Loss of Bace2 in zebrafish affects melanocyte migration and is distinct from Bace1 knock out phenotypes

Alzheimer's disease is the most frequent dementia. Pathologically, Alzheimer's disease is characterized by the accumulation of senile plaques composed of amyloid β‐peptide (Aβ). Two proteases, β‐ and γ‐secretase proteolytically generate Aβ from its precursor, the ß‐amyloid precursor protein (APP). Inhibition of β‐secretase, also referred to as b eta‐site A PP c leaving e nzyme (BACE1) or γ‐secretase is therefore of prime interest for the development of amyloid‐lowering drugs. To assess the in vivo function of zebrafish Bace1 (zBace1), we generated zBace1 knock out fish by zinc finger nuclease‐mediated genome editing. bace1 mutants (bace1?/?) are hypomyelinated in the PNS while the CNS is not affected. Moreover, the number of mechanosensory neuromasts is elevated in bace1?/?. Mutations in zebrafish Bace2 (zBace2) revealed a distinct melanocyte migration phenotype, which is not observed in bace1?/?. Double homozygous bace1?/?; bace2?/? fish do not enhance the single mutant phenotypes indicating non‐redundant distinct physiological functions. Single homozygous bace1 mutants as well as double homozygous bace1 and bace2 mutants are viable and fertile suggesting that Bace1 is a promising drug target without major side effects. The identification of a specific bace2 ?/? associated phenotype further allows improving selective Bace1 inhibitors and to distinguish between Bace 1 and Bace 2 inhibition in vivo.

     

Bace1 and Neuregulin‐1 cooperate to control formation and maintenance of muscle spindles

The protease β‐secretase 1 (Bace1) was identified through its critical role in production of amyloid‐β peptides (Aβ), the major component of amyloid plaques in Alzheimer's disease. Bace1 is considered a promising target for the treatment of this pathology, but processes additional substrates, among them Neuregulin‐1 (Nrg1). Our biochemical analysis indicates that Bace1 processes the Ig‐containing β1 Nrg1 (IgNrg1β1) isoform. We find that a graded reduction in IgNrg1 signal strength in vivo results in increasingly severe deficits in formation and maturation of muscle spindles, a proprioceptive organ critical for muscle coordination. Further, we show that Bace1 is required for formation and maturation of the muscle spindle. Finally, pharmacological inhibition and conditional mutagenesis in adult animals demonstrate that Bace1 and Nrg1 are essential to sustain muscle spindles and to maintain motor coordination. Our results assign to Bace1 a role in the control of coordinated movement through its regulation of muscle spindle physiology, and implicate IgNrg1‐dependent processing as a molecular mechanism.     

Loss of Bace1 in Mice Does Not Alter the Severity of Caerulein Induced Pancreatitis

ContextBeta-site alpha-amyloid protein cleaving enzyme1 (BACE1) plays a key role in the pathogenesis of Alzheimer’s disease. Additional to its moderate expression in the brain, high levels of BACE1 mRNA were found in the pancreas. Murine Bace1 has been immunohistochemicaly detected at the apical pole of acinar cells within the exocrine pancreas of mice and Bace1 activity was observed in pancreatic juice. In vitro experiments revealed enteropeptidase as a putative substrate for Bace1 suggesting a role in acute pancreatitis.ObjectiveThe aim of this study was to address a protective mechanism of Bace1 in acute experimental pancreatitis in mice.MethodsAcute experimental pancreatitis was induced by intraperitoneal injection of caerulein in homozygote Bace1^-/- mice and wild type mice. Serum and tissue analyses were carried out after 4 h, 8 h and 24 h. Measurement of plasma amylase and lipase was performed to confirm pancreatitis induction. In order to assess the severity of pancreatitis H&E stained pancreatic sections were examined regarding edema, inflammation and apoptosis. Immunohistochemical detection of myeloperoxidase (MPO) positive cells was carried out to further quantify the extent of inflammation. Expression of Bace2 within the pancreas was analyzed by immunohistochemistry and RT-qPCR.ResultsWe demonstrate that total loss of Bace1 in mice leads to no alterations in the course of acute experimental caerulein-pancreatitis. Bace1^-/- mice develop a moderate pancreatitis that is comparable in histomorphological and serological features with those seen in wild type mice.DiscussionWe discuss the results in the context of the applied caerulein induced edematous pancreatitis model and possible compensatory mechanisms via Bace2 that might be responsible for the observed results.     

