Protocol for optical clearing and imaging of fluorescently labeled ex vivo rat brain slices.

Tissue clearing is commonly used for whole-brain imaging but seldom used for brain slices. Here, we present a simple protocol to slice, immunostain, and clear sections of adult rat brains for subsequent high-resolution confocal imaging. The protocol does not require toxic reagents or specialized equipment. We also provide instructions for culturing of rat brain slices free floating on permeable culture inserts, maintained in regular CO2 incubators, and handled only at media change.

Exploring the K isotope composition of Göttingen minipig brain regions, and implications for Alzheimer's disease.

Natural stable metal isotopes have shown utility in differentiation between healthy and diseased brain states (e.g. Alzheimer's disease, AD). While the AD brain accumulates some metals, it purges others, namely K (accompanied by increased serum K, suggesting brain-blood transferal). Here, K isotope compositions of Göttingen minipig brain regions for two AD models at midlife are reported. Results indicate heavy K isotope enrichment where amyloid beta (Aß) accumulation is observed, and this enrichment correlates with relative K depletion. These results suggest preferential efflux of isotopically light K+ from the brain, a linkage between brain K concentrations and isotope compositions, and linkage to Aß (previously shown to purge cellular brain K+). Brain K isotope compositions differ from that for serum and brain K is much more abundant than in serum, suggesting that changes in brain K may transfer a measurable K isotope excursion to serum, thereby generating an early AD biomarker.

Directly Reprogrammed Neurons Express MAPT and APP Splice Variants Pertinent to Ageing and Neurodegeneration.

Neurons produced by reprogramming of other cell types are used to study cellular mechanisms of age-related neurodegenerative diseases. To model Alzheimer's disease and other tauopathies, it is essential that alternative splicing of the MAPT transcript in these neurons produces the relevant tau isoforms. Human neurons derived from induced pluripotent stem cells, however, express tau isoform compositions characteristic of foetal neurons rather than of adult neurons unless cultured in vitro for extended time periods. In this study, we characterised the dynamics of the MAPT and APP alternative splicing during a developmental time-course of porcine and murine cerebral cortices. We found age-dependent and species-specific isoform composition of MAPT, including 3R and 4R isoforms in the porcine adult brain similar to that of the adult human brain. We converted adult and embryonic fibroblasts directly into induced neurons and found similar developmental patterns of isoform composition, notably, the 3R and 4R isoforms relevant to the pathogenesis of Alzheimer's disease. Also, we observed cell-type-specific isoform expression of APP transcripts during the conversion. The approach was further used to generate induced neurons from transgenic pigs carrying Alzheimer's disease-causing mutations. We show that such neurons authentically model the first crucial steps in AD pathogenesis.

MicroRNAs and Ascl1 facilitate direct conversion of porcine fibroblasts into induced neurons.

Direct neuronal conversion describes the process of generating induced neurons from somatic cells such as fibroblasts by overexpressing cell type-specific transcription factors, microRNAs or by culturing in the presence of small molecules. This was first achieved by expressing Brn2, Ascl1 and Myt1L in mouse fibroblasts, and was later achieved in human cells by the inclusion of additional factors such as NeuroD1. Here, we present the first protocol for directly converting porcine fibroblasts into induced neurons. We used lentivirus-mediated delivery of previously identified neuron-specifying transcription factors and microRNAs and evaluated morphology and neuron marker expression after ten days of conversion. We found that Ascl1 and microRNAs, miR-9/9* and miR-124 together generated more neuronal cells than other conditions tested. The porcine induced neurons expressed common mature markers such as MAP2 and Synaptophysin after four weeks of conversion. Transcriptomic analysis revealed that fibroblast-specific signatures were silenced early in the conversion process, while the neuron-specific genes became more abundant during conversion. We generated a heterogeneous population of glutamatergic and GABAergic neurons.

Rapid generation of regionally specified CNS neurons by sequential patterning and conversion of human induced pluripotent stem cells.

