👤 Stefan F Lichtenthaler

The β-secretase BACE1 (β-site amyloid precursor (APP) cleaving enzyme 1) is a major drug target for Alzheimer's disease (AD), as it catalyzes the first step in amyloid β (Aβ) generation, but has additional substrates and functions, in particular in the brain. Several advanced clinical trials with BACE1 inhibitors were stopped because of an adverse event, a mild cognitive worsening. The underlying mechanism is not yet known but may result from co-inhibition of the BACE1-homolog BACE2. While a cerebrospinal fluid (CSF) biomarker for measuring BACE2 activity is not yet established, VCAM-1 has been suggested as such a biomarker, but has not yet been tested upon prolonged dosing in vivo. Using CSF pharmacoproteomics and a subchronic dosing paradigm in non-human primates, we demonstrate that compound 89, a BACE inhibitor not yet tested in humans, and the clinically tested drug elenbecestat inhibit BACE1 in vivo, with little or no effect on BACE2, as seen with a reduction of substrates of BACE1, but not of the BACE2 substrate VCAM-1. As a control, verubecestat, which inhibits both BACE2 and BACE1, reduced CSF abundance of BACE1 substrates as well as of VCAM-1. This study demonstrates the suitability of VCAM-1 as a pharmacodynamic biomarker for measuring BACE2 target engagement in CSF. Show less

The β-secretase BACE1 has become a prime target in Alzheimer's disease (AD) therapy, because it drives the production of pathogenic amyloid β peptides. However, clinical trials with BACE1-targeting drugs were halted due to adverse effects on cognitive performance. We propose here that cognitive impairment by BACE1 inhibitors may be a corollary of a higher function of BACE1 related to proper sleep regulation. To address non-enzymatic effects of BACE1 on ion channels likely involved in the sleep-wake cycle, we analyze sleep patterns in both BACE1-KO mice and a newly generated transgenic line expressing a proteolysis-deficient BACE1 variant (BACE1-KI). We find that BACE1-KI and BACE1-KO mice display common and distinct sleep-wake disturbances. Compared with their respective wild-type littermates, both mutant lines sleep less during the light phase (when they preferentially rest). Furthermore, transition rates between wake and sleep states are altered, as are sleep spindles and EEG power spectra mainly in the gamma range. Thus, a better understanding of how BACE1 interferes with sleep-modulated behaviors is needed if clinical trials with BACE1-targeted inhibitors are to resume. Show less

The β-secretase β-site APP cleaving enzyme 1 (BACE1) is a major drug target for Alzheimer's disease (AD). Clinically tested BACE1 inhibitors induced unexpected cognitive side effects that may stem from their cross-inhibition of the homologous protease BACE2. Yet, little is known about BACE2 functions and substrates in vivo, and no biomarker is available to monitor the extent of BACE2 inhibition in vivo, particularly in cerebrospinal fluid (CSF). To identify a potential CSF biomarker for monitoring BACE2 activity, we analyzed the CSF proteome changes in non-human primates after treatment with a BACE1-selective inhibitor (a brain-targeted monoclonal antibody) in comparison to verubecestat, a clinically tested small-molecule drug inhibiting both BACE1 and BACE2. Acute treatment with either the antibody or verubecestat similarly reduced CSF abundance of the cleavage products of several known BACE1 substrates, including SEZ6, gp130, and CACHD1, demonstrating similar target engagement in vivo. One CSF protein, vascular cell adhesion protein 1 (VCAM-1), was only reduced upon inhibition with verubecestat, but not upon BACE1-selective inhibition with the antibody. We conclude that VCAM-1 is a promising biomarker candidate for monitoring BACE2 inhibition in CSF, which is instrumental for the development of BACE1-selective inhibitors for the prevention of AD. Show less

The pleiotropic cytokine IL-6 regulates numerous processes in the body, including neuronal functions. IL-6 either binds to membrane-bound receptor (mIL-6R) and triggers signaling via heteromerization with the signal transducer gp130 (classical signaling), or binds to its soluble form (sIL-6R) to act on cells that do not express mIL-6R (trans-signaling). The ß-secretase BACE1 can cleave gp130 as well as IL-6R and we hypothesized that BACE1 may alter neuron activity and synaptic transmission via modulation of IL-6 signaling. We used multielectrode array (MEA) recordings to monitor electrical activity of neuronal networks in acute cerebellar slices as well as long-term potentiation (LTP) induced by high-frequency stimulation in the hippocampus and to assess how exposure to IL-6 affects these processes. A pharmacological approach was applied to elucidate the contribution of trans-signaling involving BACE1. Spontaneous neuronal activity in cerebellar slices significantly decreased upon perfusion with IL-6 but not LIF and recovered during wash out. BACE1 inhibitors verubecestat or AZD3839 abolished the inhibitory effects of IL-6. Furthermore, IL-6 and LIF reversibly inhibited LTP in hippocampal slices, and in contrast to cerebellar neurons, BACE1 inhibitors verubecestat or AZD3839 did not abolish the inhibitory effect of IL-6 on LTP. Interestingly, a dramatic rebound effect on excitatory postsynaptic potentials was observed with BACE1 inhibitor AZD3839 but not verubecestat during wash out. Our results support relevant and differential roles of IL-6, LIF and BACE1 in pathways modulating neuronal discharge activity in the cerebellum and the synaptic plasticity in the hippocampus, and a possible involvement of this interaction in deficits of memory and learning. Show less

