OPENPichia: licence-free Komagataella phaffii chassis strains and toolkit for protein expression.

The industrial yeast Komagataella phaffii (formerly named Pichia pastoris) is commonly used to synthesize recombinant proteins, many of which are used as human therapeutics or in food. However, the basic strain, named NRRL Y-11430, from which all commercial hosts are derived, is not available without restrictions on its use. Comparative genome sequencing leaves little doubt that NRRL Y-11430 is derived from a K. phaffii type strain deposited in the UC Davis Phaff Yeast Strain Collection in 1954. We analysed four equivalent type strains in several culture collections and identified the NCYC 2543 strain, from which we started to develop an open-access Pichia chassis strain that anyone can use to produce recombinant proteins to industry standards. NRRL Y-11430 is readily transformable, which we found to be due to a HOC1 open-reading-frame truncation that alters cell-wall mannan. We introduced the HOC1 open-reading-frame truncation into NCYC 2543, which increased the transformability and improved secretion of some but not all of our tested proteins. We provide our genome-sequenced type strain, the hoc1tr derivative that we named OPENPichia as well as a synthetic, modular expression vector toolkit under liberal end-user distribution licences as an unencumbered OPENPichia resource for the microbial biotechnology community.

Resin comparison for serial block face scanning volume electron microscopy.

Serial Block Face Scanning Electron Microscopy (SBF-SEM) is one of several volume electron microscopy (vEM) techniques whose purpose is to reveal the nanostructure of cells and tissues in three dimensions. As one of the earliest, and possibly most widely adopted of the disruptive vEM techniques there have been hundreds of publications using the method, although very few comparative studies of specimen preparation parameters. While some studies have focused on staining and specimen acquisition no comparison of resin embedding has yet been conducted. To this end we have surveyed the SBF-SEM literature to determine which resins are commonly used and compared them in both cellular and fixed tissue samples in an attempt to optimize sample preparation for: effectiveness of resin infiltration, resistance to charging and beam damage and clarity of image in the resulting data set. Here we present the results and discuss the various factors that go into optimizing specimen preparation for SBF-SEM.

Light triggered nanoscale biolistics for efficient intracellular delivery of functional macromolecules in mammalian cells.

Biolistic intracellular delivery of functional macromolecules makes use of dense microparticles which are ballistically fired onto cells with a pressurized gun. While it has been used to transfect plant cells, its application to mammalian cells has met with limited success mainly due to high toxicity. Here we present a more refined nanotechnological approach to biolistic delivery with light-triggered self-assembled nanobombs (NBs) that consist of a photothermal core particle surrounded by smaller nanoprojectiles. Upon irradiation with pulsed laser light, fast heating of the core particle results in vapor bubble formation, which propels the nanoprojectiles through the cell membrane of nearby cells. We show successful transfection of both adherent and non-adherent cells with mRNA and pDNA, outperforming electroporation as the most used physical transfection technology by a factor of 5.5-7.6 in transfection yield. With a throughput of 104-105 cells per second, biolistic delivery with NBs offers scalable and highly efficient transfections of mammalian cells.

Involvement of the Choroid Plexus in the Pathogenesis of Niemann-Pick Disease Type C.

