Lars O Tjernberg

Karolinska Institutet, Solna, Stockholm, Sweden

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Publications (89)608.39 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The transmembrane protease complex γ-secretase is a key enzyme for Alzheimer disease pathogenesis since it liberates the neurotoxic amyloid β-peptide (Aβ) but still, the regulation of its activity in different cell types and subcellular compartments is largely unknown. Several γ-secretase inhibitors have been developed but none of them have been launched due to side effects that seem to arise from reduced processing of Notch, one of many γ-secretase substrates. Hence, it is desirable to specifically inhibit Aβ production. In our previous studies, we have from brain identified several novel γ-secretase associated proteins (GSAPs), which affect Aβ production without causing any major effects on Notch processing. In the present study we have, in detergent resistant membranes prepared from brain, identified four novel GSAPs that affect Aβ production to a larger extent than Notch processing. We studied these and selected GSAPs from this and our previous studies, and evaluated the interaction between the GSAPs and γ-secretase in different cell types and studied their mRNA expression in different human organs. By in situ proximity ligation assay, we demonstrated that many GSAPs showed considerably more prominent interaction with γ-secretase in neurons than in HEK-APP cells, and the mRNA expression study showed that several GSAPs are highly expressed in human brain. This study underscores the importance of studying protein-protein interactions in relevant cell types and suggests that reducing Aβ production by interfering with brain or neuron-specific γ-secretase/GSAP interactions could reduce the risk of unwanted side effects associated with treatment of Alzheimer disease. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    FEBS Journal 04/2015; DOI:10.1111/febs.13303 · 3.99 Impact Factor
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    ABSTRACT: Dysfunctional Omi/HtrA2, a mitochondrial serine protease, has been implicated in various neurodegenerative disorders. Despite the wealth of evidence on the roles of Omi/HtrA2 in apoptosis, little is known about its cytosolic targets, the cleavage of which could account for the observed morphological changes such as cytoskeletal reorganizations in axons. By proteomic analysis, vimentin was identified as a substrate for Omi/HtrA2 and we have reported increased Omi/HtrA2 protease activity in Alzheimer disease (AD) brain. Here, we investigated a possible link between Omi/HtrA2 and vimentin cleavage, and consequence of this cleavage on mitochondrial distribution in neurons. In vitro protease assays showed vimentin to be cleaved by Omi/HtrA2 protease, and proximity ligation assay demonstrated an increased interaction between Omi/HtrA2 and vimentin in human primary neurons upon stress stimuli. Using differentiated neuroblastoma SH-SY5Y cells, we showed that Omi/HtrA2 under several different stress conditions induces cleavage of vimentin in wild-type as well as SH-SY5Y cells transfected with amyloid precursor protein with the Alzheimer disease-associated Swedish mutation. After stress treatment, inhibition of Omi/HtrA2 protease activity by the Omi/HtrA2 specific inhibitor, Ucf-101, reduced the cleavage of vimentin in wild-type cells. Following altered vimentin filaments integrity by stress stimuli, mitochondria was redistributed in differentiated SH-SY5Y cells and human primary neurons. In summary, the findings outlined in this paper suggest a role of Omi/HtrA2 in modulation of vimentin filamentous structure in neurons. Our results provide important findings for understanding the biological role of Omi/HtrA2 activity during stress conditions, and give knowledge of interplay between Omi/HtrA2 and vimentin which might affect mitochondrial distribution in neurons.
    Molecular Neurobiology 10/2014; DOI:10.1007/s12035-014-8906-3 · 5.29 Impact Factor
  • Alzheimer's and Dementia 07/2014; 10(4):P298. DOI:10.1016/j.jalz.2014.04.497 · 17.47 Impact Factor
  • Alzheimer's and Dementia 07/2014; 10(4):P491. DOI:10.1016/j.jalz.2014.05.732 · 17.47 Impact Factor
  • Alzheimer's and Dementia 07/2014; 10(4):P217. DOI:10.1016/j.jalz.2014.04.295 · 17.47 Impact Factor
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    ABSTRACT: Synaptic degeneration is one of the earliest hallmarks of Alzheimer disease. The molecular mechanism underlying this degeneration is not fully elucidated but one key player appears to be the synaptotoxic amyloid β-peptide (Aβ). The exact localization of the production of Aβ and the mechanisms whereby Aβ is released remain elusive. We have earlier shown that Aβ can be produced in crude synaptic vesicles and it has been reported that increased synaptic activity results in increased secreted but decreased intracellular Aβ levels. Therefore, we considered whether Aβ could be produced in synaptic vesicles and/or released through the same mechanisms as neurotransmitters in synaptic vesicle exocytosis. Small amounts of Aβ were found to be produced in pure synaptic vesicle preparations. We also studied the release of glutamate and Aβ from rat cortical nerve terminals (synaptosomes). We found that large amounts of Aβ were secreted from non-stimulated synaptosomes, from which glutamate was not released. On the contrary, we could not detect any differences in Aβ release between non-stimulated synaptosomes and synaptosomes stimulated with KCl or 4-aminopyridine, whereas glutamate release was readily inducible in this system. To conclude, our results indicate that the major release mechanism of Aβ from isolated nerve terminals differs from the synaptic release of glutamate and that the activity-dependent increase of secreted Aβ, reported by several groups using intact cells, is likely dependent on post-synaptic events, trafficking and/or protein synthesis mechanisms.