Label‐free quantitative analysis of the membrane proteome of Bace1 protease knock‐out zebrafish brains

The aspartyl protease BACE1 cleaves neuregulin 1 and is involved in myelination and is a candidate drug target for Alzheimer's disease, where it acts as the β‐secretase cleaving the amyloid precursor protein. However, little is known about other substrates in vivo. Here, we provide a proteomic workflow for BACE1 substrate identification from whole brains, combining filter‐aided sample preparation, strong‐anion exchange fractionation, and label‐free quantification. We used bace1‐deficient zebrafish and quantified differences in protein levels between wild‐type and bace1 ?/? zebrafish brains. Over 4500 proteins were identified with at least two unique peptides and quantified in both wild‐type and bace1 ?/? zebrafish brains. The majority of zebrafish membrane proteins did not show altered protein levels, indicating that Bace1 has a restricted substrate specificity. Twenty‐four membrane proteins accumulated in the bace1 ?/? brains and thus represent candidate Bace1 substrates. They include several known BACE1 substrates, such as the zebrafish homologs of amyloid precursor protein and the cell adhesion protein L1, which validate the proteomic workflow. Additionally, several candidate substrates with a function in neurite outgrowth and axon guidance, such as plexin A3 and glypican‐1 were identified, pointing to a function of Bace1 in neurodevelopment. Taken together, our study provides the first proteomic analysis of knock‐out zebrafish tissue and demonstrates that combining gene knock‐out models in zebrafish with quantitative proteomics is a powerful approach to address biomedical questions.     

Tmem27 dimerization, deglycosylation, plasma membrane depletion, and the extracellular Phe-Phe motif are negative regulators of cleavage by Bace2

The pancreatic β-cell surface protein Tmem27 is promotes the preservation of functional β-cell mass. It is a selective substrate of the protease Bace2, yet the intramolecular features of Tmem27 that regulate its processing by this sheddase have not been characterized. In particular, the importance of homodimerization, glycosylation, trafficking to the plasma membrane (PM), the existence of multiple cleavage sites, and the amino acid residues that govern these features are currently unknown. Using Tmem27 mutational analysis and multiple biochemical approaches, we here show that Tmem27 dimerization is a dynamic process mediated by its intracellular cysteine residue and that prevents Tmem27 cleavage, that extracellular asparagine glycosylation is essential for Tmem27 trafficking to the PM and its processing by Bace2, that the amount of Tmem27 at the PM is proportional to its total cell levels upon glucose stimulation and Bace2 inhibition, and that the double phenylalanine motif in the Tmem27 cleavage site is an intramolecular Bace2 inhibitor. These findings define structural properties of Tmem27 that affect the susceptibility to its protease Bace2 and have implications for the efficiency with which Tmem27 and other Bace2 substrates are cleaved in normal and disease states.     

Overexpression of the key virulence proteases Bace16 and Bae16 in Bacillus nematocida B16 to improve its nematocidal activity

Proteases Bace16 and Bae16, an alkaline serine protease and a neutral protease, respectively, in the nematocidal bacterium Bacillus nematocida B16, have been identified as two key virulence factors and shown to have remarkable nematotoxic activities against the free-living nematode Panagrellus redivius and the plant parasite nematode Bursaphelenchus xylophilus. To facilitate the successful biological control application of this organism in the field, we genetically altered the strain B. nematocida B16 and optimized its growth condition to overexpress these two pathogenic proteases. The recombinant integration vectors of pAX01-Bace16 and pAX01-Bae16 for overexpressing the two proteases were constructed and successfully transformed into competent cells of the bacterium B. nematocida B16. The optimal induction condition for overexpressing Bace16 is 2% xylose at 37°C for 48 h. Our analyses showed that the proteolytic activity and nematocidal activity of the strain overexpressing Bace16 increased by about 62 and 80%, respectively, over the wild-type strain. However, our tested induction conditions could not significantly improve either the proteolytic activity or the nematocidal activity of the Bae16 overexpression mutant.     

