Proteogenomic profiling uncovers differential therapeutic vulnerabilities between TCF3::PBX1 and TCF3::HLF translocated B-cell acute lymphoblastic leukemia.

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Integrative multi-omics reveals two biologically distinct groups of pilocytic astrocytoma.

Pilocytic astrocytoma (PA), the most common pediatric brain tumor, is driven by aberrant mitogen-activated protein kinase signaling most commonly caused by BRAF gene fusions or activating mutations. While 5-year overall survival rates exceed 95%, tumor recurrence or progression constitutes a major clinical challenge in incompletely resected tumors. Here, we used similarity network fusion (SNF) analysis in an integrative multi-omics approach employing RNA transcriptomic and mass spectrometry-based proteomic profiling to molecularly characterize PA tissue samples from 62 patients. Thereby, we uncovered that PAs segregated into two molecularly distinct groups, namely, Group 1 and Group 2, which were validated in three non-overlapping cohorts. Patients with Group 1 tumors were significantly younger and showed worse progression-free survival compared to patients with group 2 tumors. Ingenuity pathways analysis (IPA) and gene set enrichment analysis (GSEA) revealed that Group 1 tumors were enriched for immune response pathways, such as interferon signaling, while Group 2 tumors showed enrichment for action potential and neurotransmitter signaling pathways. Analysis of immune cell-related gene signatures showed an enrichment of infiltrating T Cells in Group 1 versus Group 2 tumors. Taken together, integrative multi-omics of PA identified biologically distinct and prognostically relevant tumor groups that may improve risk stratification of this single pathway driven tumor type.

Tacedinaline (CI-994), a class I HDAC inhibitor, targets intrinsic tumor growth and leptomeningeal dissemination in MYC-driven medulloblastoma while making them susceptible to anti-CD47-induced macrophage phagocytosis via NF-kB-TGM2 driven tumor inflammation.

BACKGROUND: While major advances have been made in improving the quality of life and survival of children with most forms of medulloblastoma (MB), those with MYC-driven tumors (Grp3-MB) still suffer significant morbidity and mortality. There is an urgent need to explore multimodal therapeutic regimens which are effective and safe for children. Large-scale studies have revealed abnormal cancer epigenomes caused by mutations and structural alterations of chromatin modifiers, aberrant DNA methylation, and histone modification signatures. Therefore, targeting epigenetic modifiers for cancer treatment has gained increasing interest, and inhibitors for various epigenetic modulators have been intensively studied in clinical trials. Here, we report a cross-entity, epigenetic drug screen to evaluate therapeutic vulnerabilities in MYC amplified MB, which sensitizes them to macrophage-mediated phagocytosis by targeting the CD47-signal regulatory protein α (SIRPα) innate checkpoint pathway. METHODS: We performed a primary screen including 78 epigenetic inhibitors and a secondary screen including 20 histone deacetylase inhibitors (HDACi) to compare response profiles in atypical teratoid/rhabdoid tumor (AT/RT, n=11), MB (n=14), and glioblastoma (n=14). This unbiased approach revealed the preferential activity of HDACi in MYC-driven MB. Importantly, the class I selective HDACi, CI-994, showed significant cell viability reduction mediated by induction of apoptosis in MYC-driven MB, with little-to-no activity in non-MYC-driven MB, AT/RT, and glioblastoma in vitro. We tested the combinatorial effect of targeting class I HDACs and the CD47-SIRPa phagocytosis checkpoint pathway using in vitro phagocytosis assays and in vivo orthotopic xenograft models. RESULTS: CI-994 displayed antitumoral effects at the primary site and the metastatic compartment in two orthotopic mouse models of MYC-driven MB. Furthermore, RNA sequencing revealed nuclear factor-kB (NF-κB) pathway induction as a response to CI-994 treatment, followed by transglutaminase 2 (TGM2) expression, which enhanced inflammatory cytokine secretion. We further show interferon-γ release and cell surface expression of engulfment ('eat-me') signals (such as calreticulin). Finally, combining CI-994 treatment with an anti-CD47 mAb targeting the CD47-SIRPα phagocytosis checkpoint enhanced in vitro phagocytosis and survival in tumor-bearing mice. CONCLUSION: Together, these findings suggest a dynamic relationship between MYC amplification and innate immune suppression in MYC amplified MB and support further investigation of phagocytosis modulation as a strategy to enhance cancer immunotherapy responses.

