Association of Complement Factors With Disability Progression in Primary Progressive Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: The complement system is known to play a role in multiple sclerosis (MS) pathogenesis. However, its contribution to disease progression remains elusive. The study investigated the role of the complement system in disability progression of patients with primary progressive MS (PPMS). METHODS: Sixty-eight patients with PPMS from 12 European MS centers were included in the study. Serum and CSF levels of a panel of complement components (CCs) were measured by multiplex enzyme-linked immunosorbent assay at a baseline time point (i.e., sampling). Mean (SD) follow-up time from baseline was 9.6 (4.8) years. Only one patient (1.5%) was treated during follow-up. Univariable and multivariable logistic regressions adjusted for age, sex, and albumin quotient were performed to assess the association between baseline CC levels and disability progression in short term (2 years), medium term (6 years), and long term (at the time of the last follow-up). RESULTS: In short term, CC played little or no role in disability progression. In medium term, an elevated serum C3a/C3 ratio was associated with a higher risk of disability progression (adjusted OR 2.30; 95% CI 1.17-6.03; p = 0.040). By contrast, increased CSF C1q levels were associated with a trend toward reduced risk of disability progression (adjusted OR 0.43; 95% CI 0.17-0.98; p = 0.054). Similarly, in long term, an elevated serum C3a/C3 ratio was associated with higher risk of disability progression (adjusted OR 1.81; 95% CI 1.09-3.40; p = 0.037), and increased CSF C1q levels predicted lower disability progression (adjusted OR 0.41; 95% CI 0.17-0.86; p = 0.025). DISCUSSION: Proteins involved in the activation of early complement cascades play a role in disability progression as risk (elevated serum C3a/C3 ratio) or protective (elevated CSF C1q) factors after 6 or more years of follow-up in patients with PPMS. The protective effects associated with C1q levels in CSF may be related to its neuroprotective and anti-inflammatory properties.

Periodic table screening for enhanced positive contrast in MRI and in vivo uptake in glioblastoma.

The quest for nanomaterial-based imaging probes that can provide positive contrast in MRI is fueled by the necessity of developing novel diagnostic applications with potential for clinical translation that current gold standard probes cannot provide. Although interest in nanomaterials for positive contrast has increased in recent years, their study is less developed than that of traditional negative contrast probes in MRI. In our search for new magnetic materials with enhanced features as positive contrast probes for MRI, we decided to explore the chemical space to comprehensively analyze the effects of different metals on the performance of iron oxide nanomaterials already able to provide positive contrast in MRI. To this end, we synthesized 30 different iron oxide-based nanomaterials. Thorough characterization was performed, including multivariate analysis, to study the effect of different variables on their relaxometric properties. Based on these results, we identified the best combination of metals for in vivo imaging and tested them in different experiments. First, we tested its performance on magnetic resonance angiography using a concentration ten times lower than that clinically approved for Gd. Finally, we studied the capability of these nanomaterials to cross the affected blood-brain barrier in a glioblastoma model. The results showed that the selected nanomaterials provided excellent positive contrast at large magnetic field and were able to accumulate at the tumor site, highlighting the affected tissue.

Integrative Single-Plaque Analysis Reveals Signature Aß and Lipid Profiles in the Alzheimer's Brain.