Functional identification of the gene <Emphasis Type="Italic">bace16</Emphasis> from nematophagous bacterium <Emphasis Type="Italic">Bacillus nematocida</Emphasis>

Bacillus nematocida is a Gram-positive bacterium capable of killing nematodes. Our recent studies identified an extracellular serine protease Bace16 in B. nematocida as a candidate of pathogenic factor in the infection against nematodes, which displayed a high similarity with the serine protease family subtilisin BPN’, and the MEROPS ID is S08.034. To further confirm the roles that bace16 played in the mechanism of nematocidal pathogenesis, recombinant mature Bace16 (rm-Bace16) was expressed in Escherichia coli strain BL21 using pET-30 vector system. Bioassay experiments demonstrated that the purified recombinant protease had the ability to degrade nematode cuticles and kill nematodes. In addition, a bace16 knockout mutant of B. nematocida constructed by homologous recombination showed considerably lower proteolytic activity and less than 50% nematocidal activity than the wild-type strain. These results confirmed that Bace16 could serve as an important virulence factor during the infectious process. Qiuhong Niu and Xiaowei Huang contributed equally to this work.     

Processing of beta-secretase by furin and other members of the proprotein convertase family

The amyloid peptide is the main constituent of the amyloid plaques in brain of Alzheimer's disease patients. This peptide is generated from the amyloid precursor protein by two consecutive cleavages. Cleavage at the N terminus is performed by the recently discovered beta-secretase (Bace). This aspartyl protease contains a propeptide that has to be removed to obtain mature Bace. Furin and other members of the furin family of prohormone convertases are involved in this process. Surprisingly, beta-secretase activity, neither at the classical Asp(1) position nor at the Glu(11) position of amyloid precursor protein, seems to be controlled by this maturation step. Furthermore, we show that Glu(11) cleavage is a function of the expression level of Bace, that it depends on the membrane anchorage of Bace, and that Asp(1) cleavage can be followed by Glu(11) cleavage. Our data suggest that pro-Bace could be active as a beta-secretase in the early biosynthetic compartments of the cell and could be involved in the generation of the intracellular pool of the amyloid peptide. We conclude that modulation of the conversion of pro-Bace to mature Bace is not a relevant drug target to treat Alzheimer's disease.     

Bace2 is a β cell-enriched protease that regulates pancreatic β cell function and mass

Decreased β cell mass and function are hallmarks of type 2 diabetes. Here we identified, through a siRNA screen, beta site amyloid precursor protein cleaving enzyme 2 (Bace2) as the sheddase of the proproliferative plasma membrane protein Tmem27 in murine and human β cells. Mice with functionally inactive Bace2 and insulin-resistant mice treated with a newly identified Bace2 inhibitor both display augmented β cell mass and improved control of glucose homeostasis due to increased insulin levels. These results implicate Bace2 in the control of β cell maintenance and provide a rational strategy to inhibit this protease?for the expansion of functional pancreatic β cell mass.     

APP Is Cleaved by Bace1 in Pre-Synaptic Vesicles and Establishes a Pre-Synaptic Interactome,via Its Intracellular Domain,with Molecular Complexes that Regulate Pre-Synaptic Vesicles Functions

Amyloid Precursor Protein (APP) is a type I membrane protein that undergoes extensive processing by secretases, including BACE1. Although mutations in APP and genes that regulate processing of APP, such as PSENs and BRI2/ITM2B, cause dementias, the normal function of APP in synaptic transmission, synaptic plasticity and memory formation is poorly understood. To grasp the biochemical mechanisms underlying the function of APP in the central nervous system, it is important to first define the sub-cellular localization of APP in synapses and the synaptic interactome of APP. Using biochemical and electron microscopy approaches, we have found that APP is localized in pre-synaptic vesicles, where it is processed by Bace1. By means of a proteomic approach, we have characterized the synaptic interactome of the APP intracellular domain. We focused on this region of APP because in vivo data underline the central funtional and pathological role of the intracellular domain of APP. Consistent with the expression of APP in pre-synaptic vesicles, the synaptic APP intracellular domain interactome is predominantly constituted by pre-synaptic, rather than post-synaptic, proteins. This pre-synaptic interactome of the APP intracellular domain includes proteins expressed on pre-synaptic vesicles such as the vesicular SNARE Vamp2/Vamp1 and the Ca²⁺ sensors Synaptotagmin-1/Synaptotagmin-2, and non-vesicular pre-synaptic proteins that regulate exocytosis, endocytosis and recycling of pre-synaptic vesicles, such as target-membrane-SNAREs (Syntaxin-1b, Syntaxin-1a, Snap25 and Snap47), Munc-18, Nsf, α/β/γ-Snaps and complexin. These data are consistent with a functional role for APP, via its carboxyl-terminal domain, in exocytosis, endocytosis and/or recycling of pre-synaptic vesicles.     

