Cuprizone Intoxication Induces Cell Intrinsic Alterations in Oligodendrocyte Metabolism Independent of Copper Chelation.

Cuprizone intoxication is a common animal model used to test myelin regenerative therapies for the treatment of diseases such as multiple sclerosis. Mice fed this copper chelator develop reversible, region-specific oligodendrocyte loss and demyelination. While the cellular changes influencing the demyelinating process have been explored in this model, there is no consensus about the biochemical mechanisms of toxicity in oligodendrocytes and about whether this damage arises from the chelation of copper in vivo. Here we have identified an oligodendroglial cell line that displays sensitivity to cuprizone toxicity and performed global metabolomic profiling to determine biochemical pathways altered by this treatment. We link these changes with alterations in brain metabolism in mice fed cuprizone for 2 and 6 weeks. We find that cuprizone induces widespread changes in one-carbon and amino acid metabolism as well as alterations in small molecules that are important for energy generation. We used mass spectrometry to examine chemical interactions that are important for copper chelation and toxicity. Our results indicate that cuprizone induces global perturbations in cellular metabolism that may be independent of its copper chelating ability and potentially related to its interactions with pyridoxal 5'-phosphate, a coenzyme essential for amino acid metabolism.

Progressive nigrostriatal terminal dysfunction and degeneration in the engrailed1 heterozygous mouse model of Parkinson's disease.

Current research on Parkinson's disease (PD) pathogenesis requires relevant animal models that mimic the gradual and progressive development of neuronal dysfunction and degeneration that characterizes the disease. Polymorphisms in engrailed 1 (En1), a homeobox transcription factor that is crucial for both the development and survival of mesencephalic dopaminergic neurons, are associated with sporadic PD. This suggests that En1 mutant mice might be a promising candidate PD model. Indeed, a mouse that lacks one En1 allele exhibits decreased mitochondrial complex I activity and progressive midbrain dopamine neuron degeneration in adulthood, both features associated with PD. We aimed to further characterize the disease-like phenotype of these En1(+/-) mice with a focus on early neurodegenerative changes that can be utilized to score efficacy of future disease modifying studies. We observed early terminal defects in the dopaminergic nigrostriatal pathway in En1(+/-) mice. Several weeks before a significant loss of dopaminergic neurons in the substantia nigra could be detected, we found that striatal terminals expressing high levels of dopaminergic neuron markers TH, VMAT2, and DAT were dystrophic and swollen. Using transmission electron microscopy, we identified electron dense bodies consistent with abnormal autophagic vacuoles in these terminal swellings. In line with these findings, we detected an up-regulation of the mTOR pathway, concurrent with a downregulation of the autophagic marker LC3B, in ventral midbrain and nigral dopaminergic neurons of the En1(+/-) mice. This supports the notion that autophagic protein degradation is reduced in the absence of one En1 allele. We imaged the nigrostriatal pathway using the CLARITY technique and observed many fragmented axons in the medial forebrain bundle of the En1(+/-) mice, consistent with axonal maintenance failure. Using in vivo electrochemistry, we found that nigrostriatal terminals in the dorsal striatum were severely deficient in dopamine release and reuptake. Our findings support a progressive retrograde degeneration of En1(+/-) nigrostriatal neurons, akin to what is suggested to occur in PD. We suggest that using the En1(+/-) mice as a model will provide further key insights into PD pathogenesis, and propose that axon terminal integrity and function can be utilized to estimate dopaminergic neuron health and efficacy of experimental PD therapies.

Immune properties of human umbilical cord Wharton's jelly-derived cells.