Three-dimensional analyses of vascular network morphology in a murine lymph node by X-ray phase-contrast tomography with a 2D Talbot array.

With growing molecular evidence for correlations between spatial arrangement of blood vasculature and fundamental immunological functions, carried out in distinct compartments of the subdivided lymph node, there is an urgent need for three-dimensional models that can link these aspects. We reconstructed such models at a 1.84 µm resolution by the means of X-ray phase-contrast imaging with a 2D Talbot array in a short time without any staining. In addition reconstructions are verified in immunohistochemistry staining as well as in ultrastructural analyses. While conventional illustrations of mammalian lymph nodes depict the hilus as a definite point of blood and lymphatic vessel entry and exit, our method revealed that multiple branches enter and emerge from an area that extends up to one third of the organ's surface. This could be a prerequisite for the drastic and location-dependent remodeling of vascularization, which is necessary for lymph node expansion during inflammation. Contrary to corrosion cast studies we identified B-cell follicles exhibiting a two times denser capillary network than the deep cortical units of the T-cell zone. In addition to our observation of high endothelial venules spatially surrounding the follicles, this suggests a direct connection between morphology and B-cell homing. Our findings will deepen the understanding of functional lymph node composition and lymphocyte migration on a fundamental basis.

Primary cilia contribute to the aggressiveness of atypical teratoid/rhabdoid tumors.

Atypical teratoid/rhabdoid tumor (AT/RT) is a highly malignant brain tumor in infants that is characterized by loss of nuclear expression of SMARCB1 or SMARCA4 proteins. Recent studies show that AT/RTs comprise three molecular subgroups, namely AT/RT-TYR, AT/RT-MYC and AT/RT-SHH. The subgroups show distinct expression patterns of genes involved in ciliogenesis, however, little is known about the functional roles of primary cilia in the biology of AT/RT. Here, we show that primary cilia are present across all AT/RT subgroups with specific enrichment in AT/RT-TYR patient samples. Furthermore, we demonstrate that primary ciliogenesis contributes to AT/RT biology in vitro and in vivo. Specifically, we observed a significant decrease in proliferation and clonogenicity following disruption of primary ciliogenesis in AT/RT cell line models. Additionally, apoptosis was significantly increased via the induction of STAT1 and DR5 signaling, as detected by proteogenomic profiling. In a Drosophila model of SMARCB1 deficiency, concomitant knockdown of several cilia-associated genes resulted in a substantial shift of the lethal phenotype with more than 20% of flies reaching adulthood. We also found significantly extended survival in an orthotopic xenograft mouse model of AT/RT upon disruption of primary ciliogenesis. Taken together, our findings indicate that primary ciliogenesis or its downstream signaling contributes to the aggressiveness of AT/RT and, therefore, may constitute a novel therapeutic target.

The HHIP-AS1 lncRNA promotes tumorigenicity through stabilization of dynein complex 1 in human SHH-driven tumors.