Niemann-Pick type C (NPC) disease, sometimes called childhood Alzheimer's, is a rare neurovisceral lipid storage disease with progressive neurodegeneration leading to premature death. The disease is caused by loss-of-function mutations in the Npc1 or Npc2 gene which both result into lipid accumulation in the late endosomes and lysosomes. Since the disease presents with a broad heterogenous clinical spectrum, the involved disease mechanisms are still incompletely understood and this hampers finding an effective treatment. As NPC patients, who carry NPC1 mutations, have shown to share several pathological features with Alzheimer's disease (AD) and we and others have previously shown that AD is associated with a dysfunctionality of the blood-cerebrospinal fluid (CSF) barrier located at choroid plexus, we investigated the functionality of this latter barrier in NPC1 pathology. Using NPC1-/- mice, we show that despite an increase in inflammatory gene expression in choroid plexus epithelial (CPE) cells, the blood-CSF barrier integrity is not dramatically affected. Interestingly, we did observe a massive increase in autophagosomes in CPE cells and enlarged extracellular vesicles (EVs) in CSF upon NPC1 pathology. Additionally, we revealed that these EVs exert toxic effects on brain tissue, in vitro as well as in vivo. Moreover, we observed that EVs derived from the supernatant of NPC1-/- choroid plexus explants are able to induce typical brain pathology characteristics of NPC1-/-, more specifically microgliosis and astrogliosis. Taken together, our data reveal for the first time that the choroid plexus and CSF EVs might play a role in the brain-related pathogenesis of NPC1.

Osteopontin Expression Identifies a Subset of Recruited Macrophages Distinct from Kupffer Cells in the Fatty Liver.

Metabolic-associated fatty liver disease (MAFLD) represents a spectrum of disease states ranging from simple steatosis to non-alcoholic steatohepatitis (NASH). Hepatic macrophages, specifically Kupffer cells (KCs), are suggested to play important roles in the pathogenesis of MAFLD through their activation, although the exact roles played by these cells remain unclear. Here, we demonstrated that KCs were reduced in MAFLD being replaced by macrophages originating from the bone marrow. Recruited macrophages existed in two subsets with distinct activation states, either closely resembling homeostatic KCs or lipid-associated macrophages (LAMs) from obese adipose tissue. Hepatic LAMs expressed Osteopontin, a biomarker for patients with NASH, linked with the development of fibrosis. Fitting with this, LAMs were found in regions of the liver with reduced numbers of KCs, characterized by increased Desmin expression. Together, our data highlight considerable heterogeneity within the macrophage pool and suggest a need for more specific macrophage targeting strategies in MAFLD.

Three-Dimensional Visualization of APEX2-Tagged Erg11 in Saccharomyces cerevisiae Using Focused Ion Beam Scanning Electron Microscopy.

The determination of the exact location of a protein in the cell is essential to the understanding of biological processes. Here, we report for the first time the visualization of a protein of interest in Saccharomyces cerevisiae using focused ion beam scanning electron microscopy (FIB-SEM). As a proof of concept, the integral endoplasmic reticulum (ER) membrane protein Erg11 has been C-terminally tagged with APEX2, which is an engineered peroxidase that catalyzes an electron-dense deposition of 3,3'-diaminobenzidine (DAB), as such marking the location of the fused protein of interest in electron microscopic images. As DAB is unable to cross the yeast cell wall to react with APEX2, cell walls have been partly removed by the formation of spheroplasts. This has resulted in a clear electron-dense ER signal for the Erg11 protein using FIB-SEM. With this study, we have validated the use of the APEX2 tag for visualization of yeast proteins in electron microscopy. Furthermore, we have introduced a methodology that enables precise and three-dimensional (3D) localization studies in yeast, with nanometer resolution and without the need for antibody staining. Because of these properties, the described technique can offer valuable information on the molecular functions of studied proteins.IMPORTANCE With this study, we have validated the use of the APEX2 tag to define the localization of proteins in the model yeast S. cerevisiae As such, FIB-SEM can identify the exact 3D location of a protein of interest in the cell with nanometer-scale resolution. Such detailed imaging could provide essential information on the elucidation of various biological processes. APEX2, which adds electron density to a fused protein of interest upon addition of the substrate DAB, originally was used in mammalian studies. With this study, we expand its use to protein localization studies in one of the most important models in molecular biology.

Stellate Cells, Hepatocytes, and Endothelial Cells Imprint the Kupffer Cell Identity on Monocytes Colonizing the Liver Macrophage Niche.

