VHHs as tools for therapeutic protein delivery to the central nervous system

Background: The blood brain barrier (BBB) limits the therapeutic perspective for central nervous system (CNS) disorders. Previously we found an anti-mouse transferrin receptor (TfR) VHH (Nb62) that was able to deliver a biologically active neuropeptide into the CNS in mice. Here, we aimed to test its potential to shuttle a therapeutic relevant cargo. Since this VHH could not recognize the human TfR and hence its translational potential is limited, we also aimed to find and validate an anti-human transferrin VHH to deliver a therapeutic cargo into the CNS. / Methods: Alpaca immunizations with human TfR, and subsequent phage selection and screening for human TfR binding VHHs was performed to find a human TfR specific VHH (Nb188). Its ability to cross the BBB was determined by fusing it to neurotensin, a neuropeptide that reduces body temperature when present in the CNS but is not able to cross the BBB on its own. Next, the anti–β-secretase 1 (BACE1) 1A11 Fab and Nb62 or Nb188 were fused to an Fc domain to generate heterodimeric antibodies (1A11AM-Nb62 and 1A11AM-Nb188). These were then administered intravenously in wild-type mice and in mice in which the murine apical domain of the TfR was replaced by the human apical domain (hAPI KI). Pharmacokinetic and pharmacodynamic (PK/PD) studies were performed to assess the concentration of the heterodimeric antibodies in the brain over time and the ability to inhibit brain-specific BACE1 by analysing the brain levels of Aβ1–40. / Results: Selections and screening of a phage library resulted in the discovery of an anti-human TfR VHH (Nb188). Fusion of Nb188 to neurotensin induced hypothermia after intravenous injections in hAPI KI mice. In addition, systemic administration 1A11AM-Nb62 and 1A11AM-Nb188 fusions were able to reduce Aβ1-40 levels in the brain whereas 1A11AM fused to an irrelevant VHH did not. A PK/PD experiment showed that this effect could last for 3 days. / Conclusion: We have discovered an anti-human TfR specific VHH that is able to reach the CNS when administered systemically. In addition, both the currently discovered anti-human TfR VHH and the previously identified mouse-specific anti-TfR VHH, are both able to shuttle a therapeutically relevant cargo into the CNS. We suggest the mouse-specific VHH as a valuable research tool in mice and the human-specific VHH as a moiety to enhance the delivery efficiency of therapeutics into the CNS in human patients

Rer1p competes with APH-1 for binding to nicastrin and regulates γ-secretase complex assembly in the early secretory pathway

The γ-secretase complex, consisting of presenilin, nicastrin, presenilin enhancer-2 (PEN-2), and anterior pharynx defective-1 (APH-1) cleaves type I integral membrane proteins like amyloid precursor protein and Notch in a process of regulated intramembrane proteolysis. The regulatory mechanisms governing the multistep assembly of this “proteasome of the membrane” are unknown. We characterize a new interaction partner of nicastrin, the retrieval receptor Rer1p. Rer1p binds preferentially immature nicastrin via polar residues within its transmembrane domain that are also critical for interaction with APH-1. Absence of APH-1 substantially increased binding of nicastrin to Rer1p, demonstrating the competitive nature of these interactions. Moreover, Rer1p expression levels control the formation of γ-secretase subcomplexes and, concomitantly, total cellular γ-secretase activity. We identify Rer1p as a novel limiting factor that negatively regulates γ-secretase complex assembly by competing with APH-1 during active recycling between the endoplasmic reticulum (ER) and Golgi. We conclude that total cellular γ-secretase activity is restrained by a secondary ER control system that provides a potential therapeutic value

Presenilin 1 mediates the turnover of telencephalin in hippocampal neurons via an autophagic degradative pathway

