Single nuclei RNA-seq reveals a medium spiny neuron glutamate excitotoxicity signature prior to the onset of neuronal death in an ovine Huntington's disease model.

Huntington's disease (HD) is a neurodegenerative genetic disorder caused by an expansion in the CAG repeat tract of the huntingtin (HTT) gene resulting in behavioural, cognitive, and motor defects. Current knowledge of disease pathogenesis remains incomplete, and no disease course-modifying interventions are in clinical use. We have previously reported the development and characterisation of the OVT73 transgenic sheep model of HD. The 73 polyglutamine repeat is somatically stable and therefore likely captures a prodromal phase of the disease with an absence of motor symptomatology even at 5-years of age and no detectable striatal cell loss. To better understand the disease-initiating events we have undertaken a single nuclei transcriptome study of the striatum of an extensively studied cohort of 5-year-old OVT73 HD sheep and age matched wild-type controls. We have identified transcriptional upregulation of genes encoding N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors in medium spiny neurons, the cell type preferentially lost early in HD. Further, we observed an upregulation of astrocytic glutamate uptake transporters and medium spiny neuron GABAA receptors, which may maintain glutamate homeostasis. Taken together, these observations support the glutamate excitotoxicity hypothesis as an early neurodegeneration cascade-initiating process but the threshold of toxicity may be regulated by several protective mechanisms. Addressing this biochemical defect early may prevent neuronal loss and avoid the more complex secondary consequences precipitated by cell death.

Preweaning Purified Fucoidan Drench: Effects on Growth, Immune Response, and Intestinal Morphology in Weaned Piglets.

Weaning stress imposes considerable physiological challenges on piglets, often manifesting in intestinal disturbances, such as inflammation and compromised barrier function, ultimately affecting growth and health outcomes. While conventional interventions, including antimicrobials, have effectively mitigated these sequelae, concerns surrounding antimicrobial resistance necessitate the exploration of alternatives. Fucoidan, derived from brown seaweed, offers promise due to its antioxidant and anti-inflammatory effects. Previous research has been limited to the in-feed supplementation of partially purified fucoidan extracted from brown seaweed. The focus of the present study is assessing the effect of a preweaning drench with highly purified (85%) fucoidan on piglet growth, immune response, and intestinal morphology post-weaning. Forty-eight male piglets at 17 ± 3 days of age (5.67 ± 0.16 kg) were assigned to a saline (control), fucoidan, or antimicrobial group, receiving treatment as a single 18 mL oral drench three days before weaning. Monitoring for seven days post-weaning included body weight measurements, blood sample collection for the inflammatory protein assay, and small intestine morphological analysis. The findings revealed that the preweaning fucoidan drench did not elicit adverse effects on piglets. However, neither fucoidan nor antimicrobial drenches significantly enhanced growth parameters, immune markers, or intestinal morphology compared to that of the control-treated piglets (p > 0.05). The lack of response may be attributed to the high health status of the experimental cohort and the limitation of a single dosage. Future research should consider a more challenging production setting to evaluate the viability and optimal application of fucoidan as an antimicrobial alternative in the pig industry.

Tools for Efficient Genome Editing; ZFN, TALEN, and CRISPR.

The last two decades have marked significant advancement in the genome editing field. Three generations of programmable nucleases (ZFNs, TALENs, and CRISPR-Cas system) have been adopted to introduce targeted DNA double-strand breaks (DSBs) in eukaryotic cells. DNA repair machinery of the cells has been exploited to introduce insertion and deletions (indels) at the targeted DSBs to study function of any gene-of-interest. The resulting indels were generally assumed to be "random" events produced by "error-prone" DNA repair pathways. However, recent advances in computational tools developed to study the Cas9-induced mutations have changed the consensus and implied the "non-randomness" nature of these mutations. Furthermore, CRISPR-centric tools are evolving at an unprecedented pace, for example, base- and prime-editors are the newest developments that have been added to the genome editing toolbox. Altogether, genome editing tools have revolutionized our way of conducting research in life sciences. Here, we present a concise overview of genome editing tools and describe the DNA repair pathways underlying the generation of genome editing outcome.

Generating a Heat-Tolerance Mouse Model.