Cerebral accumulation of amyloid-ß (Aß) initiates molecular and cellular cascades that lead to Alzheimer's disease (AD). However, amyloid deposition does not invariably lead to dementia. Amyloid-positive but cognitively unaffected (AP-CU) individuals present widespread amyloid pathology, suggesting that molecular signatures more complex than the total amyloid burden are required to better differentiate AD from AP-CU cases. Motivated by the essential role of Aß and the key lipid involvement in AD pathogenesis, we applied multimodal mass spectrometry imaging (MSI) and machine learning (ML) to investigate amyloid plaque heterogeneity, regarding Aß and lipid composition, in AP-CU versus AD brain samples at the single-plaque level. Instead of focusing on a population mean, our analytical approach allowed the investigation of large populations of plaques at the single-plaque level. We found that different (sub)populations of amyloid plaques, differing in Aß and lipid composition, coexist in the brain samples studied. The integration of MSI data with ML-based feature extraction further revealed that plaque-associated gangliosides GM2 and GM1, as well as Aß1-38, but not Aß1-42, are relevant differentiators between the investigated pathologies. The pinpointed differences may guide further fundamental research investigating the role of amyloid plaque heterogeneity in AD pathogenesis/progression and may provide molecular clues for further development of emerging immunotherapies to effectively target toxic amyloid assemblies in AD therapy. Our study exemplifies how an integrative analytical strategy facilitates the unraveling of complex biochemical phenomena, advancing our understanding of AD from an analytical perspective and offering potential avenues for the refinement of diagnostic tools.

Apo and Aß46-bound γ-secretase structures provide insights into amyloid-ß processing by the APH-1B isoform.

Deposition of amyloid-ß (Aß) peptides in the brain is a hallmark of Alzheimer's disease. Aßs are generated through sequential proteolysis of the amyloid precursor protein by the γ-secretase complexes (GSECs). Aß peptide length, modulated by the Presenilin (PSEN) and APH-1 subunits of GSEC, is critical for Alzheimer's pathogenesis. Despite high relevance, mechanistic understanding of the proteolysis of Aß, and its modulation by APH-1, remain incomplete. Here, we report cryo-EM structures of human GSEC (PSEN1/APH-1B) reconstituted into lipid nanodiscs in apo form and in complex with the intermediate Aß46 substrate without cross-linking. We find that three non-conserved and structurally divergent APH-1 regions establish contacts with PSEN1, and that substrate-binding induces concerted rearrangements in one of the identified PSEN1/APH-1 interfaces, providing structural basis for APH-1 allosteric-like effects. In addition, the GSEC-Aß46 structure reveals an interaction between Aß46 and loop 1PSEN1, and identifies three other H-bonding interactions that, according to functional validation, are required for substrate recognition and efficient sequential catalysis.

Dysregulated Lipid Metabolism Networks Modulate T-cell Function in People with Relapsing Remitting Multiple Sclerosis.

Altered cholesterol, oxysterol, sphingolipid, and fatty acid concentrations are reported in blood, cerebrospinal fluid, and brain tissue of people with relapsing remitting multiple sclerosis (RRMS) and are linked to disease progression and treatment responses. CD4+ T cells are pathogenic in RRMS, and defective T cell function could be mediated in part by liver X receptors (LXRs) - nuclear receptors that regulate lipid homeostasis and immunity. RNA-sequencing and pathway analysis identified that genes within the 'lipid metabolism' and 'signalling of nuclear receptors' pathways were dysregulated in CD4+ T cells isolated from RRMS patients compared with healthy donors. While LXRB and genes associated with cholesterol metabolism were upregulated, other T cell LXR-target genes, including genes involved in cellular lipid uptake (inducible degrader of the LDL receptor, IDOL), and the rate-limiting enzyme for glycosphingolipid biosynthesis (UDP-glucosylceramide synthase, UGCG) were downregulated in T cells from patients with RRMS compared to healthy donors. Correspondingly, plasma membrane glycosphingolipids were reduced, and cholesterol levels increased in RRMS CD4+ T cells, an effect partially recapitulated in healthy T cells by in vitro culture with T cell receptor stimulation in the presence of serum from RRMS patients. Notably, stimulation with LXR-agonist GW3965 normalised membrane cholesterol levels, and reduced proliferation and IL17 cytokine production in RRMS CD4+ T-cells. Thus, LXR-mediated lipid metabolism pathways were dysregulated in T cells from patients with RRMS and could contribute to RRMS pathogenesis. Therapies that modify lipid metabolism could help restore immune cell function.

Blood metabolomic and transcriptomic signatures stratify patient subgroups in multiple sclerosis according to disease severity.

