Fast 3-D Imaging of Brain Organoids With a New Single-Objective Planar-Illumination Two-Photon Microscope.

Human inducible pluripotent stem cells (hiPSCs) hold a large potential for disease modeling. hiPSC-derived human astrocyte and neuronal cultures permit investigations of neural signaling pathways with subcellular resolution. Combinatorial cultures, and three-dimensional (3-D) embryonic bodies (EBs) enlarge the scope of investigations to multi-cellular phenomena. The highest level of complexity, brain organoids that-in many aspects-recapitulate anatomical and functional features of the developing brain permit the study of developmental and morphological aspects of human disease. An ideal microscope for 3-D tissue imaging at these different scales would combine features from both confocal laser-scanning and light-sheet microscopes: a micrometric optical sectioning capacity and sub-micrometric spatial resolution, a large field of view and high frame rate, and a low degree of invasiveness, i.e., ideally, a better photon efficiency than that of a confocal microscope. In the present work, we describe such an instrument that uses planar two-photon (2P) excitation. Its particularity is that-unlike two- or three-lens light-sheet microscopes-it uses a single, low-magnification, high-numerical aperture objective for the generation and scanning of a virtual light sheet. The microscope builds on a modified Nipkow-Petrán spinning-disk scheme for achieving wide-field excitation. However, unlike the Yokogawa design that uses a tandem disk, our concept combines micro lenses, dichroic mirrors and detection pinholes on a single disk. This new design, advantageous for 2P excitation, circumvents problems arising with the tandem disk from the large wavelength difference between the infrared excitation light and visible fluorescence. 2P fluorescence excited by the light sheet is collected with the same objective and imaged onto a fast sCMOS camera. We demonstrate 3-D imaging of TO-PRO3-stained EBs and of brain organoids, uncleared and after rapid partial transparisation with triethanolamine formamide (RTF) and we compare the performance of our instrument to that of a confocal laser-scanning microscope (CLSM) having a similar numerical aperture. Our large-field 2P-spinning disk microscope permits one order of magnitude faster imaging, affords less photobleaching and permits better depth penetration than a confocal microscope with similar spatial resolution.

Inclusions of R6/2 Mice Are Not Amyloid and Differ Structurally from Those of Huntington Disease Brain.

R6/2 mice contain an N-terminal fragment of human huntingtin with an expanded polyQ and develop a neurological disease resembling Huntington disease. Although the brain of R6/2 mice contains numerous inclusions, there is very little neuronal death. In that respect, R6/2 mice differ from patients with Huntington disease whose striatum and cerebral cortex develop inclusions associated with extensive neuronal loss. We have previously demonstrated using synchrotron-based infrared microspectroscopy that the striatum and the cortex of patients with Huntington disease contained inclusions specifically enriched in amyloid ß-sheets. We had concluded that the presence of an amyloid motif conferred toxicity to the inclusions. We demonstrate here by synchrotron based infrared microspectroscopy in transmission and attenuated total reflectance mode that the inclusions of R6/2 mice possess no detectable amyloid and are composed of proteins whose structure is not distinguishable from that of the surrounding soluble proteins. The difference in structure between the inclusions of patients affected by Huntington disease and those of R6/2 mice might explain why the former but not the latter cause neuronal death.

Identification of brain substrates of transglutaminase by functional proteomics supports its role in neurodegenerative diseases.

Transglutaminases are calcium-dependent enzymes that catalyze the formation of ε-(γ-glutamyl)lysine isopeptide bonds between specific glutamine and lysine residues. Some transglutaminase isoforms are present in the brain and are thought to participate in the protein aggregation characteristic of neurological diseases such as Huntington, Alzheimer's and Parkinson's disease. We have developed a functional proteomics strategy in which biotinylated amine-donor and amine-acceptor probes were used to identify the transglutaminase substrates present in brain. Bioinformatics analyses revealed that most of the 166 brain substrates identified interacted with huntingtin, the amyloid precursor protein or α-synuclein and that neurological disease was the most significant canonical pathway associated with the substrates. The physiological relevance of the substrates identified by mass spectrometry was confirmed by the fact that three of them (actin, ß-tubulin and a neurofilament subunit) were polymerized in neuronal cells when cytosolic calcium concentration was raised. We also showed by in-situ immunolabeling that some of the substrates were part of the protein aggregates found in neurological diseases. These results strongly support the idea that the crosslinking activity of brain transglutaminase participates in the formation of the protein aggregates found in diseases of the central nervous system.