Most lncRNAs display species-specific expression patterns suggesting that animal models of cancer may only incompletely recapitulate the regulatory crosstalk between lncRNAs and oncogenic pathways in humans. Among these pathways, Sonic Hedgehog (SHH) signaling is aberrantly activated in several human cancer entities. We unravel that aberrant expression of the primate-specific lncRNA HedgeHog Interacting Protein-AntiSense 1 (HHIP-AS1) is a hallmark of SHH-driven tumors including medulloblastoma and atypical teratoid/rhabdoid tumors. HHIP-AS1 is actively transcribed from a bidirectional promoter shared with SHH regulator HHIP. Knockdown of HHIP-AS1 induces mitotic spindle deregulation impairing tumorigenicity in vitro and in vivo. Mechanistically, HHIP-AS1 binds directly to the mRNA of cytoplasmic dynein 1 intermediate chain 2 (DYNC1I2) and attenuates its degradation by hsa-miR-425-5p. We uncover that neither HHIP-AS1 nor the corresponding regulatory element in DYNC1I2 are evolutionary conserved in mice. Taken together, we discover an lncRNA-mediated mechanism that enables the pro-mitotic effects of SHH pathway activation in human tumors.

Intratumoral heterogeneity of MYC drives medulloblastoma metastasis and angiogenesis.

BACKGROUND: Intratumoral heterogeneity is crucially involved in metastasis, resistance to therapy, and cancer relapse. Amplifications of the proto-oncogene MYC display notable heterogeneity at the single-cell level and are associated with a particularly dismal prognosis in high-risk medulloblastomas (MBs). The aim of this study was to establish the relevance of interclonal cross-talk between MYC-driven and non-MYC-driven MB cells. METHODS: We used fluorescence in situ hybridization, single-cell transcriptomics, and immunohistochemistry, in vitro isogenic cell models, non-targeted proteomics, mass spectrometry-based metabolite quantification, HUVECs tube formation assay, and orthotopic in vivo experiments to investigate interclonal cross-talk in MB. RESULTS: We found that the release of lactate dehydrogenase A (LDHA) from MYC-driven cells facilitates metastatic seeding and outgrowth, while secretion of dickkopf WNT signaling pathway inhibitor 3 from non-MYC-driven cells promotes tumor angiogenesis. This tumor-supporting interaction between both subclones was abrogated by targeting the secretome through pharmacological and genetic inhibition of LDHA, which significantly suppressed tumor cell migration. CONCLUSION: Our study reveals the functional relevance of clonal diversity and highlights the therapeutic potential of targeting the secretome to interrupt interclonal communication and progression in high-risk MB.

The long non-coding RNA HOTAIRM1 promotes tumor aggressiveness and radiotherapy resistance in glioblastoma.

Glioblastoma is the most common malignant primary brain tumor. To date, clinically relevant biomarkers are restricted to isocitrate dehydrogenase (IDH) gene 1 or 2 mutations and O6-methylguanine DNA methyltransferase (MGMT) promoter methylation. Long non-coding RNAs (lncRNAs) have been shown to contribute to glioblastoma pathogenesis and could potentially serve as novel biomarkers. The clinical significance of HOXA Transcript Antisense RNA, Myeloid-Specific 1 (HOTAIRM1) was determined by analyzing HOTAIRM1 in multiple glioblastoma gene expression data sets for associations with prognosis, as well as, IDH mutation and MGMT promoter methylation status. Finally, the role of HOTAIRM1 in glioblastoma biology and radiotherapy resistance was characterized in vitro and in vivo. We identified HOTAIRM1 as a candidate lncRNA whose up-regulation is significantly associated with shorter survival of glioblastoma patients, independent from IDH mutation and MGMT promoter methylation. Glioblastoma cell line models uniformly showed reduced cell viability, decreased invasive growth and diminished colony formation capacity upon HOTAIRM1 down-regulation. Integrated proteogenomic analyses revealed impaired mitochondrial function and determination of reactive oxygen species (ROS) levels confirmed increased ROS levels upon HOTAIRM1 knock-down. HOTAIRM1 knock-down decreased expression of transglutaminase 2 (TGM2), a candidate protein implicated in mitochondrial function, and knock-down of TGM2 mimicked the phenotype of HOTAIRM1 down-regulation in glioblastoma cells. Moreover, HOTAIRM1 modulates radiosensitivity of glioblastoma cells both in vitro and in vivo. Our data support a role for HOTAIRM1 as a driver of biological aggressiveness, radioresistance and poor outcome in glioblastoma. Targeting HOTAIRM1 may be a promising new therapeutic approach.