Creating mouse models of human genetic disease (Gurumurthy and Lloyd, Dis Models Mech 12(1):dmm029462, 2019) and livestock trait (Schering et al. Arch Physiol Biochem 121(5):194-205, 2015; Habiela et al. J Gen Virol 95 (Pt 11):2329-2345, 2014) have been proven to be a useful tool for understanding the mechanism behind the phenotypes and fundamental and applied research in livestock. A single base pair deletion of prolactin receptor (PRLR) has an impact on hair morphology phenotypes beyond its classical roles in lactation in cattle, the so-called slick cattle (Littlejohn et al. Nat Commun 5:5861, 2014). Here, we generate a knock-in mouse model by targeting the specific locus of PRLR gene using Cas9-mediated genome editing via homology-directed repair (HDR) in mouse zygotes. The mouse model carrying the identical PRLR mutation in slick cattle may provide a useful animal model to study the pathway of thermoregulation and the mechanism of heat-tolerance in the livestock.

Evaluation of miRNA as Biomarkers of Emotional Valence in Pigs.

The ability to assess the welfare of animals is dependent on our ability to accurately determine their emotional (affective) state, with particular emphasis being placed on the identification of positive emotions. The challenge remains that current physiological and behavioral indices are either unable to distinguish between positive and negative emotional states, or they are simply not suitable for a production environment. Therefore, the development of novel measures of animal emotion is a necessity. Here we investigated the efficacy of microRNA (miRNA) in the brain and blood as biomarkers of emotional state in the pig. Female Large White × Landrace pigs (n = 24) were selected at weaning and trained to perform a judgment bias test (JBT), before being exposed for 5 weeks to either enriched (n = 12) or barren housing (n = 12) conditions. Pigs were tested on the JBT once prior to treatment, and immediately following treatment. MiRNA and neurotransmitters were analyzed in blood and brain tissue after euthanasia. Treatment had no effect on the outcomes of the JBT. There was also no effect of treatment on miRNA expression in blood or the brain (FDR p > 0.05). However, pigs exposed to enriched housing had elevated dopamine within the striatum compared to pigs in barren housing (p = 0.02). The results imply that either (a) miRNAs are not likely to be valid biomarkers of a positive affective state, at least under the type of conditions employed in this study, or (b) that the study design used to modify affective state was not able to create differential affective states, and therefore establish the validity of miRNA as biomarkers.

A Multi-Omic Huntington's Disease Transgenic Sheep-Model Database for Investigating Disease Pathogenesis.

BACKGROUND: The pathological mechanism of cellular dysfunction and death in Huntington's disease (HD) is not well defined. Our transgenic HD sheep model (OVT73) was generated to investigate these mechanisms and for therapeutic testing. One particular cohort of animals has undergone focused investigation resulting in a large interrelated multi-omic dataset, with statistically significant changes observed comparing OVT73 and control 'omic' profiles and reported in literature. OBJECTIVE: Here we make this dataset publicly available for the advancement of HD pathogenic mechanism discovery. METHODS: To enable investigation in a user-friendly format, we integrated seven multi-omic datasets from a cohort of 5-year-old OVT73 (n = 6) and control (n = 6) sheep into a single database utilising the programming language R. It includes high-throughput transcriptomic, metabolomic and proteomic data from blood, brain, and other tissues. RESULTS: We present the 'multi-omic' HD sheep database as a queryable web-based platform that can be used by the wider HD research community (https://hdsheep.cer.auckland.ac.nz/). The database is supported with a suite of simple automated statistical analysis functions for rapid exploratory analyses. We present examples of its use that validates the integrity relative to results previously reported. The data may also be downloaded for user determined analysis. CONCLUSION: We propose the use of this online database as a hypothesis generator and method to confirm/refute findings made from patient samples and alternate model systems, to expand our understanding of HD pathogenesis. Importantly, additional tissue samples are available for further investigation of this cohort.

Mitochondrial DNA Depletion in Granulosa Cell Derived Nuclear Transfer Tissues.