The differentiation of patient-specific induced pluripotent stem cells (iPSCs) into specific neuronal subtypes has been exploited as an approach for modeling a variety of neurological disorders. However, achieving a highly pure population of neurons is challenging when using directed differentiation methods, especially for neuronal subtypes generated by complex and protracted protocols. In this study, we efficiently produced highly pure populations of regionally specified CNS neurons by using a modified NGN2-Puromycin direct conversion protocol. The protocol is amenable across a range of iPSC lines, with more than 95% of cells at day 21 positive for the neuronal marker MAP2. We found that conversion from pluripotent stem cells resulted in neurons from the central and peripheral nervous system; however, by incorporating a short CNS patterning step, we eliminated these peripheral neurons. Furthermore, we used the patterning step to control the rostral-caudal identity. This approach of sequential patterning and conversion produced pure populations of forebrain neurons, when patterned with SMAD inhibitors. Additionally, when SMAD inhibitors and WNT agonists were applied, the approach produced anterior hindbrain excitatory neurons and resulted in a neuronal population containing VSX2/SHOX2 V2a interneurons. Overall, this sequential patterning and conversion protocol can be used for the production of a variety of CNS excitatory neurons from patient-derived iPSCs, and is a highly versatile system for investigating early disease events for a range of neurological disorders including Alzheimer's disease, motor neurons disease and spinal cord injury.

Longitudinal biometal accumulation and Ca isotope composition of the Göttingen minipig brain.

Biometals play a critical role in both the healthy and diseased brain's functioning. They accumulate in the normal aging brain, and are inherent to neurodegenerative disorders and their associated pathologies. A prominent example of this is the brain accumulation of metals such as Ca, Fe and Cu (and more ambiguously, Zn) associated with Alzheimer's disease (AD). The natural stable isotope compositions of such metals have also shown utility in constraining biological mechanisms, and in differentiating between healthy and diseased states, sometimes prior to conventional methods. Here we have detailed the distribution of the biologically relevant elements Mg, P, K, Ca, Fe, Cu and Zn in brain regions of Göttingen minipigs ranging in age from three months to nearly six years, including control animals and both a single- and double-transgenic model of AD (PS1, APP/PS1). Moreover, we have characterized the Ca isotope composition of the brain for the first time. Concentration data track rises in brain biometals with age, namely for Fe and Cu, as observed in the normal ageing brain and in AD, and biometal data point to increased soluble amyloid beta (Aß) load prior to AD plaque identification via brain imaging. Calcium isotope results define the brain as the isotopically lightest permanent reservoir in the body, indicating that brain Ca dyshomeostasis may induce measurable isotopic disturbances in accessible downstream reservoirs such as biofluids.

Transcriptomic profiling of porcine pluripotency identifies species-specific reprogramming requirements for culturing iPSCs.

Porcine embryonic and induced pluripotent stem cells (ESCs; iPSCs) have proven difficult to derive and maintain in vitro. This may be due to inappropriate culturing conditions and incomplete activation of proper pluripotency networks. To this end, we characterized the transcriptome of porcine inner cell mass, epiblast, and transgene-dependent iPSCs in relation to human and mouse embryonic and epiblast stem cells. We found that porcine inner cell mass has a unique pluripotency transcriptome distinct from human and mouse ESCs but shares more features with human naïve-like than primed stem cell states, as illustrated by their expression of KLF17 but not KLF2. Our data further show that current reprogramming strategies fail to silence parental fibroblast-specific genes and to activate specific signalling pathways that may be important for porcine pluripotency. Accordingly, we used human naïve culturing conditions to improve reprogramming efficiencies of porcine embryonic fibroblasts and enable essential naïve stem cell markers such as NANOG, KLF17 and CDH1to be expressed. The resultant porcine iPSC-like cells display a transcriptomic signature more closely resembling an inner cell mass state. These results represent new important steps towards generating bona fide porcine iPSCs and their great potential in translational medicine.

Examining the homeostatic distribution of metals and Zn isotopes in Göttingen minipigs.