Aberrant ERBB4-SRC Signaling as a Hallmark of Group 4 Medulloblastoma Revealed by Integrative Phosphoproteomic Profiling.

The current consensus recognizes four main medulloblastoma subgroups (wingless, Sonic hedgehog, group 3 and group 4). While medulloblastoma subgroups have been characterized extensively at the (epi-)genomic and transcriptomic levels, the proteome and phosphoproteome landscape remain to be comprehensively elucidated. Using quantitative (phospho)-proteomics in primary human medulloblastomas, we unravel distinct posttranscriptional regulation leading to highly divergent oncogenic signaling and kinase activity profiles in groups 3 and 4 medulloblastomas. Specifically, proteomic and phosphoproteomic analyses identify aberrant ERBB4-SRC signaling in group 4. Hence, enforced expression of an activated SRC combined with p53 inactivation induces murine tumors that resemble group 4 medulloblastoma. Therefore, our integrative proteogenomics approach unveils an oncogenic pathway and potential therapeutic vulnerability in the most common medulloblastoma subgroup.

Methylphenidate enhances neuronal differentiation and reduces proliferation concomitant to activation of Wnt signal transduction pathways.

Methylphenidate (Ritalin) is the most commonly prescribed drug in the treatment of attention-deficit hyperactivity disorder. It is suggested that in vivo, methylphenidate treatment supports cortical maturation, however, the molecular and cellular mechanisms are not well understood. This study aimed to explore the potential effect of methylphenidate on cell proliferation and maturation in various cellular models, hypothesizing its interaction with the Wnt-signaling. The termination of cell proliferation concomitant to neuronal maturation following methylphenidate treatment was observed in all of the cell-models tested: murine neural stem-, rat PC12- and the human SH-SY5Y-cells. Inhibition of Wnt-signaling in SH-SY5Y cells with Dkk1 30 min before methylphenidate treatment suppressed neuronal differentiation but enhanced proliferation. The possible involvement of the dopamine-transporter in cell differentiation was discounted following the observation of opposing results after GBR-12909 treatment. Moreover, Wnt-activation via methylphenidate was confirmed in Wnt-luciferase-reporter assay. These findings reveal a new mechanism of action of methylphenidate that might explain long-term effects.

Congenital embryonal rhabdomyosarcoma caused by heterozygous concomitant PTCH1 and PTCH2 germline mutations.

The sonic hedgehog (SHH) signaling pathway has been shown to play important roles in embryogenesis, cell proliferation as well as in cell differentiation. It is aberrantly activated in various common cancers in adults, but also in pediatric neoplasms, such as rhabdomyosarcoma (RMS) and atypical teratoid/rhabdoid tumors (AT/RTs). Dysregulation and germline mutation in PATCHED1 (PTCH1), a receptor for SHH, is responsible for the Gorlin Syndrome, a familial cancer predisposing syndrome including RMS. Here, we report a newborn diagnosed with congenital embryonal RMS. Whole-exome sequencing (WES) identified the presence of two heterozygous germline mutations in two target genes of the SHH signaling pathway. The PTCH1 mutation p.(Gly38Glu) is inherited from the mother, whereas the PTCH2 p.(His622Tyr) mutation is transmitted from the father. Quantitative RT-PCR expression analysis of GLI and SMO, key players of the SHH pathway, showed significantly increase in the tumor tissue of the patient and also enrichment in the germline sample in comparison to the parents indicating activation of the SHH pathway in the patient. These findings demonstrate that SHH pathway activity seems to play a role in eRMS as evidenced by high expression levels of GLI1 RNA transcripts. We speculate that PTCH2 modulates tumorigenesis linked to the PTCH1 mutation and is likely associated with the congenital onset of the RMS observed in our patient.