    Neuroscience Letters 03/2014; DOI:10.1016/j.neulet.2014.02.050 · 2.06 Impact Factor
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    ABSTRACT: The enzyme complex γ-secretase generates amyloid β-peptide (Aβ), a 37–43-residue peptide associated with Alzheimer disease (AD). Mutations in presenilin 1 (PS1), the catalytical subunit of γ-secretase, result in familial AD (FAD). A unifying theme among FAD mutations is an alteration in the ratio Aβ species produced (the Aβ42/Aβ40 ratio), but the molecular mechanisms responsible remain elusive. In this report we have studied the impact of several different PS1 FAD mutations on the integration of selected PS1 transmembrane domains and on PS1 active site conformation, and whether any effects translate to a particular amyloid precursor protein (APP) processing phenotype. Most mutations studied caused an increase in the Aβ42/Aβ40 ratio, but via different mechanisms. The mutations that caused a particular large increase in the Aβ42/Aβ40 ratio did also display an impaired APP intracellular domain (AICD) formation and a lower total Aβ production. Interestingly, seven mutations close to the catalytic site caused a severely impaired integration of proximal transmembrane/hydrophobic sequences into the membrane. This structural defect did not correlate to a particular APP processing phenotype. Six selected FAD mutations, all of which exhibited different APP processing profiles and impact on PS1 transmembrane domain integration, were found to display an altered active site conformation. Combined, our data suggest that FAD mutations affect the PS1 structure and active site differently, resulting in several complex APP processing phenotypes, where the most aggressive mutations in terms of increased Aβ42/Aβ40 ratio are associated with a decrease in total γ-secretase activity.
    01/2014; 4. DOI:10.1016/j.fob.2014.04.006
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    Sophia Schedin-Weiss, Bengt Winblad, Lars O Tjernberg
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    ABSTRACT: Glycosylation is one of the most common, and the most complex, forms of post-translational modification of proteins. This review serves to highlight the role of protein glycosylation in Alzheimer disease (AD), a topic that has not been thoroughly investigated, although glycosylation defects have been observed in AD patients. The major pathological hallmarks in AD are neurofibrillary tangles and amyloid plaques. Neurofibrillary tangles are composed of phosphorylated tau, and the plaques are composed of amyloid β-peptide (Aβ), which is generated from amyloid precursor protein (APP). Defects in glycosylation of APP, tau and other proteins have been reported in AD. Another interesting observation is that the two proteases required for the generation of amyloid β-peptide (Aβ), i.e. γ-secretase and β-secretase, also have roles in protein glycosylation. For instance, γ-secretase and β-secretase affect the extent of complex N-glycosylation and sialylation of APP, respectively. These processes may be important in AD pathogenesis, as proper intracellular sorting, processing and export of APP are affected by how it is glycosylated. Furthermore, lack of one of the key components of γ-secretase, presenilin, leads to defective glycosylation of many additional proteins that are related to AD pathogenesis and/or neuronal function, including nicastrin, reelin, butyrylcholinesterase, cholinesterase, neural cell adhesion molecule, v-ATPase, and tyrosine-related kinase B. Improved understanding of the effects of AD on protein glycosylation, and vice versa, may therefore be important for improving the diagnosis and treatment of AD patients.
    FEBS Journal 10/2013; 281(1). DOI:10.1111/febs.12590 · 3.99 Impact Factor
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    ABSTRACT: Here, we present a highly sensitive method to study protein-protein interactions and subcellular location selectively for active multicomponent enzymes. We apply the method on γ-secretase, the enzyme complex that catalyzes the cleavage of the amyloid precursor protein (APP) to generate amyloid β-peptide (Aβ), the major causative agent in Alzheimer disease (AD). The novel assay is based on proximity ligation, which can be used to study protein interactions in situ with very high sensitivity. In traditional proximity ligation assay (PLA), primary antibody recognition is typically accompanied by oligonucleotide-conjugated secondary antibodies as detection probes. Here, we first performed PLA experiments using antibodies against the γ-secretase components presenilin 1 (PS1), containing the catalytic site residues, and nicastrin, suggested to be involved in substrate recognition. To selectively study the interactions of active γ-secretase, we replaced one of the primary antibodies with a photoreactive γ-secretase inhibitor containing a PEG linker and a biotin group (GTB), and used oligonucleotide-conjugated streptavidin as a probe. Interestingly, significantly fewer interactions were detected with the latter, novel, assay, which is a reasonable finding considering that a substantial portion of PS1 is inactive. In addition, the PLA signals were located more peripherally when GTB was used instead of a PS1 antibody, suggesting that γ-secretase matures distal from the perinuclear ER region. This novel technique thus enables highly sensitive protein interaction studies, determines the subcellular location of the interactions, and differentiates between active and inactive γ-secretase in intact cells. We suggest that similar PLA assays using enzyme inhibitors could be useful also for other enzyme interaction studies.