The importance of basonuclin 2 in adult mice and its relation to basonuclin 1.

BNC2 is an extremely conserved zinc finger protein with important functions in the development of craniofacial bones and male germ cells. Because disruption of the Bnc2 gene in mice causes neonatal lethality, the function of the protein in adult animals has not been studied. Until now BNC2 was considered to have a wider tissue distribution than its paralog, BNC1, but the precise cell types expressing Bnc2 are largely unknown. We identify here the cell types containing BNC2 in the mouse and we show the unexpected presence of BNC1 in many BNC2-containing cells. BNC1 and BNC2 are colocalized in male and female germ cells, ovarian epithelial cells, sensory neurons, hair follicle keratinocytes and connective cells of organ capsules. In many cell lineages, the two basonuclins appear and disappear synchronously. Within the male germ cell lineage, BNC1 and BNC2 are found in prospermatogonia and undifferentiated spermatogonia, and disappear abruptly from differentiating spermatogonia. During oogenesis, the two basonuclins accumulate specifically in maturing oocytes. During the development of hair follicles, BNC1 and BNC2 concentrate in the primary hair germs. As follicle morphogenesis proceeds, cells possessing BNC1 and BNC2 invade the dermis and surround the papilla. During anagen, BNC1 and BNC2 are largely restricted to the basal layer of the outer root sheath and the matrix. During catagen, the compartment of cells possessing BNC1 and BNC2 regresses, and in telogen, the two basonuclins are confined to the secondary hair germ. During the next anagen, the BNC1/BNC2-containing cell population regenerates the hair follicle. By examining Bnc2(-/-) mice that have escaped the neonatal lethality usually associated with lack of BNC2, we demonstrate that BNC2 possesses important functions in many of the cell types where it resides. Hair follicles of postnatal Bnc2(-/-) mice do not fully develop during the first cycle and thereafter remain blocked in telogen. It is concluded that the presence of BNC2 in the secondary hair germ is required to regenerate the transient segment of the follicle. Postnatal Bnc2(-/-) mice also show severe dwarfism, defects in oogenesis and alterations of palatal rugae. Although the two basonuclins possess very similar zinc fingers and are largely coexpressed, BNC1 cannot substitute for BNC2. This is shown incontrovertibly in knockin mice expressing Bnc1 instead of Bnc2 as these mice invariably die at birth with craniofacial abnormalities undistinguishable from those of Bnc2(-/-) mice. The function of the basonuclins in the secondary hair germ is of particular interest.

Polyglutamine Aggregation in Huntington Disease: Does Structure Determine Toxicity?

Huntington disease is a dominantly inherited disease of the central nervous system. The mutational expansion of polyglutamine beyond a critical length produces a toxic gain of function in huntingtin and results in neuronal death. In the course of the disease, expanded huntingtin is proteolyzed, becomes abnormally folded, and accumulates in oligomers, fibrils, and microscopic inclusions. The aggregated forms of the expanded protein are structurally diverse. Structural heterogeneity may explain why polyglutamine-containing aggregates could paradoxically be either toxic or neuroprotective. When defined, the toxic structures could then specifically be targeted by prophylactic or therapeutic drugs aimed at inhibiting polyglutamine aggregation.

The zinc-finger protein basonuclin 2 is required for proper mitotic arrest, prevention of premature meiotic initiation and meiotic progression in mouse male germ cells.