There are no blood-based biomarkers distinguishing patients with relapsing-remitting (RRMS) from secondary progressive multiple sclerosis (SPMS) although evidence supports metabolomic changes according to MS disease severity. Here machine learning analysis of serum metabolomic data stratified patients with RRMS from SPMS with high accuracy and a putative score was developed that stratified MS patient subsets. The top differentially expressed metabolites between SPMS versus patients with RRMS included lipids and fatty acids, metabolites enriched in pathways related to cellular respiration, notably, elevated lactate and glutamine (gluconeogenesis-related) and acetoacetate and bOHbutyrate (ketone bodies), and reduced alanine and pyruvate (glycolysis-related). Serum metabolomic changes were recapitulated in the whole blood transcriptome, whereby differentially expressed genes were also enriched in cellular respiration pathways in patients with SPMS. The final gene-metabolite interaction network demonstrated a potential metabolic shift from glycolysis toward increased gluconeogenesis and ketogenesis in SPMS, indicating metabolic stress which may trigger stress response pathways and subsequent neurodegeneration.

Functional and topological analysis of PSENEN, the fourth subunit of the γ-secretase complex.

The γ-secretase complexes are intramembrane cleaving proteases involved in the generation of the Aß peptides in Alzheimer's disease. The complex consists of four subunits, with Presenilin harboring the catalytic site. Here, we study the role of the smallest subunit, PSENEN or Presenilin enhancer 2, encoded by the gene Psenen, in vivo and in vitro. We find a profound Notch deficiency phenotype in Psenen-/- embryos confirming the essential role of PSENEN in the γ-secretase complex. We used Psenen-/- fibroblasts to explore the structure-function of PSENEN by the scanning cysteine accessibility method. Glycine 22 and proline 27, which border the membrane domains 1 and 2 of PSENEN, are involved in complex formation and stabilization of γ-secretase. The hairpin structured hydrophobic membrane domains 1 and 2 are exposed to a water-containing cavity in the complex, while transmembrane domain 3 is not water exposed. We finally demonstrate the essential role of PSENEN for the cleavage activity of the complex. PSENEN is more than a structural component of the γ-secretase complex and might contribute to the catalytic mechanism of the enzyme.

Serum biomarker levels predict disability progression in patients with primary progressive multiple sclerosis.

BACKGROUND: We aimed to investigate the potential of serum biomarker levels to predict disability progression in a multicentric real-world cohort of patients with primary progressive multiple sclerosis (PPMS). METHODS: A total of 141 patients with PPMS from 18 European MS centres were included. Disability progression was investigated using change in Expanded Disability Status Scale (EDSS) score over three time intervals: baseline to 2 years, 6 years and to the last follow-up. Serum levels of neurofilament light chain (sNfL), glial fibrillar acidic protein (sGFAP) and chitinase 3-like 1 (sCHI3L1) were measured using single-molecule array assays at baseline. Correlations between biomarker levels, and between biomarkers and age were quantified using Spearman's r. Univariable and multivariable linear models were performed to assess associations between biomarker levels and EDSS change over the different time periods. RESULTS: Median (IQR) age of patients was 52.9 (46.4-58.5) years, and 58 (41.1%) were men. Median follow-up time was 9.1 (7.0-12.6) years. Only 8 (5.7%) patients received treatment during follow-up. sNfL and sGFAP levels were moderately correlated (r=0.43) and both weakly correlated with sCHI3L1 levels (r=0.19 and r=0.17, respectively). In multivariable analyses, levels of the three biomarkers were associated with EDSS changes across all time periods. However, when analysis was restricted to non-inflammatory patients according to clinical and radiological parameters (n=64), only sCHI3L1 levels remained associated with future EDSS change. CONCLUSIONS: Levels of sNfL, sGFAP and sCHI3L1 are prognostic biomarkers associated with disability progression in patients with PPMS, being CHI3L1 findings less dependent on the inflammatory component associated with disease progression.

Age of onset predicted by Aß profiling in a novel PSEN1 (I180F) mutation.