The role of metals in biologic systems is manifold, and understanding their behaviour in bodily processes, especially those relating to neurodegenerative diseases, is at the forefront of medical science. The function(s) of metals - such as the transition metals - and their utility in both the diagnosis and treatment of diseases in human beings, is often examined via the characterization of their distribution in animal models, with porcine models considered exceptional proxies for human physiology. To this end, we have investigated the homeostatic distribution of numerous metals (Mg, K, Ca, Mn, Fe, Cu, Zn, Rb and Mo), the non-metal P, and Zn isotopes in the organs and blood (red blood cells, plasma) of Göttingen minipigs. These results represent the first set of data outlining the homeostatic distribution of metals and Zn isotopes in Göttingen minipigs, and indicate a relatively homogeneous distribution of alkali/alkaline earth metals and P among the organs, with generally lower levels in the blood, while indicating more heterogeneous and systematic abundance patterns for transition metals. In general, the distribution of all elements analysed is similar to that found in humans. Our elemental abundance data, together with data reported for humans in the literature, suggest that element-to-element ratios, e.g. Cu/Mg, show potential as simple diagnostics for diseases such as Alzheimer's. Isotopic data indicate a heterogeneous distribution of Zn isotopes among the organs and blood, with the liver, heart and brain being the most depleted in heavy Zn isotopes, and the blood the most enriched, consistent with observations in other animal models and humans. The Zn isotopic composition of Göttingen minipigs displays a systematic offset towards lighter δ66Zn values relative to mice and sheep models, suggesting physiology that is more closely aligned with that of humans. Cumulatively, these observations strongly suggest that Göttingen minipigs are an excellent animal model for translational research involving metals, and these data provide a strong foundation for future research.

Central and Peripheral Nervous System Progenitors Derived from Human Pluripotent Stem Cells Reveal a Unique Temporal and Cell-Type Specific Expression of PMCAs.

The P-type ATPases family consists of ion and lipid transporters. Their unique diversity in function and expression is critical for normal development. In this study we investigated human pluripotent stem cells (hPSC) and different neural progenitor states to characterize the expression of the plasma membrane calcium ATPases (PMCAs) during human neural development and in mature mesencephalic dopaminergic (mesDA) neurons. Our RNA sequencing data identified a dynamic change in ATPase expression correlating with the differentiation time of the neural progenitors, which was independent of the neuronal progenitor type. Expression of ATP2B1 and ATP2B4 were the most abundantly expressed, in accordance with their main role in Ca2+ regulation and we observed all of the PMCAs to have a subcellular punctate localization. Interestingly in hPSCs ATP2B1 and ATP2B3 were highly expressed in a cell cycle specific manner and ATP2B2 and ATP2B4 were highly expressed in a hPSC sub-population. In neural rosettes a strong apical PMCA expression was identified in the luminal region. Lastly, we confirmed all PMCAs to be expressed in mesDA neurons, however at varying levels. Our results reveal that PMCA expression dynamically changes during stem cell differentiation and highlights the diverging needs of cell populations to regulate and properly integrate Ca2+ changes, which can ultimately correspond to changes in specific stem cell transcription states.

Expression of the Alzheimer's Disease Mutations AßPP695sw and PSEN1M146I in Double-Transgenic Göttingen Minipigs.

Mutations in the amyloid-ß protein precursor gene (AßPP), the presenilin 1 gene (PSEN1) or the presenilin 2 gene (PSEN2) that increase production of the AßPP-derived peptide Aß42 cause early-onset Alzheimer's disease. Rodent models of the disease show that further increase in Aß42 production and earlier brain pathology can be obtained by coexpressing AßPP and PSEN1 mutations. To generate such elevated Aß42 level in a large animal model, we produced Göttingen minipigs carrying in their genome one copy of a human PSEN1 cDNA with the Met146Ile (PSEN1M146I) mutation and three copies of a human AßPP695 cDNA with the Lys670Asn/Met671Leu (AßPPsw) double-mutation. Both transgenes were expressed in fibroblasts and in the brain, and their respective proteins were processed normally. Immunohistochemical staining with Aß42-specific antibodies detected intraneuronal accumulation of Aß42 in brains from a 10- and an 18-month-old pig. Such accumulation may represent an early event in the pathogenesis of Alzheimer's disease.