Absence of mitosis and meiosis are distinguishing properties of male germ cells during late fetal and early neonatal periods. Repressors of male germ cell meiosis have been identified, but mitotic repressors are largely unknown, and no protein repressing both meiosis and mitosis is known. We demonstrate here that the zinc-finger protein BNC2 is present in male but not in female germ cells. In testis, BNC2 exists as several spliced isoforms and presumably binds to DNA. Within the male germ cell lineage, BNC2 is restricted to prospermatogonia and undifferentiated spermatogonia. Fetal prospermatogonia that lack BNC2 multiply excessively on embryonic day (E)14.5 and reenter the cell cycle prematurely. Mutant prospermatogonia also engage in abnormal meiosis; on E17.5, Bnc2(-/-) prospermatogonia start synthesizing the synaptonemal protein SYCP3, and by the time of birth, many Bnc2(-/-) prospermatogonia have accumulated large amounts of nonfilamentous SYCP3, thus appearing to be blocked at leptonema. Bnc2(-/-) prospermatogonia do not undergo proper male differentiation, as they lack almost all the mRNA for the male-specific methylation protein DNMT3L and have increased levels of mRNAs that encode meiotic proteins, including STRA8. Bnc2(-/-) prospermatogonia can produce spermatogonia, but these enter meiosis prematurely and undergo massive apoptotic death during meiotic prophase. This study identifies BNC2 as a major regulator of male germ stem cells, which is required for repression of meiosis and mitosis in prospermatogonia, and for meiosis progression during spermatogenesis. In view of the extreme evolutionary conservation of BNC2, the findings described here are likely to apply to many species.

Monomeric, oligomeric and polymeric proteins in huntington disease and other diseases of polyglutamine expansion.

Huntington disease and other diseases of polyglutamine expansion are each caused by a different protein bearing an excessively long polyglutamine sequence and are associated with neuronal death. Although these diseases affect largely different brain regions, they all share a number of characteristics, and, therefore, are likely to possess a common mechanism. In all of the diseases, the causative protein is proteolyzed, becomes abnormally folded and accumulates in oligomers and larger aggregates. The aggregated and possibly the monomeric expanded polyglutamine are likely to play a critical role in the pathogenesis and there is increasing evidence that the secondary structure of the protein influences its toxicity. We describe here, with special attention to huntingtin, the mechanisms of polyglutamine aggregation and the modulation of aggregation by the sequences flanking the polyglutamine. We give a comprehensive picture of the characteristics of monomeric and aggregated polyglutamine, including morphology, composition, seeding ability, secondary structure, and toxicity. The structural heterogeneity of aggregated polyglutamine may explain why polyglutamine-containing aggregates could paradoxically be either toxic or neuroprotective.

Structure of inclusions of Huntington's disease brain revealed by synchrotron infrared microspectroscopy: polymorphism and relevance to cytotoxicity.

Huntington's disease is caused by a polyglutamine expansion in huntingtin. Affected brain regions contain characteristic aggregates of the misfolded expanded protein. Studies in cells and animals show that aggregates are polymorphic and that the secondary structure of the aggregates is likely to condition their cytotoxicity. Therefore knowing the structure of aggregates is important as neurotoxic secondary structures may be specifically targeted during the search for prophylactic or therapeutic drugs. The structure of aggregates in the brain of patients is still unknown. Using synchrotron based infrared microspectroscopy we demonstrate that the brains of patients with Huntington disease contain putative oligomers and various kinds of microscopic aggregates (inclusions) that can be distinguished by their differential absorbance at 1627 cm(-1) (amyloid ß sheets) and 1639 cm(-1) (ß sheets/unordered). We also describe the parallel/antiparallel organization of the ß strands. As the inclusions enriched in both ß sheets and ß sheets/unordered structures are characteristic of severely affected brain regions, we conclude that this kind of amyloid inclusions is likely to be particularly toxic to neurons.

[Basonuclins and DISCO proteins: regulators of development in vertebrates and insects]. / Basonuclines et protéines DISCO: des régulateurs du développement des vertébrés et des insectes.

Basonuclins 1 and 2 are nuclear proteins specific to vertebrates. They have recently been shown to be the orthologs of the DISCO proteins of insects. All of these proteins have an essential function in embryonic development. It is likely that they are transcription factors with multiple gene targets, some of which are transcribed by DNA polymerase I, and others by polymerase II. It remains to be found which targets are common to the two basonuclins and even to the DISCO proteins, and why certain cell types possess either basonuclin 1 or basonuclin 2, while others possess both. The implication of the basonuclins in several human diseases adds to their interest as regulatory proteins.