We describe a novel I180F mutation in PSEN1 in which biomarker-supported Alzheimer's disease (AD) segregated in two affected family members. The affected amino acid is highly conserved across species and in silico models predict pathogenicity for AD. The mean age of onset was 56 which was reasonably predicted by the pattern of Aß species produced in an in vitro model.

How Modulator Binding at the Amyloidß-γ-Secretase Interface Enhances Substrate Binding and Attenuates Membrane Distortion.

Inhibition of γ-secretase, an intramembrane protease, to reduce secretion of Amyloid-ß (Aß) peptides has been considered for treating Alzheimer's disease. However, γ-secretase inhibitors suffer from severe side effects. As an alternative, γ-secretase modulators (GSM) reduce the generation of toxic peptides by enhancing the cleavage processivity without diminishing the enzyme activity. Starting from a known γ-secretase structure without substrate but in complex with an E2012 GSM, we generated a structural model that included a bound Aß43 peptide and studied interactions among enzyme, substrate, GSM, and lipids. Our result suggests that E2012 binding at the enzyme-substrate-membrane interface attenuates the membrane distortion by shielding the substrate-membrane interaction. The model predicts that the E2012 modulation is charge-dependent and explains the preserved hydrogen acceptor and the aromatic ring observed in many imidazole-based GSM. Predicted effects of γ-secretase mutations on E2012 modulation were confirmed experimentally. We anticipate that the study will facilitate the future development of effective GSMs.

Systemic lupus erythematosus patients have unique changes in serum metabolic profiles across age associated with cardiometabolic risk.

OBJECTIVES: Cardiovascular disease through accelerated atherosclerosis is a leading cause of mortality for patients with systemic lupus erythematosus (SLE), likely due to increased chronic inflammation and cardiometabolic defects over age. We investigated age-associated changes in metabolomic profiles of SLE patients and healthy controls (HCs). METHODS: Serum NMR metabolomic profiles from female SLE patients (n = 164, age = 14-76) and HCs (n = 123, age = 13-72) were assessed across age by linear regression and by age group between patients/HCs (Group-1, age ≤ 25, n = 62/46; Group-2, age = 26-49, n = 50/46; Group-3, age ≥ 50, n = 52/31) using multiple t-tests. The impact of inflammation, disease activity and treatments were assessed, and UK Biobank disease-wide association analysis of metabolites was performed. RESULTS: Age-specific metabolomic profiles were identified in SLE patients vs HCs, including reduced amino acids (Group-1), increased very-low-density lipoproteins (Group-2), and increased low-density lipoproteins (Group-3). Twenty-five metabolites were significantly altered in all SLE age groups, dominated by decreased atheroprotective high-density lipoprotein (HDL) subsets, HDL-bound apolipoprotein(Apo)A1 and increased glycoprotein acetyls (GlycA). Furthermore, ApoA1 and GlycA were differentially associated with disease activity and serological measures, as well as atherosclerosis incidence and myocardial infarction mortality risk through disease-wide association. Separately, glycolysis pathway metabolites (acetone/citrate/creatinine/glycerol/lactate/pyruvate) uniquely increased with age in SLE, significantly influenced by prednisolone (increased pyruvate/lactate) and hydroxychloroquine (decreased citrate/creatinine) treatment and associated with type-1 and type-2 diabetes by disease-wide association. CONCLUSIONS: Increasing HDL (ApoA1) levels through therapeutic/nutritional intervention, whilst maintaining low disease activity, in SLE patients from a young age could improve cardiometabolic disease outcomes. Biomarkers from the glycolytic pathway could indicate adverse metabolic effects of current therapies.

Optimization of iron oxide nanoparticles for MRI-guided magnetic hyperthermia tumor therapy: reassessing the role of shape in their magnetocaloric effect.