    PLoS ONE 05/2013; 8(5):e63962. DOI:10.1371/journal.pone.0063962 · 3.53 Impact Factor
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    ABSTRACT: [This corrects the article on p. e55847 in vol. 8.].
    PLoS ONE 05/2013; 8(5). DOI:10.1371/annotation/b4d7aee1-6578-4e73-b15d-67aef9b3da8c · 3.53 Impact Factor
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    ABSTRACT: The amyloid-cascade hypothesis posits that the role of amyloid β-peptide (Aβ) 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 Aβ are produced, of which the 40 and 42 residue variants (Aβ40 and Aβ42) are the most common. Aβ42 has a strong tendency to form neurotoxic aggregates and is involved in AD pathogenesis. Longer Aβ isoforms, like the less studied Aβ43, are gaining attention for their higher propensity to aggregate into neurotoxic oligomers. To further investigate Aβ43 in AD, we conducted a quantitative study on Aβ43 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 Aβ43, as well as Aβ40 and Aβ42, were quantified using ELISA. We compared quantitative data showing Aβ levels in occipital and frontal cortex from sporadic (SAD) and familial (FAD) AD cases, as well as non-demented (ND) controls. Results showed Aβ43 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. Aβ42 and Aβ43 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 Aβ43 in human brain are low compared to Aβ40 and Aβ42, we suggest that Aβ43 could initiate the formation of oligomers and amyloid plaques and thereby be crucial to AD pathogenesis.
    PLoS ONE 02/2013; 8(2):e55847. DOI:10.1371/journal.pone.0055847 · 3.53 Impact Factor
  • Molecular Neurodegeneration 01/2013; 8(Suppl 1):P69. DOI:10.1186/1750-1326-8-S1-P69 · 5.29 Impact Factor
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    ABSTRACT: The transmembrane protease complex γ-secretase is responsible for the generation of the neurotoxic amyloid β-peptide (Aβ) from its precursor (APP). Aβ has a causative role in Alzheimer disease, and thus, γ-secretase is a therapeutic target. However, since there are more than 70 γ-secretase substrates besides APP, selective inhibition of APP processing is required. Recent data indicates the existence of several γ-secretase associated proteins (GSAPs) that affect the selection and processing of substrates. Here, we use a γ-secretase inhibitor for affinity purification of γ-secretase and associated proteins from microsomes and detergent resistant membranes (DRMs) prepared from rat or human brain. By tandem mass spectrometry we identified a novel brain GSAP; erlin-2. This protein was recently reported to reside in DRMs in the ER. A proximity ligation assay, as well as co-immunoprecipitation, confirmed the association of erlin-2 with γ-secretase. We found that a higher proportion of erlin-2 was associated with γ-secretase in DRMs than in soluble membranes. siRNA experiments indicated that reduced levels of erlin-2 resulted in a decreased Aβ production, whereas the effect on Notch processing was limited. In summary, we have found a novel brain GSAP, erlin-2, that resides in DRMs and affects Aβ production.
    Biochemical and Biophysical Research Communications 07/2012; 424(3):476-81. DOI:10.1016/j.bbrc.2012.06.137 · 2.28 Impact Factor
  • Alzheimer's and Dementia 07/2012; 8(4):P655. DOI:10.1016/j.jalz.2012.05.1761 · 17.47 Impact Factor
  • Alzheimer's and Dementia 07/2012; 8(4):P311. DOI:10.1016/j.jalz.2012.05.2150 · 17.47 Impact Factor
  • Alzheimer's and Dementia 07/2012; 8(4):P310. DOI:10.1016/j.jalz.2012.05.2147 · 17.47 Impact Factor
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    ABSTRACT: 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 β-peptide (Aβ), which is formed from its precursor protein by two sequential cleavages mediated by β- and γ-secretase. We have earlier shown that γ-secretase activity is enriched in synaptic compartments, suggesting that the synaptotoxic Aβ is produced locally. Proteins that interact with γ-secretase at the synapse and regulate the production of Aβ 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 Aβ levels. Silencing of NADH dehydrogenase [ubiquinone] iron-sulfur protein 7 (NDUFS7) resulted in a decrease in Aβ levels whereas silencing of tubulin polymerization promoting protein (TPPP) resulted in an increase in Aβ 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 Aβ levels without affecting Notch cleavage.