Control of gene expression by the retinoic acid-related orphan receptor alpha in HepG2 human hepatoma cells.

Retinoic acid-related Orphan Receptor alpha (RORα; NR1F1) is a widely distributed nuclear receptor involved in several (patho)physiological functions including lipid metabolism, inflammation, angiogenesis, and circadian rhythm. To better understand the role of this nuclear receptor in liver, we aimed at displaying genes controlled by RORα in liver cells by generating HepG2 human hepatoma cells stably over-expressing RORα. Genes whose expression was altered in these cells versus control cells were displayed using micro-arrays followed by qRT-PCR analysis. Expression of these genes was also altered in cells in which RORα was transiently over-expressed after adenoviral infection. A number of the genes found were involved in known pathways controlled by RORα, for instance LPA, NR1D2 and ADIPOQ in lipid metabolism, ADIPOQ and PLG in inflammation, PLG in fibrinolysis and NR1D2 and NR1D1 in circadian rhythm. This study also revealed that genes such as G6PC, involved in glucose homeostasis, and AGRP, involved in the control of body weight, are also controlled by RORα. Lastly, SPARC, involved in cell growth and adhesion, and associated with liver carcinogenesis, was up-regulated by RORα. SPARC was found to be a new putative RORα target gene since it possesses, in its promoter, a functional RORE as evidenced by EMSAs and transfection experiments. Most of the other genes that we found regulated by RORα also contained putative ROREs in their regulatory regions. Chromatin immunoprecipitation (ChIP) confirmed that the ROREs present in the SPARC, PLG, G6PC, NR1D2 and AGRP genes were occupied by RORα in HepG2 cells. Therefore these genes must now be considered as direct RORα targets. Our results open new routes on the roles of RORα in glucose metabolism and carcinogenesis within cells of hepatic origin.

Human balanced translocation and mouse gene inactivation implicate Basonuclin 2 in distal urethral development.

We studied a man with distal hypospadias, partial anomalous pulmonary venous return, mild limb-length inequality and a balanced translocation involving chromosomes 9 and 13. To gain insight into the etiology of his birth defects, we mapped the translocation breakpoints by high-resolution comparative genomic hybridization (CGH), using chromosome 9- and 13-specific tiling arrays to analyze genetic material from a spontaneously aborted fetus with unbalanced segregation of the translocation. The chromosome 13 breakpoint was ∼400 kb away from the nearest gene, but the chromosome 9 breakpoint fell within an intron of Basonuclin 2 (BNC2), a gene that encodes an evolutionarily conserved nuclear zinc-finger protein. The BNC2/Bnc2 gene is abundantly expressed in developing mouse and human periurethral tissues. In all, 6 of 48 unrelated subjects with distal hypospadias had nine novel nonsynonymous substitutions in BNC2, five of which were computationally predicted to be deleterious. In comparison, two of 23 controls with normal penile urethra morphology, each had a novel nonsynonymous substitution in BNC2, one of which was predicted to be deleterious. Bnc2(-/-) mice of both sexes displayed a high frequency of distal urethral defects; heterozygotes showed similar defects with reduced penetrance. The association of BNC2 disruption with distal urethral defects and the gene's expression pattern indicate that it functions in urethral development.

Basonuclins and disco: Orthologous zinc finger proteins essential for development in vertebrates and arthropods.

Basonuclin 1 and the recently discovered basonuclin 2 are vertebrate proteins with multiple paired C(2)H(2) zinc fingers. It has long been known that the zinc fingers of basonuclin 1 closely resembled those of the Drosophila disconnected and discorelated proteins, two proteins essential for head development, but the relation between the basonuclins and the disco proteins has remained unclear because the putative function of basonuclin 1 in the control of keratinocyte growth potential appeared unrelated to that of disco and there was no resemblance between basonuclin 1 and Drosophila disco outside of the zinc fingers. The recent generation of a basonuclin-2 knockout has demonstrated that basonuclin 2 shares with disco a function in head development and the availability of new arthropod genome sequences has shown that the basonuclins are the vertebrate orthologs of the insect disco proteins. All these proteins are thought to be transcription factors, and it will have to be determined to what extent they share similar targets.