Somatic cell nuclear transfer (SCNT) is a key technology with broad applications that range from production of cloned farm animals to derivation of patient-matched stem cells or production of humanized animal organs for xenotransplantation. However, effects of aberrant epigenetic reprogramming on gene expression compromise cell and organ phenotype, resulting in low success rate of SCNT. Standard SCNT procedures include enucleation of recipient oocytes before the nuclear donor cell is introduced. Enucleation removes not only the spindle apparatus and chromosomes of the oocyte but also the perinuclear, mitochondria rich, ooplasm. Here, we use a Bos taurus SCNT model with in vitro fertilized (IVF) and in vivo conceived controls to demonstrate a ∼50% reduction in mitochondrial DNA (mtDNA) in the liver and skeletal muscle, but not the brain, of SCNT fetuses at day 80 of gestation. In the muscle, we also observed significantly reduced transcript abundances of mtDNA-encoded subunits of the respiratory chain. Importantly, mtDNA content and mtDNA transcript abundances correlate with hepatomegaly and muscle hypertrophy of SCNT fetuses. Expression of selected nuclear-encoded genes pivotal for mtDNA replication was similar to controls, arguing against an indirect epigenetic nuclear reprogramming effect on mtDNA amount. We conclude that mtDNA depletion is a major signature of perturbations after SCNT. We further propose that mitochondrial perturbation in interaction with incomplete nuclear reprogramming drives abnormal epigenetic features and correlated phenotypes, a concept supported by previously reported effects of mtDNA depletion on the epigenome and the pleiotropic phenotypic effects of mtDNA depletion in humans. This provides a novel perspective on the reprogramming process and opens new avenues to improve SCNT protocols for healthy embryo and tissue development.

Targeting epigenetic nuclear reprogramming in aggregated cloned equine embryos.

Epigenetic perturbations during the reprogramming process have been described as the primary cause of the low efficiency of somatic cell nuclear transfer (SCNT). In this study, we tested three strategies targeting nuclear reprogramming to investigate effects on equine SCNT. First, we evaluated the effect of treating somatic cells with chetomin, a fungal secondary metabolite reported to inhibit the trimethylation on histone 3 lysine 9 (H3K9 me3). Second, caffeine was added to the culture medium during the enucleation of oocytes and before activation of reconstructed embryos as a protein phosphatase inhibitor to improve nuclear reprogramming. Third, we tested the effects of the histone deacetylase inhibitor trichostatin A (TSA) added during both activation and early embryo culture. Although none of these treatments significantly improved the developmental rates of the invitro aggregated cloned equine embryos, the first equine cloned foal born in Australia was produced with somatic cells treated with chetomin. The present study describes the use of chetomin, caffeine and TSA for the first time in horses, serving as a starting point for the establishment of future protocols to target epigenetic reprogramming for improving the efficiency of equine cloning. Cloning is an expensive and inefficient process, but has gained particular interest in the equine industry. In this study we explored different strategies to improve cloning efficiency and produced the first cloned foal born in Australia. Our data serve as a starting point for the establishment of future protocols for improving equine cloning efficiency.

GSK3 inhibition, but not epigenetic remodeling, mediates efficient derivation of germline embryonic stem cells from nonobese diabetic mice.

The nonobese diabetic (NOD) mouse strain is a predominant animal model of type 1 diabetes. However, this mouse strain is considered to be non-permissive for embryonic stem cell (ESC) derivation using conventional methods. We examined small molecule inhibition of glycogen synthase kinase 3 (GSK3) to block spontaneous cell differentiation and promote pluripotency persistence. Here we show a single pharmacological GSK3 inhibitor, 6-bromoindirubin-3'-oxime (BIO), in combination with leukemia inhibition factor (LIF), promoted generation of stable NOD ESC lines at >80% efficiency. Significantly, expansion of the established NOD ESC lines no longer required treatment with BIO. These NOD ESC lines contributed to chimeric mice and transmitted to germline progeny that spontaneously developed diabetes. By contrast, 5-aza-2'-deoxycytidine (AZA), a small molecule inhibitor of DNA methylation, and trichostatin A (TSA) and valproic acid (VPA), small molecule inhibitors of histone deacetylase, could not promote generation of NOD ESCs by epigenetic remodeling. These combined findings provide strategic insights for imposing pluripotency in cells isolated from a non-permissive strain.