Superparamagnetic iron oxide nanoparticles have hogged the limelight in different fields of nanotechnology. Surprisingly, notwithstanding the prominent role played as agents in magnetic hyperthermia treatments, the effects of nanoparticle size and shape on the magnetic hyperthermia performance have not been entirely elucidated yet. Here, spherical or cubical magnetic nanoparticles synthesized by a thermal decomposition method with the same magnetic and hyperthermia properties are evaluated. Interestingly, spherical nanoparticles displayed significantly higher magnetic relaxivity than cubic nanoparticles; however, comparable differences were not observed in specific absorption rate (SAR), pointing out the need for additional research to better understand the connection between these two parameters. Additionally, the as-synthetized spherical nanoparticles showed negligible cytotoxicity and, therefore, were tested in vivo in tumor-bearing mice. Following intratumoral administration of these spherical nanoparticles and a single exposure to alternating magnetic fields (AMF) closely mimicking clinical conditions, a significant delay in tumor growth was observed. Although further in vivo experiments are warranted to optimize the magnetic hyperthermia conditions, our findings support the great potential of these nanoparticles as magnetic hyperthermia mediators for tumor therapy.

The circadian clock time tunes axonal regeneration.

Nerve injuries cause permanent neurological disability due to limited axonal regeneration. Injury-dependent and -independent mechanisms have provided important insight into neuronal regeneration, however, common denominators underpinning regeneration remain elusive. A comparative analysis of transcriptomic datasets associated with neuronal regenerative ability revealed circadian rhythms as the most significantly enriched pathway. Subsequently, we demonstrated that sensory neurons possess an endogenous clock and that their regenerative ability displays diurnal oscillations in a murine model of sciatic nerve injury. Consistently, transcriptomic analysis showed a time-of-day-dependent enrichment for processes associated with axonal regeneration and the circadian clock. Conditional deletion experiments demonstrated that Bmal1 is required for neuronal intrinsic circadian regeneration and target re-innervation. Lastly, lithium enhanced nerve regeneration in wild-type but not in clock-deficient mice. Together, these findings demonstrate that the molecular clock fine-tunes the regenerative ability of sensory neurons and propose compounds affecting clock pathways as a novel approach to nerve repair.

APP substrate ectodomain defines amyloid-ß peptide length by restraining γ-secretase processivity and facilitating product release.

Sequential proteolysis of the amyloid precursor protein (APP) by γ-secretases generates amyloid-ß (Aß) peptides and defines the proportion of short-to-long Aß peptides, which is tightly connected to Alzheimer's disease (AD) pathogenesis. Here, we study the mechanism that controls substrate processing by γ-secretases and Aß peptide length. We found that polar interactions established by the APPC99 ectodomain (ECD), involving but not limited to its juxtamembrane region, restrain both the extent and degree of γ-secretases processive cleavage by destabilizing enzyme-substrate interactions. We show that increasing hydrophobicity, via mutation or ligand binding, at APPC99 -ECD attenuates substrate-driven product release and rescues the effects of Alzheimer's disease-associated pathogenic γ-secretase and APP variants on Aß length. In addition, our study reveals that APPC99 -ECD facilitates the paradoxical production of longer Aßs caused by some γ-secretase inhibitors, which act as high-affinity competitors of the substrate. These findings assign a pivotal role to the substrate ECD in the sequential proteolysis by γ-secretases and suggest it as a sweet spot for the potential design of APP-targeting compounds selectively promoting its processing by these enzymes.

MYC directly transactivates CR2/CD21, the receptor of the Epstein-Barr virus, enhancing the viral infection of Burkitt lymphoma cells.

MYC is an oncogenic transcription factor dysregulated in about half of total human tumors. While transcriptomic studies reveal more than 1000 genes regulated by MYC, a much smaller fraction of genes is directly transactivated by MYC. Virtually all Burkitt lymphoma (BL) carry chromosomal translocations involving MYC oncogene. Most endemic BL and a fraction of sporadic BL are associated with Epstein-Barr virus (EBV) infection. The currently accepted mechanism is that EBV is the BL-causing agent inducing MYC translocation. Herein we show that the EBV receptor, CR2 (also called CD21), is a direct MYC target gene. This is based on several pieces of evidence: MYC induces CR2 expression in both proliferating and arrested cells and in the absence of protein synthesis, binds the CR2 promoter and transactivates CR2 in an E-box-dependent manner. Moreover, using mice with conditional MYC ablation we show that MYC induces CR2 in primary B cells. Importantly, modulation of MYC levels directly correlates with EBV's ability of infection in BL cells. Altogether, in contrast to the widely accepted hypothesis for the correlation between EBV and BL, we propose an alternative hypothesis in which MYC dysregulation could be the first event leading to the subsequent EBV infection.