Macrophages are strongly adapted to their tissue of residence. Yet, little is known about the cell-cell interactions that imprint the tissue-specific identities of macrophages in their respective niches. Using conditional depletion of liver Kupffer cells, we traced the developmental stages of monocytes differentiating into Kupffer cells and mapped the cellular interactions imprinting the Kupffer cell identity. Kupffer cell loss induced tumor necrosis factor (TNF)- and interleukin-1 (IL-1) receptor-dependent activation of stellate cells and endothelial cells, resulting in the transient production of chemokines and adhesion molecules orchestrating monocyte engraftment. Engrafted circulating monocytes transmigrated into the perisinusoidal space and acquired the liver-associated transcription factors inhibitor of DNA 3 (ID3) and liver X receptor-α (LXR-α). Coordinated interactions with hepatocytes induced ID3 expression, whereas endothelial cells and stellate cells induced LXR-α via a synergistic NOTCH-BMP pathway. This study shows that the Kupffer cell niche is composed of stellate cells, hepatocytes, and endothelial cells that together imprint the liver-specific macrophage identity.

Targeted Studies Using Serial Block Face and Focused Ion Beam Scan Electron Microscopy.

This protocol allows for the efficient and effective imaging of cell or tissue samples in three dimensions at the resolution level of electron microscopy. For many years electron microscopy (EM) has remained an inherently two-dimensional technique. With the advent of serial scanning electron microscope imaging techniques (volume EM), using either an integrated microtome or focused ion beam to slice then view embedded tissues, the third dimension becomes easily accessible. Serial block face scanning electron microscopy (SBF-SEM) uses an ultramicrotome enclosed in the SEM chamber. It has the capability to handle large specimens (1,000 µm x 1,000 µm) and image large fields of view at small X,Y pixel size, but is limited in the Z dimension by the diamond knife. Focused ion beam SEM (FIB-SEM) is not limited in 3D resolution, (isotropic voxels of ≤5 nm are achievable), but the field of view is much more limited. This protocol demonstrates a workflow for combining the two techniques to allow for finding individual regions of interest (ROIs) in a large field and then imaging the subsequent targeted volume at high isotropic voxel resolution. Preparing fixed cells or tissues is more demanding for volume EM techniques due to the extra contrasting needed for efficient signal generation in SEM imaging. Such protocols are time consuming and labor intensive. This protocol also incorporates microwave assisted tissue processing facilitating the penetration of reagents, which reduces the time needed for the processing protocol from days to hours.

Serial block face-scanning electron microscopy for volume electron microscopy.

There are different technologies that can be used to obtain a 3D image at nanometer resolution. Over the past decade, there has been a growing interest in applying Serial Block Face Scanning Electron Microscopy (SBF-SEM) in different fields of life science research. This technology has the advantage that it can cover a range of volumes, going from monolayers to multiple tissue layers in all three dimensions. SBF-SEM was originally used in neuroscience and then expanded to other research domains. The whole process of sample preparation for SBF-SEM is very long and consists of many steps, which makes adjustment of a given workflow very challenging. Here we describe the SBF-SEM workflow and those steps in the process that can be tweaked for any sample.

Combining serial block face and focused ion beam scanning electron microscopy for 3D studies of rare events.

Volume electron microscopy allows for the automated acquisition of serial-section imaging data that can be reconstructed in three-dimensions (3D) to provide a detailed, geometrically accurate view of cellular ultrastructure. Two, volume electron microscopy (EM) techniques, serial block face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM), use a similar slice-and-view approach but differ in their fields of view and 3D resolution. This chapter highlights a workflow where the ability of SBF-SEM to image a large field of view is combined with the precise sectioning capability of FIB-SEM to first locate a rare cellular event in a large tissue volume and then inspect the event with higher resolution. Using these two EM platforms in synergy is a powerful technique and can be useful for both simple structural studies as well as correlative studies using both light and electron microscopy.

Longitudinal assessment of cerebral perfusion and vascular response to hypoventilation in a bigenic mouse model of Alzheimer's disease with amyloid and tau pathology.