Brain urea increase is an early Huntington's disease pathogenic event observed in a prodromal transgenic sheep model and HD cases.

The neurodegenerative disorder Huntington's disease (HD) is typically characterized by extensive loss of striatal neurons and the midlife onset of debilitating and progressive chorea, dementia, and psychological disturbance. HD is caused by a CAG repeat expansion in the Huntingtin (HTT) gene, translating to an elongated glutamine tract in the huntingtin protein. The pathogenic mechanism resulting in cell dysfunction and death beyond the causative mutation is not well defined. To further delineate the early molecular events in HD, we performed RNA-sequencing (RNA-seq) on striatal tissue from a cohort of 5-y-old OVT73-line sheep expressing a human CAG-expansion HTT cDNA transgene. Our HD OVT73 sheep are a prodromal model and exhibit minimal pathology and no detectable neuronal loss. We identified significantly increased levels of the urea transporter SLC14A1 in the OVT73 striatum, along with other important osmotic regulators. Further investigation revealed elevated levels of the metabolite urea in the OVT73 striatum and cerebellum, consistent with our recently published observation of increased urea in postmortem human brain from HD cases. Extending that finding, we demonstrate that postmortem human brain urea levels are elevated in a larger cohort of HD cases, including those with low-level neuropathology (Vonsattel grade 0/1). This elevation indicates increased protein catabolism, possibly as an alternate energy source given the generalized metabolic defect in HD. Increased urea and ammonia levels due to dysregulation of the urea cycle are known to cause neurologic impairment. Taken together, our findings indicate that aberrant urea metabolism could be the primary biochemical disruption initiating neuropathogenesis in HD.

Alzheimer's disease markers in the aged sheep (Ovis aries).

This study reports the identification and characterization of markers of Alzheimer's disease (AD) in aged sheep (Ovis aries) as a preliminary step toward making a genetically modified large animal model of AD. Importantly, the sequences of key proteins involved in AD pathogenesis are highly conserved between sheep and human. The processing of the amyloid-ß (Aß) protein is conserved between sheep and human, and sheep Aß1-42/Aß1-40 ratios in cerebrospinal fluid (CSF) are also very similar to human. In addition, total tau and neurofilament light levels in CSF are comparable with those found in human. The presence of neurofibrillary tangles in aged sheep brain has previously been established; here, we report for the first time that plaques, the other pathologic hallmark of AD, are also present in the aged sheep brain. In summary, the biological machinery to generate the key neuropathologic features of AD is conserved between the human and sheep, making the sheep a good candidate for future genetic manipulation to accelerate the condition for use in pathophysiological discovery and therapeutic testing.

Inhibition of JAK-STAT ERK/MAPK and Glycogen Synthase Kinase-3 Induces a Change in Gene Expression Profile of Bovine Induced Pluripotent Stem Cells.

Pluripotent stem cells (PSCs) fall in two states, one highly undifferentiated, the naïve state, and the primed state, characterized by the inability to contribute to germinal lineage. Several reports have demonstrated that these states can be modified by changes to the cell culture conditions. With the advent of nuclear reprogramming, bovine induced pluripotent stem cells (biPSCs) have been generated. These cells represent examples of a transient-intermediate state of pluripotency with remarkable characteristics and biotechnological potential. Herein, we generated and characterized biPSC. Next, we evaluated different culture conditions for the ability to affect the expression of the set of core pluripotent transcription factors in biPSC. It was found that the use of 6-bromoindirubin-3-oxime and Sc1 inhibitors alone or in combination with 5-AzaC induced significantly higher levels of expression of endogenous REX1, OCT4, NANOG, and SOX2. Furthermore, LIF increased the levels of expression of OCT4 and REX1, compared with those cultured with LIF + bFGF. By contrast, bFGF decreased the levels of expression for both REX1 and OCT4. These results demonstrate that the biPSC gene expression profile is malleable by modification of the cell culture conditions well after nuclear reprogramming, and the culture conditions may determine their differentiation potential.

Bone morphogenetic protein 4 and retinoic acid trigger bovine VASA homolog expression in differentiating bovine induced pluripotent stem cells.