Alzheimer's disease linked Aß42 exerts product feedback inhibition on γ-secretase impairing downstream cell signaling.

Amyloid ß (Aß) peptides accumulating in the brain are proposed to trigger Alzheimer's disease (AD). However, molecular cascades underlying their toxicity are poorly defined. Here, we explored a novel hypothesis for Aß42 toxicity that arises from its proven affinity for γ-secretases. We hypothesized that the reported increases in Aß42, particularly in the endolysosomal compartment, promote the establishment of a product feedback inhibitory mechanism on γ-secretases, and thereby impair downstream signaling events. We show that human Aß42 peptides, but neither murine Aß42 nor human Aß17-42 (p3), inhibit γ-secretases and trigger accumulation of unprocessed substrates in neurons, including C-terminal fragments (CTFs) of APP, p75 and pan-cadherin. Moreover, Aß42 treatment dysregulated cellular homeostasis, as shown by the induction of p75-dependent neuronal death in two distinct cellular systems. Our findings raise the possibility that pathological elevations in Aß42 contribute to cellular toxicity via the γ-secretase inhibition, and provide a novel conceptual framework to address Aß toxicity in the context of γ-secretase-dependent homeostatic signaling.

ERK2 stimulates MYC transcription by anchoring CDK9 to the MYC promoter in a kinase activity-independent manner.

The transcription factor MYC regulates cell proliferation, transformation, and survival in response to growth factor signaling that is mediated in part by the kinase activity of ERK2. Because ERK2 can also bind to DNA to modify gene expression, we investigated whether it more directly regulates MYC transcription. We identified ERK2 binding sites in the MYC promoter and detected ERK2 at the promoter in various serum-stimulated cell types. Expression of nuclear-localized ERK2 constructs in serum-starved cells revealed that ERK2 in the nucleus-regardless of its kinase activity-increased MYC mRNA expression and MYC protein abundance. ERK2 bound to the promoter through its amino-terminal insert domain and to the cyclin-dependent kinase CDK9 (which activates RNA polymerase II) through its carboxyl-terminal conserved docking domain. Both interactions were essential for ERK2-induced MYC expression, and depleting ERK impaired CDK9 occupancy and RNA polymerase II progression at the MYC promoter. Artificially tethering CDK9 to the MYC promoter by fusing it to the ERK2 insert domain was sufficient to stimulate MYC expression in serum-starved cells. Our findings demonstrate a role for ERK2 at the MYC promoter acting as a kinase-independent anchor for the recruitment of CDK9 to promote MYC expression.

Enzyme-substrate hybrid ß-sheet controls geometry and water access to the γ-secretase active site.

γ-Secretase is an aspartyl intramembrane protease that cleaves the amyloid precursor protein (APP) involved in Alzheimer's disease pathology and other transmembrane proteins. Substrate-bound structures reveal a stable hybrid ß-sheet immediately following the substrate scissile bond consisting of ß1 and ß2 from the enzyme and ß3 from the substrate. Molecular dynamics simulations and enhanced sampling simulations demonstrate that the hybrid ß-sheet stability is strongly correlated with the formation of a stable cleavage-compatible active geometry and it also controls water access to the active site. The hybrid ß-sheet is only stable for substrates with 3 or more C-terminal residues beyond the scissile bond. The simulation model allowed us to predict the effect of Pro and Phe mutations that weaken the formation of the hybrid ß-sheet which were confirmed by experimental testing. Our study provides a direct explanation why γ-secretase preferentially cleaves APP in steps of 3 residues and how the hybrid ß-sheet facilitates γ-secretase proteolysis.