Alzheimer's disease is the most common neurodegenerative disease, and many patients also present with vascular dysfunction. In this study, we aimed to assess cerebral blood flow (CBF) and cerebrovascular response (CVR) as early, pre-symptomatic (3 months of age), imaging markers in a bigenic model of Alzheimer's disease (APP.V717IxTau.P301L, biAT) and in the monogenic parental strains. We further developed our previously published combination of pulsed arterial spin labeling perfusion MRI and hypo-ventilation paradigm, which allows weaning of the mice from the ventilator. Furthermore, the commonly used isoflurane anesthesia induces vasodilation and is thereby inherently a vascular challenge. We therefore assessed perfusion differences in the mouse models under free-breathing isoflurane conditions. We report (i) that we can determine CBF and hypoventilation-based CVR under ketamine/midazolam anesthesia and wean mice from the ventilator, making it a valuable tool for assessment of CBF and CVR in mice, (ii) that biAT mice exhibit lower cortical CBF than wild-type mice at age 3 months, (iii) that CVR was increased in both biAT and APP.V717I mice but not in Tau.P301L mice, identifying the APP genotype as a strong influencer of brain CVR and (iv) that perfusion differences at baseline are masked by the widely used isoflurane anesthesia.

Novel Phospho-Tau Monoclonal Antibody Generated Using a Liposomal Vaccine, with Enhanced Recognition of a Conformational Tauopathy Epitope.

The microtubule-associated protein Tau is an intrinsically unfolded, very soluble neuronal protein. Under still unknown circumstances, Tau protein forms soluble oligomers and insoluble aggregates that are closely linked to the cause and progression of various brain pathologies, including Alzheimer's disease. Previously we reported the development of liposome-based vaccines and their efficacy and safety in preclinical mouse models for tauopathy. Here we report the use of a liposomal vaccine for the generation of a monoclonal antibody with particular characteristics that makes it a valuable tool for fundamental studies as well as a candidate antibody for diagnostic and therapeutic applications. The specificity and affinity of antibody ACI-5400 were characterized by a panel of methods: (i) measuring the selectivity for a specific phospho-Tau epitope known to be associated with tauopathy, (ii) performing a combination of peptide and protein binding assays, (iii) staining of brain sections from mouse preclinical tauopathy models and from human subjects representing six different tauopathies, and (iv) evaluating the selective binding to pathological epitopes on extracts from tauopathy brains in non-denaturing sandwich assays. We conclude that the ACI-5400 antibody binds to protein Tau phosphorylated at S396 and favors a conformation that is typically present in the brain of tauopathy patients, including Alzheimer's disease.

Positive and negative early life experiences differentially modulate long term survival and amyloid protein levels in a mouse model of Alzheimer's disease.

Stress has been implicated as a risk factor for the severity and progression of sporadic Alzheimer's disease (AD). Early life experiences determine stress responsivity in later life, and modulate age-dependent cognitive decline. Therefore, we examined whether early life experiences influence AD outcome in a bigenic mouse model which progressively develops combined tau and amyloid pathology (biAT mice).Mice were subjected to either early life stress (ELS) or to 'positive' early handling (EH) postnatally (from day 2 to 9). In biAT mice, ELS significantly compromised long term survival, in contrast to EH which increased life expectancy. In 4 month old mice, ELS-reared biAT mice displayed increased hippocampal Aß levels, while these levels were reduced in EH-reared biAT mice. No effects of ELS or EH were observed on the brain levels of APP, protein tau, or PSD-95. Dendritic morphology was moderately affected after ELS and EH in the amygdala and medial prefrontal cortex, while object recognition memory and open field performance were not affected. We conclude that despite the strong transgenic background, early life experiences significantly modulate the life expectancy of biAT mice. Parallel changes in hippocampal Aß levels were evident, without affecting cognition of young adult biAT mice.