Despite numerous studies on fetal therapy for myelomeningoceles (MMC), the pathophysiology of this malformation remains poorly understood. This study aimed to analyze the biochemical profile and proteome of amniotic fluid (AF) supernatants from MMC fetuses to explore the prenatal pathophysiology. Biochemical analysis of 61 AF samples from MMC fetuses was compared with 45 healthy fetuses' samples. Proteome analysis was conducted in 18 MMC and 18 healthy singleton fetuses, and in 5 dichorionic pregnancies with MMC fetuses and their healthy co-twins. ELISA tests were used to validate proteome results. Biochemical analysis revealed anal incontinence in 37 % of MMC cases, absent in controls (p < 0.0001). Proteomics identified 2453 quantified proteins with 39 significantly up-regulated and 10 down-regulated in the MMC group. Up-regulated proteins included ectodomains of CHL1, APLP1, SEZ6, SEZ6L, known targets of the protease BACE1. We explored the overlap of neonatal cerebrospinal fluid (CSF) and AF proteome and highlighted 411 proteins in common, mostly upregulated in MMC AF compared to controls. Our study thoroughly characterizes the AF proteome and reveals numerous proteins to be changed as a consequence of MMC. Many of these proteins are typical constituents of CSF. No difference in AF inflammation markers were observed between MMC and healthy fetuses. SIGNIFICANCE: This study provides good evidence that neuroepithelial destruction in MMC is independent of inflammation or presumed meconium toxicity. Show less

The β-secretase β-site APP cleaving enzyme (BACE1) is a central drug target for Alzheimer's disease. Clinically tested, BACE1-directed inhibitors also block the homologous protease BACE2. Yet little is known about physiological BACE2 substrates and functions in vivo. Here, we identify BACE2 as the protease shedding the lymphangiogenic vascular endothelial growth factor receptor 3 (VEGFR3). Inactivation of BACE2, but not BACE1, inhibited shedding of VEGFR3 from primary human lymphatic endothelial cells (LECs) and reduced release of the shed, soluble VEGFR3 (sVEGFR3) ectodomain into the blood of mice, nonhuman primates, and humans. Functionally, BACE2 inactivation increased full-length VEGFR3 and enhanced VEGFR3 signaling in LECs and also in vivo in zebrafish, where enhanced migration of LECs was observed. Thus, this study identifies BACE2 as a modulator of lymphangiogenic VEGFR3 signaling and demonstrates the utility of sVEGFR3 as a pharmacodynamic plasma marker for BACE2 activity in vivo, a prerequisite for developing BACE1-selective inhibitors for safer prevention of Alzheimer's disease. Show less

Loss-of-function mutations in CLN3 cause juvenile Batten disease, featuring neurodegeneration and early-stage neuroinflammation. How loss of CLN3 function leads to early neuroinflammation is not yet understood. Here, we have comprehensively studied microglia from Cln3 Show less

β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) is considered a therapeutic target to combat Alzheimer's disease by reducing β-amyloid in the brain. To date, all clinical trials involving the inhibition of BACE1 have been discontinued due to a lack of efficacy or undesirable side effects such as cognitive worsening. The latter could have been the result of the inhibition of BACE at the synapse where it is expressed in high amounts. We have previously shown that prolonged inhibition of BACE interferes with structural synaptic plasticity, most likely due to the diminished processing of the physiological BACE substrate Seizure protein 6 (Sez6) which is exclusively processed by BACE1 and is required for dendritic spine plasticity. Given that BACE1 has significant amino acid similarity with its homolog BACE2, the inhibition of BACE2 may cause some of the side effects, as most BACE inhibitors do not discriminate between the two. In this study, we used newly developed BACE inhibitors that have a different chemotype from previously developed inhibitors and a high selectivity for BACE1 over BACE2. By using longitudinal in vivo two-photon microscopy, we investigated the effect on dendritic spine dynamics of pyramidal layer V neurons in the somatosensory cortex in mice treated with highly selective BACE1 inhibitors. Treatment with those inhibitors showed a reduction in soluble Sez6 (sSez6) levels to 27% (elenbecestat, Biogen, Eisai Co., Ltd., Tokyo, Japan), 17% (Shionogi compound Show less

The protease BACE1 is a major drug target for Alzheimer's disease, but chronic BACE1 inhibition is associated with non-progressive cognitive worsening that may be caused by modulation of unknown physiological BACE1 substrates. To identify in vivo-relevant BACE1 substrates, we applied pharmacoproteomics to non-human-primate cerebrospinal fluid (CSF) after acute treatment with BACE inhibitors. Besides SEZ6, the strongest, dose-dependent reduction was observed for the pro-inflammatory cytokine receptor gp130/IL6ST, which we establish as an in vivo BACE1 substrate. Gp130 was also reduced in human CSF from a clinical trial with a BACE inhibitor and in plasma of BACE1-deficient mice. Mechanistically, we demonstrate that BACE1 directly cleaves gp130, thereby attenuating membrane-bound gp130 and increasing soluble gp130 abundance and controlling gp130 function in neuronal IL-6 signaling and neuronal survival upon growth-factor withdrawal. BACE1 is a new modulator of gp130 function. The BACE1-cleaved, soluble gp130 may serve as a pharmacodynamic BACE1 activity marker to reduce the occurrence of side effects of chronic BACE1 inhibition in humans. Show less