Presenilin 1 (PS1) interacts with telencephalin (TLN) and the amyloid precursor protein via their transmembrane domain (Annaert, W.G., C. Esselens, V. Baert, C. Boeve, G. Snellings, P. Cupers, K. Craessaerts, and B. De Strooper. 2001. Neuron. 32:579–589). Here, we demonstrate that TLN is not a substrate for γ-secretase cleavage, but displays a prolonged half-life in PS1−/− hippocampal neurons. TLN accumulates in intracellular structures bearing characteristics of autophagic vacuoles including the presence of Apg12p and LC3. Importantly, the TLN accumulations are suppressed by adenoviral expression of wild-type, FAD-linked and D257A mutant PS1, indicating that this phenotype is independent from γ-secretase activity. Cathepsin D deficiency also results in the localization of TLN to autophagic vacuoles. TLN mediates the uptake of microbeads concomitant with actin and PIP2 recruitment, indicating a phagocytic origin of TLN accumulations. Absence of endosomal/lysosomal proteins suggests that the TLN-positive vacuoles fail to fuse with endosomes/lysosomes, preventing their acidification and further degradation. Collectively, PS1 deficiency affects in a γ-secretase–independent fashion the turnover of TLN through autophagic vacuoles, most likely by an impaired capability to fuse with lysosomes

Selective inhibitors of the PSEN1–gamma-secretase complex

Clinical development of γ-secretases, a family of intramembrane cleaving proteases, as therapeutic targets for a variety of disorders including cancer and Alzheimer’s disease was aborted because of serious mechanism-based side effects in the phase III trials of unselective inhibitors. Selective inhibition of specific γ-secretase complexes, containing either PSEN1 or PSEN2 as the catalytic subunit and APH1A or APH1B as supporting subunits, does provide a feasible therapeutic window in preclinical models of these disorders. We explore here the pharmacophoric features required for PSEN1 versus PSEN2 selective inhibition. We synthesized a series of brain penetrant 2-azabicyclo[2,2,2]octane sulfonamides and identified a compound with low nanomolar potency and high selectivity (>250-fold) toward the PSEN1–APH1B subcomplex versus PSEN2 subcomplexes. We used modeling and site-directed mutagenesis to identify critical amino acids along the entry part of this inhibitor into the catalytic site of PSEN1. Specific targeting one of the different γ-secretase complexes might provide safer drugs in the future

The γ-secretase substrate proteome and its role in cell signaling regulation

γ-Secretases mediate the regulated intramembrane proteolysis (RIP) of more than 150 integral membrane proteins. We developed an unbiased γ-secretase substrate identification (G-SECSI) method to study to what extent these proteins are processed in parallel. We demonstrate here parallel processing of at least 85 membrane proteins in human microglia in steady-state cell culture conditions. Pharmacological inhibition of γ-secretase caused substantial changes of human microglial transcriptomes, including the expression of genes related to the disease-associated microglia (DAM) response described in Alzheimer disease (AD). While the overall effects of γ-secretase deficiency on transcriptomic cell states remained limited in control conditions, exposure of mouse microglia to AD-inducing amyloid plaques strongly blocked their capacity to mount this putatively protective DAM cell state. We conclude that γ-secretase serves as a critical signaling hub integrating the effects of multiple extracellular stimuli into the overall transcriptome of the cell.</p

Selective inhibitors of the PSEN1-gamma-secretase complex

Clinical development of Y-secretases, a family of intramembrane cleaving proteases, as therapeutic targets for a variety of disorders including cancer and Alzheimer’s disease was aborted because of serious mechanism-based side effects in the phase III trials of unselective inhibitors. Selective inhibition of specific Y-secretase complexes, containing either PSEN1 or PSEN2 as the catalytic subunit and APH1A or APH1B as supporting subunits, does provide a feasible therapeutic window in preclinical models of these disorders. We explore here the pharmacophoric features required for PSEN1 versus PSEN2 selective inhibition. We synthesized a series of brain penetrant 2-azabicyclo[2,2,2]octane sulfonamides and identified a compound with low nanomolar potency and high selectivity (>250-fold) toward the PSEN1–APH1B subcomplex versus PSEN2 subcomplexes. We used modeling and site-directed mutagenesis to identify critical amino acids along the entry part of this inhibitor into the catalytic site of PSEN1. Specific targeting one of the different Y-secretase complexes might provide safer drugs in the future.The work was supported by an AIO-project (no. HBC.2016.0884). This project received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation Programme (grant agreement no. ERC-834682 CELLPHASE_AD). This work was supported by the Flanders Institute for Biotechnology (VIB vzw), a Methusalem grant from KU Leuven and the Flemish Government, the Fonds voor Wetenschappelijk Onderzoek, KU Leuven, The Queen Elisabeth Medical Foundation for Neurosciences, the Opening the Future campaign of the Leuven Universitair Fonds, the Belgian Alzheimer Research Foundation (SAO-FRA), and the Alzheimer’s Association USA.Peer ReviewedPostprint (published version

