell lines from primary fibroblasts having conditional (floxed) TPPII alleles, transformed them with both the c-myc and H-ras oncogenes, and deleted TPPII using retroviral self-deleting Cre recombinase. Clonally derived TPPIIflox/flox and TPPII-/- transformed fibroblasts showed no influences of TPPII expression on basal cell survival and proliferation, nor on radiation-induced p53 activation, p21 induction, cell cycle arrest, apoptosis, or clonogenic cell death. Thus, our results do not support a generally important role of TPPII for viability and proliferation of transformed cells or their p53-mediated DNA damage response.

Tripeptidyl peptidase II plays a role in the radiation response of selected primary cell types but not based on nuclear translocation and p53 stabilization.

The giant cytosolic protease tripeptidyl peptidase II (TPPII) was recently proposed to play a role in the DNA damage response. Shown were nuclear translocation of TPPII after gamma-irradiation, lack of radiation-induced p53 stabilization in TPPII-siRNA-treated cells, and complete tumor regression in mice after gamma-irradiation when combined with TPPII-siRNA silencing or a protease inhibitor reported to inhibit TPPII. This suggested that TPPII could be a novel target for tumor radiosensitization and prompted us to study radiation responses using TPPII-knockout mice. Neither the sensitivity to total body irradiation nor the radiosensitivity of resting lymphoid cells, which both strongly depend on p53, was altered in the absence of TPPII. Functional integrity of p53 in TPPII-knockout cells is further shown by a proper G(1) arrest and by the accumulation of p53 and its transcriptional targets, p21, Bax, and Fas, on gamma-irradiation. Furthermore, we could not confirm radiation-induced nuclear translocation of TPPII. Nevertheless, after gamma-irradiation, we found slightly increased mitotic catastrophe of TPPII-deficient primary fibroblasts and increased apoptosis of TPPII-deficient activated CD8(+) T cells. The latter was accompanied by delayed resolution of the DNA double-strand break marker gammaH2AX. This could, however, be due to increased apoptotic DNA damage rather than reduced DNA damage repair. Our data do not confirm a role for TPPII in the DNA damage response based on nuclear TPPII translocation and p53 stabilization but nevertheless do show increased radiation-induced cell death of selected nontransformed cell types in the absence of the TPPII protease.

Activation of cellular death programs associated with immunosenescence-like phenotype in TPPII knockout mice.

The giant cytosolic protease tripeptidyl peptidase II (TPPII) has been implicated in the regulation of proliferation and survival of malignant cells, particularly lymphoma cells. To address its functions in normal cellular and systemic physiology we have generated TPPII-deficient mice. TPPII deficiency activates cell type-specific death programs, including proliferative apoptosis in several T lineage subsets and premature cellular senescence in fibroblasts and CD8(+) T cells. This coincides with up-regulation of p53 and dysregulation of NF-kappaB. Prominent degenerative alterations at the organismic level were a decreased lifespan and symptoms characteristic of immunohematopoietic senescence. These symptoms include accelerated thymic involution, lymphopenia, impaired proliferative T cell responses, extramedullary hematopoiesis, and inflammation. Thus, TPPII is important for maintaining normal cellular and systemic physiology, which may be relevant for potential therapeutic applications of TPPII inhibitors.

Analysis of direct and cross-presentation of antigens in TPPII knockout mice.

Tripeptidyl peptidase II (TPPII) is an oligopeptidase forming giant complexes in the cytosol that have high exo-, but also, endoproteolytic activity. Immunohistochemically, the complexes appear as distinct foci in the cytosol. In part controversial biochemical and functional studies have suggested that TPPII contributes, on the one hand, positively to Ag processing by generating epitope carboxyl termini or by trimming epitope precursors, and, on the other, negatively by destroying potentially antigenic peptides. To clarify which of these roles is predominant, we generated and analyzed TPPII-deficient mice. Cell surface levels of MHC class I peptide complexes tended to be increased on most cell types of these mice. Although presentation of three individual epitopes derived from lymphocytic choriomeningitis virus was not elevated on TPPII-/- cells, that of the immunodominant OVA epitope SIINFEKL was significantly enhanced. Consistent with this, degradation of a synthetic peptide corresponding to the OVA epitope and of another corresponding to a precursor thereof, both being proteasomally generated OVA fragments, was delayed in TPPII-deficient cytosolic extracts. In addition, dendritic cell cross-presentation of phagocytosed OVA and of OVA internalized as an immune complex was increased to about the same level as direct presentation of the Ag. The data suggest a moderate, predominantly destructive role of TPPII in class I Ag processing, in line with our finding that TPPII is not induced by IFN-gamma, which up-regulates numerous, predominantly constructive components of the Ag processing and presentation machinery.

Inducible gene expression with the Tet-on system in CD4+ T cells and thymocytes of mice.

CD4+ T cells with their growing list of effector and regulatory subpopulations have vital functions within the immunohematopoietic system. We report here on the first mouse lines that allow temporally and quantitatively controlled expression of transgenes specifically in CD4+ thymocytes and T cells. These were constructed using the Tet-on system. The rtTA2(S)-M2 version of the reverse tetracycline-dependent transactivator was placed under control of all known CD4 regulatory elements. Reporter transgene expression in mice expressing these constructs is highly specific for CD4+ cells, is strictly dependent on the tetracycline derivative doxycycline, and can be regulated by up to five logs depending on the doxycycline concentration. Moreover, we demonstrate that these mice can be used for noninvasive in vivo imaging of a coexpressed luciferase reporter. These new mouse lines should be highly valuable for studying and manipulating numerous aspects of CD4+ T cell development, biology, and function.

The role of endoplasmic reticulum-associated aminopeptidase 1 in immunity to infection and in cross-presentation.

Endoplasmic reticulum-associated aminopeptidase 1 (ERAP1) is involved in the final processing of endogenous peptides presented by MHC class I molecules to CTLs. We generated ERAP1-deficient mice and analyzed cytotoxic responses upon infection with three viruses, including lymphocytic choriomeningitis virus, which causes vigorous T cell activation and is controlled by CTLs. Despite pronounced effects on the presentation of selected epitopes, the in vivo cytotoxic response was altered for only one of several epitopes tested. Moreover, control of lymphocytic choriomeningitis virus was not impaired in the knockout mice. Thus, we conclude that lack of ERAP1 has little influence on antiviral immunohierarchies and antiviral immunity in the infections studied. We also focused on the role of ERAP1 in cross-presentation. We demonstrate that ERAP1 is required for efficient cross-presentation of cell-associated Ag and of OVA/anti-OVA immunocomplexes. Surprisingly, however, ERAP1 deficiency has no effect on cross-presentation of soluble OVA, suggesting that for soluble exogenous proteins, final processing may not take place in an environment containing active ERAP1.