Basonuclin 2 has a function in the multiplication of embryonic craniofacial mesenchymal cells and is orthologous to disco proteins.

Basonuclin 2 is a recently discovered zinc finger protein of unknown function. Its paralog, basonuclin 1, is associated with the ability of keratinocytes to multiply. The basonuclin zinc fingers are closely related to those of the Drosophila proteins disco and discorelated, but the relation between disco proteins and basonuclins has remained elusive because the function of the disco proteins in larval head development seems to have no relation to that of basonuclin 1 and because the amino acid sequence of disco, apart from the zinc fingers, also has no similarity to that of the basonuclins. We have generated mice lacking basonuclin 2. These mice die within 24 h of birth with a cleft palate and abnormalities of craniofacial bones and tongue. In the embryonic head, expression of the basonuclin 2 gene is restricted to mesenchymal cells in the palate, at the periphery of the tongue, and in the mesenchymal sheaths that surround the brain and the osteocartilagineous structures. In late embryos, the rate of multiplication of these mesenchymal cells is greatly diminished. Therefore, basonuclin 2 is essential for the multiplication of craniofacial mesenchymal cells during embryogenesis. Non-Drosophila insect databases available since 2008 reveal that the basonuclins and the disco proteins share much more extensive sequence and gene structure similarity than noted when only Drosophila sequences were examined. We conclude that basonuclin 2 is both structurally and functionally the vertebrate ortholog of the disco proteins. We also note the possibility that some human craniofacial abnormalities are due to a lack of basonuclin 2.

Misfolding of proteins with a polyglutamine expansion is facilitated by proteasomal chaperones.

Deposition of misfolded proteins with a polyglutamine expansion is a hallmark of Huntington disease and other neurodegenerative disorders. Impairment of the proteolytic function of the proteasome has been reported to be both a cause and a consequence of polyglutamine accumulation. Here we found that the proteasomal chaperones that unfold proteins to be degraded by the proteasome but also have non-proteolytic functions co-localized with huntingtin inclusions both in primary neurons and in Huntington disease patients and formed a complex independently of the proteolytic particle. Overexpression of Rpt4 or Rpt6 facilitated aggregation of mutant huntingtin and ataxin-3 without affecting proteasomal degradation. Conversely, reducing Rpt6 or Rpt4 levels decreased the number of inclusions in primary neurons, indicating that endogenous Rpt4 and Rpt6 facilitate inclusion formation. In vitro reconstitution experiments revealed that purified 19S particles promote mutant huntingtin aggregation. When fused to the ornithine decarboxylase destabilizing sequence, proteins with expanded polyglutamine were efficiently degraded and did not aggregate. We propose that aggregation of proteins with expanded polyglutamine is not a consequence of a proteolytic failure of the 20S proteasome. Rather, aggregation is elicited by chaperone subunits of the 19S particle independently of proteolysis.

Ancient origin of the gene encoding involucrin, a precursor of the cross-linked envelope of epidermis and related epithelia.

The cross-linked (cornified) envelope is a characteristic product of terminal differentiation in the keratinocyte of the epidermis and related epithelia. This envelope contains many proteins of which involucrin was the first to be discovered and shown to become cross-linked by a cellular transglutaminase. Involucrin has evolved greatly in placental mammals, but retains the glutamine repeats that make it a good substrate for the transglutaminase. Until recently, it has been impossible to detect involucrin outside the placental mammals, but analysis of the GenBank and Ensembl databases that have become available since 2006 reveals the existence of involucrin in marsupials and birds. We describe here the properties of these involucrins and the ancient history of their evolution.

[Nepsilon-(gamma-glutamyl) lysine] as a potential biomarker in neurological diseases: new detection method and fragmentation pathways.