MEG3 activates necroptosis in human neuron xenografts modeling Alzheimer’s disease

Neuronal cell loss is a defining feature of Alzheimer’s disease (AD), but the underlying mechanisms remain unclear. We xenografted human or mouse neurons into the brain of a mouse model of AD. Only human neurons displayed tangles, Gallyas silver staining, granulovacuolar neurodegeneration (GVD), phosphorylated tau blood biomarkers, and considerable neuronal cell loss. The long noncoding RNA MEG3 was strongly up-regulated in human neurons. This neuron-specific long noncoding RNA is also up-regulated in AD patients. MEG3 expression alone was sufficient to induce necroptosis in human neurons in vitro. Down-regulation of MEG3 and inhibition of necroptosis using pharmacological or genetic manipulation of receptor-interacting protein kinase 1 (RIPK1), RIPK3, or mixed lineage kinase domain-like protein (MLKL) rescued neuronal cell loss in xenografted human neurons. This model suggests potential therapeutic approaches for AD and reveals a human-specific vulnerability to AD

Differential biological contribution and functional properties of the three Aph1 genes to y-secretase activity

Dit thesisproject beoogt de analyse van de in vivo functie van de primaire moleculaire processen die de ziekte van Alzheimer veroorzaken. De pathologie van de ziekte van Alzheimer onderscheidt zich door de neerslag van amyloide plakken en neurofibrillaire kluwens in de hersenen. De plakken bestaan hoofdzakelijk uit abeta-peptiden (Abeta) en volgens de amyloid cascade hypothesis is het neurotoxische Abeta eiwit causaal voor de ziekte. Abeta ontstaat na de proteolytische splitsing door beta-secretase en gamma-secretase, uit het amyloïde voorlopereiwit (APP). Doordat gamma-secretase klieving gebeurt op verschillende plaatsen in de transmembranaire regio van APP vinden we Abeta peptiden van verschillende lengte (37- 49 aminozuren). Familiale vormen van de ziekte veroorzaakt door mutaties in de preseniline genen resulteren in een verhoogde Abeta42/Abeta40 ratio. gamma-secretase is een eiwitcomplex bestaande uit minimaal vier eenheden: Psen1 of Psen2, Aph1A of Aph1B, Pen2 en Nct. Verschillende associaties van de Psen en Aph1 homologen maken dat gamma-secretasen structurele heterogene complexen zijn. Psen1 of Psen2 bevat het actieve centrum van het complex terwijl de juiste functie van de andere 3 eiwitten minder duidelijk is. Gamma-secretase is ook betrokken bij levensbelangrijke signaalcascaden zoals de gereguleerde intramembranaire splitsing van verschillende type I eiwitten (bv Notch) Tot op heden is het niet volledig duidelijk hoe de verschillende gamma-secretasen bijdragen tot de verschillende biologische functies en of deze heterogeniteit resulteert in enzymen specificiteit. Om deze vragen te beantwoorden hebben we knock-out modellen van de verschillende Aph1 genen gegenereerd. Het onmisbare belang van Aph1A bleek uit het embryonaal lethaal fenotype dat sterke gelijkenissen vertoont met het Notch fenotype terwijl Aph1B/C mogelijk een belangrijke biologische functie in de hersenen heeft. Deze bevindingen suggereerden ons om de kinetische parameters van de verschillende Aph1 gamma-secretase te analyseren in een in vitro activiteitstest. Deze strategie heeft veel