Phosphorylation of protein Tau by GSK3ß prolongs survival of bigenic Tau.P301L×GSK3ß mice by delaying brainstem tauopathy.

Tau.P301L transgenic mice suffer precocious mortality between ages 8 and 11 months, resulting from upper airway defects caused by tauopathy in autonomic brainstem circuits that control breathing (Dutschmann et al., 2010). In individual mice, the clinical phenotype evolves progressively and rapidly (3-6weeks) from clasping, over general motor impairment to severe reduction in body-weight into the terminal phase that announces imminent death (<3days). Surprisingly, co-expression of GSK3ß with Tau.P301L significantly prolonged survival of bigenic biGT mice (Terwel et al., 2008), which we here assign to delayed development of brainstem tauopathy. Eventually, brainstem tauopathy became as prominent in old biGT mice in the specified brainstem nuclei as in the parental Tau.P301L mice, resulting in similar clinical deterioration and terminal phase preceding death, although at later age. Biochemically, in both genotypes the pathway to neurofibrillary tangles and neuropil threads was similar: phosphorylation of protein Tau and formation of soluble oligomers and insoluble aggregates, ending in the typical tangles and threads of tauopathy. The extra GSK3ß activity led to expected increased phosphorylation of protein Tau, particularly at residues S262 and S396, which we must conclude to delay the aggregation of protein Tau in the brainstem of aging biGT mice. The unexpected, paradoxical alleviation of the brainstem problems in biGT mice allowed them to grow older and thereby develop more severe tauopathy in forebrain than Tau.P301L mice, which succumb at younger age.

Terminal hypothermic Tau.P301L mice have increased Tau phosphorylation independently of glycogen synthase kinase 3α/ß.

The microtubule-associated protein Tau is responsible for a large group of neurodegenerative disorders, known as tauopathies, including Alzheimer's disease. Tauopathy result from augmented and/or aberrant phosphorylation of Tau. Besides aging and various genetic and epigenetic defects that remain largely unknown, an important non-genetic agent that contributes is hypothermia, eventually caused by anesthesia. Remarkably, tauopathy in brains of hibernating mammals is not pathogenic, and, because it is fully reversible, is even considered to be neuroprotective. Here, we assessed the terminal phase of Tau.P301L mice and bigenic crosses with mice lacking glycogen synthase kinase 3 (GSK3)α completely, or GSK3ß specifically in neurons. We also analysed biGT bigenic mice that co-express Tau.P301L with GSK3ß.S9A and develop severe forebrain tauopathy with age. We found that the precocious mortality of Tau.P301L mice was typified by hypothermia that aggravated Tau phosphorylation, but, surprisingly, independently of GSK3α/ß. The important contribution of hypothermia at the time of death of mice with tauopathy suggests that body temperature should be included as a parameter in the analysis of pre-clinical models, and, by extension, in patients suffering from tauopathy.

Early structural and functional defects in synapses and myelinated axons in stratum lacunosum moleculare in two preclinical models for tauopathy.

The stratum lacunosum moleculare (SLM) is the connection hub between entorhinal cortex and hippocampus, two brain regions that are most vulnerable in Alzheimer's disease. We recently identified a specific synaptic deficit of Nectin-3 in transgenic models for tauopathy. Here we defined cognitive impairment and electrophysiological problems in the SLM of Tau.P301L mice, which corroborated the structural defects in synapses and dendritic spines. Reduced diffusion of DiI from the ERC to the hippocampus indicated defective myelinated axonal pathways. Ultrastructurally, myelinated axons in the temporoammonic pathway (TA) that connects ERC to CA1 were damaged in Tau.P301L mice at young age. Unexpectedly, the myelin defects were even more severe in bigenic biGT mice that co-express GSK3ß with Tau.P301L in neurons. Combined, our data demonstrate that neuronal expression of protein Tau profoundly affected the functional and structural organization of the entorhinal-hippocampal complex, in particular synapses and myelinated axons in the SLM. White matter pathology deserves further attention in patients suffering from tauopathy and Alzheimer's disease.