The role of amyloid precursor protein processing by BACE1, the beta-secretase, in Alzheimer disease pathophysiology

Amyloid plaques, composed of the amyloid beta-protein (Abeta), are hallmark neuropathological lesions in Alzheimer disease (AD) brain. Abeta fulfills a central role in AD pathogenesis, and reduction of Abeta levels should prove beneficial for AD treatment. Abeta generation is initiated by proteolysis of amyloid precursor protein (APP) by the beta-secretase enzyme BACE1. Bace1 knockout (Bace1(-/-)) mice have validated BACE1 as the authentic beta-secretase in vivo. BACE1 is essential for Abeta generation and represents a suitable drug target for AD therapy, especially because this enzyme is up-regulated in AD. However, although initial data indicated that Bace1(-/-) mice lack an overt phenotype, the BACE1-mediated processing of APP and other substrates may be important for specific biological processes. In this minireview, topics range from the initial identification of BACE1 to the fundamental knowledge gaps that remain in our understanding of this protease. We address pertinent questions such as putative causes of BACE1 elevation in AD and discuss why, nine years since the identification of BACE1, treatments that address the underlying pathological mechanisms of AD are still lacking.     

Effect of memantine on expression of Bace1-as and Bace1 genes in STZ-induced Alzheimeric rats

Molecular Biology Reports - Recent studies have showed that the long non-coding RNAs (lncRNAs) expression is dysregulated in different neurodegenerative disorders like Alzheimer’s disease...     

Tau hyperphosphorylation and increased BACE1 and RAGE levels in the cortex of PPARβ/δ-null mice

The role of peroxisome proliferator activator receptor (PPAR)β/δ in the pathogenesis of Alzheimer's disease has only recently been explored through the use of PPARβ/δ agonists. Here we evaluated the effects of PPARβ/δ deficiency on the amyloidogenic pathway and tau hyperphosphorylation. PPARβ/δ-null mice showed cognitive impairment in the object recognition task, accompanied by enhanced DNA-binding activity of NF-κB in the cortex and increased expression of IL-6. In addition, two NF-κB-target genes involved in β-amyloid (Aβ) synthesis and deposition, the β site APP cleaving enzyme 1 (Bace1) and the receptor for advanced glycation endproducts (Rage), respectively, increased in PPARβ/δ-null mice compared to wild type animals. The protein levels of glial fibrillary acidic protein (GFAP) increased in the cortex of PPARβ/δ-null mice, which would suggest the presence of astrogliosis. Finally, tau hyperphosphorylation at Ser199 and enhanced levels of PHF-tau were associated with increased levels of the tau kinases CDK5 and phospho-ERK1/2 in the cortex of PPARβ/δ^?/? mice.Collectively, our findings indicate that PPARβ/δ deficiency results in cognitive impairment associated with enhanced inflammation, astrogliosis and tau hyperphosphorylation in the cortex.     

Brain-specific knockdown of miR-29 results in neuronal cell death and ataxia in mice

Several microRNAs have been implicated in neurogenesis, neuronal differentiation, neurodevelopment, and memory. Development of miRNA-based therapeutics, however, needs tools for effective miRNA modulation, tissue-specific delivery, and in vivo evidence of functional effects following the knockdown of miRNA. Expression of miR-29a is reduced in patients and animal models of several neurodegenerative disorders, including Alzheimer''s disease, Huntington''s disease, and spinocerebellar ataxias. The temporal expression pattern of miR-29b during development also correlates with its protective role in neuronal survival. Here, we report the cellular and behavioral effect of in vivo, brain-specific knockdown of miR-29. We delivered specific anti-miRNAs to the mouse brain using a neurotropic peptide, thus overcoming the blood-brain-barrier and restricting the effect of knockdown to the neuronal cells. Large regions of the hippocampus and cerebellum showed massive cell death, reiterating the role of miR-29 in neuronal survival. The mice showed characteristic features of ataxia, including reduced step length. However, the apoptotic targets of miR-29, such as Puma, Bim, Bak, or Bace1, failed to show expected levels of up-regulation in mice, following knockdown of miR-29. In contrast, another miR-29 target, voltage-dependent anion channel1 (VDAC1), was found to be induced several fold in the hippocampus, cerebellum, and cortex of mice following miRNA knockdown. Partial restoration of apoptosis was achieved by down-regulation of VDAC1 in miR-29 knockdown cells. Our study suggests that regulation of VDAC1 expression by miR-29 is an important determinant of neuronal cell survival in the brain. Loss of miR-29 results in dysregulation of VDAC1, neuronal cell death, and an ataxic phenotype.     