voordelen omdat deze productie meet zonder beïnvloed te worden door dynamische evenwichtsreacties zoals afbraak en neerslag in onoplosbare plakken. Deze in vitro activiteitstest laat toe om het effect van de verschillende Aph1 homologen te meten niet enkel op gamma-secretase uit cellen maar ook uit weefsels zoals hersenen wat een meer fysiologische analyse toelaat. Aan de hand van deze analyse hebben we vastgesteld dat het Aph1 eiwit bijdraagt tot de enzymatische activiteit van het gamma-secretase. We vinden geen effect op de eerste fysiologische belangrijke klieving ter hoogte van de epsilon-plaats maar de volgende klievingen worden wel beïnvloed door Aph1. Namelijk Aph1B gamma-secretasen produceren relatief meer langer Abeta (Abeta&#8805;42) en minder korter Abeta (Abeta&#8804;40) peptiden dan Aph1A gamma-secretasen. Dit resulteert in een twee maal verhoogde Abeta42/Abeta40 verhouding. Deze resultaten tonen aan dat het Aph1B eiwit kan bijdragen tot een pathologische functie van gamma-secretase door de vorming van langere waarschijnlijk meer pathogene Abeta peptiden.Onze onderzoeksgroep heeft aangetoond dat specifieke inactivatie van Aph1B/C gamma-secretase complexen in een AD muizen model een verbetering geeft van de symptomen zonder dat er Notch nevenwerkingen aangetoond konden worden. Onze onderzoeksresultaten suggereren dat de verschillende Aph1 gamma-secretasen bijdragen tot differentiële fysiologische en pathologische functies en openen nieuwe perspectieven voor het gebruik van meer specifieke en minder toxische gamma-secretase inhibitoren in de behandeling van Alzheimer dementie.Table of contents 1 List of Abbreviations 3 Chapter 1: Introduction 5 1.1 Alzheimer’s Disease 5 1.2 The Amyloid beta Hypothesis 6 1.3 Enzymes involved in proteolytic processing of APP 7 1.3.1 Gamma-secretase: the non amyloidogenic pathway 7 1.3.2 Gamma-secretase: gate of the amyloidogenic pathway 8 1.3.3 Gamma-secretase: an unusual multiprotein complex 8 1.3.3.1 Presenilin: the catalytic unit 9 1.3.3.2 Nicastrin: gatekeeper of the gamma-secretase complex 10 1.3.3.3 Pen2: facilitates Psen endoproteolysis 11 1.3.3.4 Aph1: forms a pre-complex with NCT 11 1.4 Gamma-secretase: heterogeneous activity 12 1.5 RIP: Regulated Intramembrane Proteolysis 13 1.6 Notch signaling: depends on gmma-secretase 13 1.7 Functional genomics using the mouse 14 Aim of the work 17 Chapter 2: Coordinated and widespread expression of gamma-secretase 19 2.1 Materials and methods 19 2.2 Results 20 2.3 Conclusions 22 Chapter 3: Differential contribution of the three Aph1 genes to gamma-secretase activity in vivo 23 3.1 Materials and methods 23 3.2 Results 28 3.3 Conclusions 39 Chapter 4: Aph1B gamma-secretase generates long Abeta peptides 43 4.1 Materials and methods 44 4.2 Results 47 4.3 Conclusions 51 Chapter 5: Conclusions 53 5.1 Different gamma-secretase complexes exist in vivo 53 5.2 Different gamma-secretase complexes have a different biological function 54 5.3 Aph1 as an essential component of the gamma-secretase complex 56 5.4 Different gamma-secretase complexes have different biochemical properties 56 5.5 Gamm-secretase: a therapeutic target for Alzheimer’s disease? 58 Summary 61 Samenvatting 63 References 65 Curriculum vitae and Publications 75status: publishe