The impact of methylphenidate and its enantiomers on dopamine synthesis and metabolism in vitro.

Methylphenidate (MPH), a psychostimulant, is an effective first-line treatment for the symptoms associated with Attention-Deficit/Hyperactivity Disorder (ADHD). Although most MPH formulations are composed of the racemic 1:1 mixture of the two enantiomers (d- and l-threo), converging lines of evidence indicate that d-threo MPH seems to be superior to the l-isomer. We aimed to investigate whether MPH racemic mixture or pure enantiomers influence the enzyme activity of tyrosine hydroxylase (TH), monoamine oxidase B (MAO-B), catechol-O-methyltransferase (COMT), and aldehyde dehydrogenase (ALDH) in vitro in homogenates of rat PC12 cells incubated with racemic, d- and l-threo MPH (1nM up to 100µM), or a vehicle for control. We could observe dose dependent enhancement of TH activity with d-threo MPH, probably due to its higher affinity to the enzyme, which we could confirm for d-threo versus l-threo MPH via docking and molecular dynamic simulations analysis. MAO-B enzyme activity was found to be enhanced when incubated with both d- and l-isomers but not with the racemic mixture. This conflicting result was hypothesized to be due to possible aggregation of the two enantiomers or other molecular conformations. Such a possible interaction was observed indirectly, when TH was incubated with constant d-threo MPH while increasing l-isomer (increasing total MPH concentrations). Hence, TH activity was slightly decreased with increased l-isomer. In conclusion, the current in vitro investigation points to the stereoselectivity of the investigated enzymes and pharmacological effects of MPH enantiomers.

Characterization of cognitive deficits in spontaneously hypertensive rats, accompanied by brain insulin receptor dysfunction.

BACKGROUND: The spontaneously hypertensive rat (SHR) has been used to model changes in the central nervous system associated with cognitive-related disorders. Recent human and animal studies indicate a possible relationship between cognitive deficits, insulin resistance and hypertension. We aimed to investigate whether cognitively impaired SHRs develop central and/or peripheral insulin resistance and how their cognitive performance is influenced by the animal's sex and age as well as strains used for comparison (Wistar and Wistar-Kyoto/WKY). METHODS: Three and seven-month-old SHR, Wistar, and WKY rats were studied for their cognitive performance using Morris Water Maze (MWM) and Passive Avoidance tests (PAT). Plasma glucose and insulin were obtained after oral glucose tolerance tests. Cerebral cortex, hippocampus, and striatum status of insulin-receptor (IR) ß-subunit and glycogen synthase kinase-3ß (GSK3ß) and their phosphorylated forms were obtained via ELISA. RESULTS: SHRs performed poorly in MWM and PAT in comparison to both control strains but more pronouncedly compared to WKY. Females performed poorer than males and 7-month-old SHRs had poorer MWM performance than 3-month-old ones. Although plasma glucose levels remained unchanged, plasma insulin levels were significantly increased in the glucose tolerance test in 7-month-old SHRs. SHRs demonstrated reduced expression and increased activity of IRß-subunit in cerebral cortex, hippocampus, and striatum with different regional changes in phospho/total GSK3ß ratio, as compared to WKYs. CONCLUSION: Results indicate that cognitive deficits in SHRs are accompanied by both central and peripheral insulin dysfunction, thus allowing for the speculation that SHRs might additionally be considered as a model of insulin resistance-induced type of dementia.

Further evidence for plasma progranulin as a biomarker in bipolar disorder.