Chronic Anatabine Treatment Reduces Alzheimer’s Disease (AD)-Like Pathology and Improves Socio-Behavioral Deficits in a Transgenic Mouse Model of AD

Anatabine is a minor tobacco alkaloid, which is also found in plants of the Solanaceae family and displays a chemical structure similarity with nicotine. We have shown previously that anatabine displays some anti-inflammatory properties and reduces microgliosis and tau phosphorylation in a pure mouse model of tauopathy. We therefore investigated the effects of a chronic oral treatment with anatabine in a transgenic mouse model (Tg PS1/APPswe) of Alzheimer’s disease (AD) which displays pathological Aβ deposits, neuroinflammation and behavioral deficits. In the elevated plus maze, Tg PS1/APPswe mice exhibited hyperactivity and disinhibition compared to wild-type mice. Six and a half months of chronic oral anatabine treatment, suppressed hyperactivity and disinhibition in Tg PS1/APPswe mice compared to Tg PS1/APPswe receiving regular drinking water. Tg PS1/APPswe mice also elicited profound social interaction and social memory deficits, which were both alleviated by the anatabine treatment. We found that anatabine reduces the activation of STAT3 and NFκB in the vicinity of Aβ deposits in Tg PS1/APPswe mice resulting in a reduction of the expression of some of their target genes including Bace1, iNOS and Cox-2. In addition, a significant reduction in microgliosis and pathological deposition of Aβ was observed in the brain of Tg PS1/APPswe mice treated with anatabine. This is the first study to investigate the impact of chronic anatabine treatment on AD-like pathology and behavior in a transgenic mouse model of AD. Overall, our data show that anatabine reduces β-amyloidosis, neuroinflammation and alleviates some behavioral deficits in Tg PS1/APPswe, supporting further exploration of anatabine as a possible disease modifying agent for the treatment of AD.     

Continuous versus cyclic progesterone exposure differentially regulates hippocampal gene expression and functional profiles

This study investigated the impact of chronic exposure to continuous (CoP4) versus cyclic progesterone (CyP4) alone or in combination with 17β-estradiol (E2) on gene expression profiles targeting bioenergetics, metabolism and inflammation in the adult female rat hippocampus. High-throughput qRT-PCR analyses revealed that ovarian hormonal depletion induced by ovariectomy (OVX) led to multiple significant gene expression alterations, which were to a great extent reversed by co-administration of E2 and CyP4. In contrast, co-administration of E2 and CoP4 induced a pattern highly resembling OVX. Bioinformatics analyses further revealed clear disparities in functional profiles associated with E2+CoP4 and E2+CyP4. Genes involved in mitochondrial energy (ATP synthase α subunit; Atp5a1), redox homeostasis (peroxiredoxin 5; Prdx5), insulin signaling (insulin-like growth factor I; Igf1), and cholesterol trafficking (liver X receptor α subtype; Nr1h3), differed in direction of regulation by E2+CoP4 (down-regulation relative to OVX) and E2+CyP4 (up-regulation relative to OVX). In contrast, genes involved in amyloid metabolism (β-secretase; Bace1) differed only in degree of regulation, as both E2+CoP4 and E2+CyP4 induced down-regulation at different efficacy. E2+CyP4-induced changes could be associated with regulation of progesterone receptor membrane component 1(Pgrmc1). In summary, results from this study provide evidence at the molecular level that differing regimens of hormone therapy (HT) can induce disparate gene expression profiles in brain. From a translational perspective, confirmation of these results in a model of natural menopause, would imply that the common regimen of continuous combined HT may have adverse consequences whereas a cyclic combined regimen, which is more physiological, could be an effective strategy to maintain neurological health and function throughout menopausal aging.