Protein aggregates are characteristic of a number of diseases of the central nervous system such as diseases of polyQ expansion. Covalent bonds formed by the action of transglutaminase are thought to participate in the stabilization of these aggregates. Transglutaminase catalyzes the formation of cross-links between the side chains of glutaminyl and lysyl residues of polypeptides. Identification of the isodipeptide N(epsilon)-(gamma-glutamyl) lysine (iEK) in terminal proteolytic digests of neuronal aggregates would demonstrate participation of transglutaminase in neurological diseases. In order to identify and quantify the iEK present in the brain of patients with neurological disease, a method combining liquid chromatography and multistep mass spectrometry was developed. Because isobaric peptides of iEK could be present in the digest of aggregated proteins, the choice of fragment diagnostic ions was crucial. These ions were identified by mass spectrometry on sodiated iEK, which was derivatized on the carboxylic functions and terminal amines in order to improve sensitivity. Deuterated molecules as well as (13)C(6)- and (15)N(2)-isotopomers were used to derive filiations in the multistep fragmentations. The main fragmentation patterns have been identified, so that two ions (m/z 396 [MH - 56-42 u](+) and 350 [MH - 56-88 u](+)) are shown to be adequate markers for quantitation experiments. In order to gain a better understanding of the fragmentation processes, detailed quantum chemical calculations have been performed at levels which are expected to provide good accuracy. A thorough study has been carried out with a reduced model in which only the 'active' part of the molecule is retained. This allowed obtaining full mechanistic details on the pathways leading to a number of observed fragments. In particular, it has been shown that losses of 87 and 88 u from A(+) = [MH - 56 u](+) are competitive. Computations on the entire derivatized isodipeptide have been used to validate the use of the smaller model in order to obtain reliable energetics and mechanisms.

Mice deficient in involucrin, envoplakin, and periplakin have a defective epidermal barrier.

The cornified envelope is assembled from transglutaminase cross-linked proteins and lipids in the outermost epidermal layers and is essential for skin barrier function. Involucrin, envoplakin, and periplakin form the protein scaffold on which the envelope assembles. To examine their combined function, we generated mice deficient in all three genes. The triple knockouts have delayed embryonic barrier formation and postnatal hyperkeratosis (abnormal accumulation of cornified cells) resulting from impaired desquamation. Cornified envelopes form but are ultrastructurally abnormal, with reduced lipid content and decreased mechanical integrity. Expression of proteases is reduced and the protease inhibitor, serpina1b, is highly upregulated, resulting in defective filaggrin processing and delayed degradation of desmoglein 1 and corneodesmosin. There is infiltration of CD4+ T cells and a reduction in resident gammadelta+ T cells, reminiscent of atopic dermatitis. Thus, combined loss of the cornified envelope proteins not only impairs the epidermal barrier, but also changes the composition of T cell subpopulations in the skin.

The human basonuclin 2 gene has the potential to generate nearly 90,000 mRNA isoforms encoding over 2000 different proteins.

The number of mRNAs and proteins that can be produced from a single gene is known to be increased by the number of start sites and by multiple splicing of products. A few genes have been found to generate extraordinarily large numbers of splicing isoforms. In the human, the largest number, nearly 2000 mRNA isoforms, has been reported for the neurexin 3alpha gene. However, the biological significance of alternative splicing often remains unclear because many alternative transcripts contain early translational stops and are thought to be rapidly degraded. We demonstrate here that human basonuclin 2 (bn2; approved gene symbol BNC2) transcripts are initiated from six promoters, are alternatively spliced at multiple positions, and are polyadenylated at four sites. Characterization of nearly 100 bn2 mRNA isoforms suggests that each promoter, splice site, and poly(A) addition site is used independently. The bn2 gene has therefore the potential to generate up to 90,000 mRNA isoforms encoding more than 2000 different proteins. Because alternative exons affect the position of the first methionine codon, the length of the coding region, and the position of the translational stop, the encoded proteins range in size from 43 to 1211 amino acids and some bear no sequence similarity to others. PCR analysis and transient expression in HeLa cells show that the major bn2 mRNA isoforms are stable and are translated into equally stable proteins, even when the mRNA bears an early translational stop.