BACKGROUND: A recent study suggested that progranulin (encoded by the fronto-temporal dementia risk gene GRN) plasma levels are decreased in bipolar disorder (BD). Replication of this finding is however lacking. METHODS: Progranulin plasma levels of bipolar patients (n=104) and healthy controls (n=80) were measured by enzyme-linked immunosorbent assay (ELISA). Participants were also genotyped for three single nucleotide polymorphisms (SNPs) in the GRN gene (rs2879096, rs4792938 and rs5848), and the effect of genetic variation on progranulin levels was examined. RESULTS: Plasma progranulin levels were decreased in BD (ANCOVA, p=0.001). Furthermore, age was significantly and positively correlated with plasma progranulin (Pearson׳s correlation, r=0.269, p<0.001). Also, lithium treatment but no other medication had a significant effect on progranulin plasma levels (ANCOVA, p=0.007). Specifically in BD, the GRN SNP rs5848 was associated with progranulin plasma levels (Kruskal-Wallis test, p<0.005). LIMITATIONS: Subgroup analysis regarding bipolar I vs. bipolar II subtype and polarity of the episode at sampling (manic vs. depressed vs. mixed vs. rapid cycling vs. euthymic) could only be performed with limited validity due to the relatively small sample size. The suitability of peripheral progranulin as a biomarker for BD is limited due to the overlap between patients and controls. CONCLUSION: The findings strengthen the evidence for progranulin being involved in pathomechanisms of bipolar disorder, and suggest a genetic determinant of progranulin concentrations that is relevant specifically in bipolar patients.

Chronic monoamine oxidase-B inhibitor treatment blocks monoamine oxidase-A enzyme activity.

Patients with Parkinson's disease receive selective irreversible monoamine oxidase (MAO)-B inhibitors, but their effects on MAO-A activity are not known during long-term application. We determined MAO-A inhibition in plasma samples from patients with MAO-B inhibitor intake or without MAO-B inhibitor treatment and from healthy controls. We detected a 70 % reduction of MAO-A activity in patients with MAO-B inhibitor therapy in comparison to the other groups. Our results suggest that treatment with MAO-B inhibitor may also influence MAO-A activity in vivo, when administered daily.

Neuron-specific alterations in signal transduction pathways associated with Alzheimer's disease.

The hallmarks of sporadic Alzheimer's disease (AD) are extracellular amyloid deposits, intracellular neurofibrillary tangles (NFTs), and neuronal death. Hyperphosphorylation of tau is a key factor in the generation of NFTs. Mitogen activated protein kinase 1 (MAPK1) and protein kinase C beta (PRKCB) are thought to play a role in hyperphosphorylation, and PRCKB is thought to be involved in hypoxic stress and vascular dysfunction, and to trigger MAPK phosphorylation pathways. We performed single-cell analyses of neurons with different vulnerabilities to AD-related changes. Using quantitative PCR (qPCR), we measured the levels of MAPK1 and PRKCB transcript in CA1 (high vulnerability), CA2 pyramidal cells from the hippocampus, granule cells from the cerebellum (low vulnerability), and neurons from the brain stem (nucleus tractus spinalis nervi trigemini, characterized by early neurophysiological deficits) at progressive Braak stages compared to age-matched controls. The highly vulnerable CA1 pyramidal neurons were characterized by age- and disease-unrelated increases in PRCKB levels and by age- and disease-related increases in MAPK1 levels. In contrast, low PRKCB levels were found in CA2 pyramidal neurons, and MAPK1 levels were elevated in controls and intermediate AD stages. Both PRKCB and MAPK1 were increased in the late AD stages. MAPK1 and PRKCB levels were low in the brainstem and cerebellum. We propose that alterations in the expression of these two genes occur early in the pathogenesis of AD in a region-specific manner. In addition, multiple signal transduction pathways need to be affected to result in AD instead of physiological aging.

Neuron-specific mitochondrial DNA deletion levels in sporadic Alzheimer's disease.