Increasing brain protein O-GlcNAc-ylation mitigates breathing defects and mortality of Tau.P301L mice.

The microtubule associated protein tau causes primary and secondary tauopathies by unknown molecular mechanisms. Post-translational O-GlcNAc-ylation of brain proteins was demonstrated here to be beneficial for Tau.P301L mice by pharmacological inhibition of O-GlcNAc-ase. Chronic treatment of ageing Tau.P301L mice mitigated their loss in body-weight and improved their motor deficits, while the survival was 3-fold higher at the pre-fixed study endpoint at age 9.5 months. Moreover, O-GlcNAc-ase inhibition significantly improved the breathing parameters of Tau.P301L mice, which underpinned pharmacologically the close correlation of mortality and upper-airway defects. O-GlcNAc-ylation of brain proteins increased rapidly and stably by systemic inhibition of O-GlcNAc-ase. Conversely, biochemical evidence for protein Tau.P301L to become O-GlcNAc-ylated was not obtained, nor was its phosphorylation consistently or markedly affected. We conclude that increasing O-GlcNAc-ylation of brain proteins improved the clinical condition and prolonged the survival of ageing Tau.P301L mice, but not by direct biochemical action on protein tau. The pharmacological effect is proposed to be located downstream in the pathological cascade initiated by protein Tau.P301L, opening novel venues for our understanding, and eventually treating the neurodegeneration mediated by protein tau.

Efficacy and safety of a liposome-based vaccine against protein Tau, assessed in tau.P301L mice that model tauopathy.

Progressive aggregation of protein Tau into oligomers and fibrils correlates with cognitive decline and synaptic dysfunction, leading to neurodegeneration in vulnerable brain regions in Alzheimer's disease. The unmet need of effective therapy for Alzheimer's disease, combined with problematic pharmacological approaches, led the field to explore immunotherapy, first against amyloid peptides and recently against protein Tau. Here we adapted the liposome-based amyloid vaccine that proved safe and efficacious, and incorporated a synthetic phosphorylated peptide to mimic the important phospho-epitope of protein Tau at residues pS396/pS404. We demonstrate that the liposome-based vaccine elicited, rapidly and robustly, specific antisera in wild-type mice and in Tau.P301L mice. Long-term vaccination proved to be safe, because it improved the clinical condition and reduced indices of tauopathy in the brain of the Tau.P301L mice, while no signs of neuro-inflammation or other adverse neurological effects were observed. The data corroborate the hypothesis that liposomes carrying phosphorylated peptides of protein Tau have considerable potential as safe and effective treatment against tauopathies, including Alzheimer's disease.

Tauopathy differentially affects cell adhesion molecules in mouse brain: early down-regulation of nectin-3 in stratum lacunosum moleculare.

Cell adhesion molecules are important structural substrates, required for synaptic plasticity and synaptogenesis. CAMs differ widely in their expression throughout different brain regions and their specific structural and functional roles in the brain remain to be elucidated. Here, we investigated selected cell adhesion molecules for alterations in expression levels and neuronal localization in validated mouse models for Alzheimer's disease that mimic the age-related progression of amyloid accumulation and tauopathy. Among the cell adhesion molecules analyzed, Nectin-3 expression was affected most and specifically in all mouse models with tauopathy. In particular was Nectin-3 depleted from the specific region of the hippocampus, known as the stratum lacunosum and moleculare, in mice that express wild-type or mutant human protein Tau, either chronically or sub-acutely. Tauopathy progresses from the entorhinal cortex to the hippocampus by unknown mechanisms that could involve transport by the myelinated axons of the temporoammonic and perforant pathways. The decreased expression of Nectin-3 in the stratum lacunosum moleculare is an early marker of impaired transport, and eventual synaptic problems, caused by beginning tauopathy.