Primordial germ cells (PGCs) are the earliest identifiable and completely committed progenitors of female and male gametes. They are obvious targets for genome editing because they assure the transmission of desirable or introduced traits to future generations. PGCs are established at the earliest stages of embryo development and are difficult to propagate in vitro--two characteristics that pose a problem for their practical application. One alternative method to enrich for PGCs in vitro is to differentiate them from pluripotent stem cells derived from adult tissues. Here, we establish a reporter system for germ cell identification in bovine pluripotent stem cells based on green fluorescent protein expression driven by the minimal essential promoter of the bovine Vasa homolog (BVH) gene, whose regulatory elements were identified by orthologous modelling of regulatory units. We then evaluated the potential of bovine induced pluripotent stem cell (biPSC) lines carrying the reporter construct to differentiate toward the germ cell lineage. Our results showed that biPSCs undergo differentiation as embryoid bodies, and a fraction of the differentiating cells expressed BVH. The rate of differentiation towards BVH-positive cells increased up to tenfold in the presence of bone morphogenetic protein 4 or retinoic acid. Finally, we determined that the expression of key PGC genes, such as BVH or SOX2, can be modified by pre-differentiation cell culture conditions, although this increase is not necessarily mirrored by an increase in the rate of differentiation.

Optimization of agitation speed in spinner flask for microcarrier structural integrity and expansion of induced pluripotent stem cells.

In recent times, the study and use of induced pluripotent stem cells (iPSC) have become important in order to avoid the ethical issues surrounding the use of embryonic stem cells. Therapeutic, industrial and research based use of iPSC requires large quantities of cells generated in vitro. Mammalian cells, including pluripotent stem cells, have been expanded using 3D culture, however current limitations have not been overcome to allow a uniform, optimized platform for dynamic culture of pluripotent stem cells to be achieved. In the current work, we have expanded mouse iPSC in a spinner flask using Cytodex 3 microcarriers. We have looked at the effect of agitation on the microcarrier survival and optimized an agitation speed that supports bead suspension and iPS cell expansion without any bead breakage. Under the optimized conditions, the mouse iPSC were able to maintain their growth, pluripotency and differentiation capability. We demonstrate that microcarrier survival and iPS cell expansion in a spinner flask are reliant on a very narrow range of spin rates, highlighting the need for precise control of such set ups and the need for improved design of more robust systems.

Synthetic mRNA Reprogramming of Human Fibroblast Cells.

Reprogramming of somatic cells, such as skin fibroblasts, to pluripotency was first achieved by forced expression of four transcription factors using integrating retroviral or lentiviral vectors, which result in integration of exogenous DNA into cellular genome and present a formidable barrier to therapeutic application of induced pluripotent stem cells (iPSCs). To facilitate the translation of iPSC technology to clinical practice, mRNA reprogramming method that generates transgene-free iPSCs is a safe and efficient method, eliminating bio-containment concerns associated with viral vectors, as well as the need for weeks of screening of cells to confirm that viral material has been completely eliminated during cell passaging.

Embryonic stem cell-somatic cell fusion and postfusion enucleation.

Embryonic stem (ES) cells are able to reprogram somatic cells following cell fusion. The resulting cell hybrids have been shown to have similar properties to pluripotent cells. It has also been shown that transcriptional changes can occur in a heterokaryon, without nuclear hybridization. However it is unclear whether these changes can be sustained following removal of the dominant ES nucleus. In this chapter, methods are described for the cell fusion of mouse tetraploid ES cells with somatic cells and enrichment of the resulting heterokaryons. We next describe the conditions for the differential removal of the ES cell nucleus, allowing for the recovery of somatic cells.

Cardiogenesis of embryonic stem cells with liquid marble micro-bioreactor.