Oxidative stress is implicated in the pathogenesis of neurodegenerative diseases, including sporadic Alzheimer´s disease (AD). Mitochondrial DNA (mtDNA) deletions are markers of oxidative damage and increase with age. To unravel the impact of mtDNA damage on AD development, we analyzed mtDNA deletion levels in diverse neuronal cell types of four brain regions (hippocampal CA1 and CA2 regions, nucleus tractus spinalis nervi trigemini, and the cerebellum) that exhibit differing levels of vulnerability to AD related changes at progressive Braak stages compared with age-matched controls. Neurons from these four brain regions were collected using laser microdissection, and analyzed using quantitative polymerase chain reaction (qPCR). Although, no correlation between mtDNA deletion levels and AD progression were found, the data revealed regional and cell type specific selective vulnerability towards mtDNA deletion levels. In conclusion, unexpected results were obtained as granule cells from the cerebellum and neurons from the nucleus tractus spinalis nervi trigemini of the brain stem displayed significant higher mtDNA deletion levels than pyramidal cells from hippocampal CA1 and CA2 region in age and AD.

Alzheimer's disease and type 2 diabetes: two diseases, one common link?

OBJECTIVES: Although Alzheimer's disease (AD) is the most common form of dementia in the elderly, its aetiology remains mostly unknown. A potential pathophysiological mechanism for AD arises from the knowledge that insulin is also synthesized independently in the central nervous system and is involved in the regulation of memory formation. AD may represent a brain-specific form of insulin resistance. METHODS: We used immunohistochemistry to investigate the numbers of cells expressing insulin receptor ß-subunit (IRß) and phosphorylated PPARγ (PPARγ(p)) in human post-mortem tissue from patients with AD; AD combined with type 2 diabetes mellitus (T2DM); just T2DM , and from aged-matched controls. These numbers were evaluated in frontal cortex and in dorsal/ventral parts of the hippocampus. RESULTS: We observed significantly lower numbers of IRß positive cells in AD cases compared to all other groups in all investigated brain regions. Also significantly more PPARγ(p) positive cells occurred in each patient group compared to control. CONCLUSIONS: T2DM and AD may not be directly linked, but may share common histological features including lower numbers of IRß positive cells and higher numbers of PPARγ(p) positive cells in all investigated brain regions. These observations may at least partially explain the increased frequency of AD in elderly diabetic patients.

Different effects of soluble and aggregated amyloid ß42 on gene/protein expression and enzyme activity involved in insulin and APP pathways.

Although Alzheimer's dementia (AD) is not characterised any longer simply as the accumulation and deposition of amyloid beta (Aß) peptides and hyperphosphorylation of tau proteins within the brain, excessive Aß(42) deposition is still considered to play a major role in this illness. Aß are able to adopt many differently aggregate forms, including amyloid fibrils as well as nonfibrillar structures (soluble Aß(42) oligomers). It is not well-established that which Aß(42) state is most responsible for AD or why. We wanted to verify which effects Aß(42) oligomers and aggregated peptides have on gene expression, protein level and enzyme activity of insulin and amyloid precursor protein (APP) pathways in vitro. Human neuroblastoma cells (SH-SY5Y) were treated with varying concentrations of soluble and aggregated Aß(42). Treatment effects on ß-secretase (BACE), glycogen synthase kinase 3α (GSK3α), glycogen synthase kinase 3ß (GSK3ß), phosphatidylinositol-3 kinase (PI-3K), insulin-degrading enzyme (IDE), insulin-receptor substrate 1 (IRS1), insulin receptor (INSR) and monoamine oxidase B (MAO-B) were investigated via quantitative-PCR, western blot, ELISA and enzyme activity assay. We could find different effects of soluble and aggregated peptides especially on gene/protein expression of GSK3ß and INSR and on GSK3ß and MAO-B activity. Soluble peptides showed significant effects leading to increased gene expression and protein amount of GSK3ß and to decreased level of gene and protein expression of INSR. MAO-B activity was enhanced after treatment with aggregated peptides and strongly inhibited after soluble Aß(42) treatment. Our data might provide insights into selective effects of specific forms of Aß(42) aggregates in AD.