    Neurochemistry International 04/2012; 61(1):108-18. DOI:10.1016/j.neuint.2012.03.016 · 2.65 Impact Factor
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    ABSTRACT: In Alzheimer disease, oligomeric amyloid β-peptide (Aβ) species lead to synapse loss and neuronal death. γ-Secretase, the transmembrane protease complex that mediates the final catalytic step that liberates Aβ from its precursor protein (APP), has a multitude of substrates, and therapeutics aimed at reducing Aβ production should ideally be specific for APP cleavage. It has been shown that APP can be processed in lipid rafts, and γ-secretase-associated proteins can affect Aβ production. Here, we use a biotinylated inhibitor for affinity purification of γ-secretase and associated proteins and mass spectrometry for identification of the purified proteins, and we identify novel γ-secretase-associated proteins in detergent-resistant membranes from brain. Furthermore, we show by small interfering RNA-mediated knockdown of gene expression that a subset of the γ-secretase-associated proteins, in particular voltage-dependent anion channel 1 (VDAC1) and contactin-associated protein 1 (CNTNAP1), reduced Aβ production (Aβ40 and Aβ42) by around 70%, whereas knockdown of presenilin 1, one of the essential γ-secretase complex components, reduced Aβ production by 50%. Importantly, these proteins had a less pronounced effect on Notch processing. We conclude that VDAC1 and CNTNAP1 associate with γ-secretase in detergent-resistant membranes and affect APP processing and suggest that molecules that interfere with this interaction could be of therapeutic use for Alzheimer disease.
    Journal of Biological Chemistry 02/2012; 287(15):11991-2005. DOI:10.1074/jbc.M111.246074 · 4.60 Impact Factor
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    ABSTRACT: Several lines of evidence suggest that aggregation of the amyloid β-peptide (Aβ) in the brain is a trigger of Alzheimer's disease (AD). Thus, quantification of Aβ in several different types of samples from brain is fundamental for understanding AD pathogenesis. For analysis of the low levels of Aβ present in microdissected neurons, a more sensitive system than the ELISAs used today would be helpful. Here, we report a novel immuno-PCR (IPCR) system in which the lowest quantitative level of Aβ₁₋₄₀ is 2 attomol/μL. We use the novel IPCR to quantify the intracellular Aβ₁₋₄₀ levels in pyramidal neurons microdissected from human brain. We show that the level of Aβ₁₋₄₀ is around 10 attomol/neuron, and thus, only 3 neurons are needed for analysis.
    Journal of neuroscience methods 02/2012; 205(2):364-7. DOI:10.1016/j.jneumeth.2012.01.015 · 1.96 Impact Factor
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    ABSTRACT: It is evident that the symptoms of Alzheimer's disease (AD) are derived from severe neuronal damage, and especially pyramidal neurons in the hippocampus are affected pathologically. Here, we analysed the proteome of hippocampal neurons, isolated from post‐mortem brains by laser capture microdissection. By using 18O labelling and mass spectrometry, the relative expression levels of 150 proteins in AD and controls were estimated. Many of the identified proteins are involved in transcription and nucleotide binding, glycolysis, heat‐shock response, microtubule stabilization, axonal transport or inflammation. The proteins showing the most altered expression in AD were selected for immunohistochemical analysis. These analyses confirmed the altered expression levels, and showed in many AD cases a pathological pattern. For comparison, we also analysed hippocampal sections by Western blot. The expression levels found by this method showed poor correlation with the neuron‐specific analysis. Hence, we conclude that cell‐specific proteome analysis reveals differences in the proteome that cannot be detected by bulk analysis.
    Journal of Cellular and Molecular Medicine 01/2012; 16(8). · 3.70 Impact Factor

Publication Stats

3k Citations
608.39 Total Impact Points


  • 1994–2014
    • Karolinska Institutet
      • • KI Alzheimer's Disease Research Center - ADRC
      • • Department of Cell and Molecular Biology
      • • Department of Medical Biochemistry and Biophysics
      • • Institutionen för klinisk neurovetenskap
      Solna, Stockholm, Sweden
  • 2012
    • Memorial Sloan-Kettering Cancer Center
      New York City, New York, United States
  • 2010
    • Dainippon Sumitomo Pharma Co., Ltd.
      Ōsaka, Ōsaka, Japan
  • 2004
    • University of Chicago
      Chicago, Illinois, United States
  • 1999
    • Karolinska University Hospital
      • Center for Molecular Medicine (CMM)
      Tukholma, Stockholm, Sweden