A liquid marble micro-bioreactor is prepared by placing a drop of murine embryonic stem cell (ESC) (Oct4B2-ESC) suspension onto a polytetrafluoroethylene (PTFE) particle bed. The Oct4B2-ESC aggregates to form embryoid bodies (EBs) with relatively uniform size and shape in a liquid marble within 3 d. For the first time, the feasibility of differentiating ESC into cardiac lineages within liquid marbles is being investigated. Without the addition of growth factors, suspended EBs from liquid marbles express various precardiac mesoderm markers including Flk-1, Gata4, and Nkx2.5. Some of the suspended EBs exhibit spontaneous contraction. These results indicate that the liquid marble provides a suitable microenvironment to induce EB formation and spontaneous cardiac mesoderm differentiation. Some of the EBs are subsequently plated onto gelatin-coated tissue culture dishes. Plated EBs express mature cardiac markers atrial myosin light chain 2a (MLC2a) and ventricular myosin light chain (MLC2v), and the cardiac structural marker α-actinin. More than 60% of the plated EBs exhibit spontaneous contraction and express mature cardiomyocyte marker cardiac troponin T (cTnT), indicating that these EBs have differentiated into functional cardiomyocytes. Together, these results demonstrate that the liquid-marble technique is an easily employed, cost effective, and efficient approach to generate EBs and facilitating their cardiogenesis.

Isolation and handling of mouse embryonic fibroblasts.

Primary mouse embryonic fibroblasts (MEFs) are the most commonly used feeder layers that help to support growth and maintain pluripotency of embryonic stem cells (ESC) in long-term culture. Feeders provide substrates/nutrients that are essential to maintain pluripotency and prevent spontaneous differentiation of ESC. Since embryonic fibroblasts stop dividing after a few passages, care must be taken to isolate them freshly. Here, we provide a protocol to derive MEFs and describe the method to inactivate the cells using mitomycin C treatment. The protocol also describes freezing, thawing, and passaging of MEFs. This basic protocol works well in our laboratory. However, it can be modified and adapted according to any user's particular requirement.

Generation of induced pluripotent stem cells from mouse adipose tissue.

The discovery that embryonic stem (ES) cell-like cells can be generated by simply over-expressing four key genes in adult somatic cells has changed the face of regenerative medicine. These induced pluripotent stem (iPS) cells have a wide range of potential uses from drug testing and in vitro disease modeling to personalized cell therapies for patients. However, prior to the realization of their potential, many issues need to be considered. One of these is the low-efficiency formation of iPSC. It has been extensively demonstrated that the somatic cell type can greatly influence reprogramming outcomes. We have shown that adipose tissue-derived cells (ADCs) can be easily isolated from adult animals and can be reprogrammed to a pluripotent state with high efficiency. Here, we describe a protocol for the high-efficiency derivation of ADCs and their subsequent use to generate mouse iPSC using Oct4, Sox2, Klf4, and cMyc retroviral vectors.

C-terminal region of MAP7 domain containing protein 3 (MAP7D3) promotes microtubule polymerization by binding at the C-terminal tail of tubulin.

MAP7 domain containing protein 3 (MAP7D3), a newly identified microtubule associated protein, has been shown to promote microtubule assembly and stability. Its microtubule binding region has been reported to consist of two coiled coil motifs located at the N-terminus. It possesses a MAP7 domain near the C-terminus and belongs to the microtubule associated protein 7 (MAP7) family. The MAP7 domain of MAP7 protein has been shown to bind to kinesin-1; however, the role of MAP7 domain in MAP7D3 remains unknown. Based on the bioinformatics analysis of MAP7D3, we hypothesized that the MAP7 domain of MAP7D3 may have microtubule binding activity. Indeed, we found that MAP7 domain of MAP7D3 bound to microtubules as well as enhanced the assembly of microtubules in vitro. Interestingly, a longer fragment MDCT that contained the MAP7 domain (MD) with the C-terminal tail (CT) of the protein promoted microtubule polymerization to a greater extent than MD and CT individually. MDCT stabilized microtubules against dilution induced disassembly. MDCT bound to reconstituted microtubules with an apparent dissociation constant of 3.0 ± 0.5 µM. An immunostaining experiment showed that MDCT localized along the length of the preassembled microtubules. Competition experiments with tau indicated that MDCT shares its binding site on microtubules with tau. Further, we present evidence indicating that MDCT binds to the C-terminal tail of tubulin. In addition, MDCT could bind to tubulin in HeLa cell extract. Here, we report a microtubule binding region in the C-terminal region of MAP7D3 that may have a role in regulating microtubule assembly dynamics.