Cells isolated from Wharton's jelly, referred to as umbilical cord matrix stromal (UCMS) cells, adhere to a tissue-culture plastic substrate, express mesenchymal stromal cell (MSC) surface markers, self-renew, and are multipotent (differentiate into bone, fat, cartilage, etc.) in vitro. These properties support the notion that UCMS cells are a member of the MSC family. Here, the immune properties of UCMS cells are characterized in vitro. The overall hypothesis is that UCMS cells possess immune properties that would be permissive to allogeneic transplantation. For example, UCMS cells will suppress of the proliferation of "stimulated" lymphocytes (immune suppression) and have reduced immunogenicity (e.g., would be poor stimulators of allogeneic lymphocyte proliferation). Hypothesis testing was as follows: first, the effect on proliferation of coculture of mitotically inactivated human UCMS cells with concanavalin-A-stimulated rat splenocytes was assessed in three different assays. Second, the effect of human UCMS cells on one-way and two-way mixed lymphocyte reaction (MLR) assays was determined. Third, the expression of human leukocyte antigen (HLA)-G was examined in human UCMS cells using reverse transcription-polymerase chain reaction, since HLA-G expression conveys immune regulatory properties at the maternal-fetal interface. Fourth, the expression of CD40, CD80, and CD86 was determined by flow cytometry. Fifth, the cytokine expression of UCMS cells was evaluated by focused gene array. The results indicate that human UCMS cells inhibit splenocyte proliferation response to concanavalin A stimulation, that they do not stimulate T-cell proliferation in a one-way MLR, and that they inhibit the proliferation of stimulated T cells in a two-way MLR. Human UCMS cells do not inhibit nonstimulated splenocyte proliferation, suggesting specificity of the response. UCMS cells express mRNA for pan-HLA-G. UCMS cells do not express the costimulatory surface antigens CD40, CD80, and CD86. UCMS cells express vascular endothelial growth factor and interleukin-6, molecules previously implicated in the immune modulation observed in MSCs. In addition, the array data indicate that UCMS cells make a cytokine and other factors that may support hematopoiesis. Together, these results support previous observations made following xenotransplantation; for example, there was no evidence of frank immune rejection of undifferentiated UCMS cells. The results suggest that human UCMS will be tolerated in allogeneic transplantation. Disclosure of potential conflicts of interest is found at the end of this article.

Therapeutic pathways of adult stem cell repair.

The use of adult stem cells as therapeutic agents to treat disease has become increasingly prevalent. During the last decade, isolated and expanded stem and progenitor cells have demonstrated the capacity to differentiate into multiple cell types. Early optimism that in vitro differentiation capacity would translate into in vivo tissue regeneration has lessened and identifying the mechanisms that underlie the benefit of stem cell repair is an emerging area of investigation. This review considers several of the pathways and mechanisms required for adult stem cell repair. These mechanisms include the mobilization and the homing of stem cells to sites of injury, immunomodulatory effect of stem cells, and the association of stem cells with increased vascularization of injured tissue. These data suggest that the unique properties of adult stem cells can be utilized to treat a wide variety of diseases that cannot be treated with existing pharmacological agents, and prompt new paradigms for the bio-pharmacokinetics of biological expressed by efficacious stem cells.

A mouse model for testing remyelinating therapies.

Used in combination with immunomodulatory therapies, remyelinating therapies are a viable therapeutic approach for treating individuals with multiple sclerosis. Studies of postmortem MS brains identified greater remyelination in demyelinated cerebral cortex than in demyelinated brain white matter and implicated reactive astrocytes as an inhibitor of white matter remyelination. An animal model that recapitulates these phenotypes would benefit the development of remyelination therapeutics. We have used a modified cuprizone protocol that causes a consistent and robust demyelination of mouse white matter and cerebral cortex. Spontaneous remyelination occurred significantly faster in the cerebral cortex than in white matter and reactive astrocytes were more abundant in white matter lesions. Remyelination of white matter and cerebral cortex was therapeutically enhanced by daily injections of thyroid hormone triiodothyronine (T3). In summary, we describe an in vivo demyelination/remyelination paradigm that can be powered to determine efficacy of therapies that enhance white matter and cortical remyelination.

Is Axonal Degeneration a Key Early Event in Parkinson's Disease?

Recent research suggests that in Parkinson's disease the long, thin and unmyelinated axons of dopaminergic neurons degenerate early in the disease process. We organized a workshop entitled 'Axonal Pathology in Parkinson's disease', on March 23rd, 2016, in Cleveland, Ohio with the goals of summarizing the state-of-the-art and defining key gaps in knowledge. A group of eight research leaders discussed new developments in clinical pathology, functional imaging, animal models, and mechanisms of degeneration including neuroinflammation, autophagy and axonal transport deficits. While the workshop focused on PD, comparisons were made to other neurological conditions where axonal degeneration is well recognized.

Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell.

Delineation of a cell's ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused throughout tissues made of diverse cell types, such as enteroendocrine cells of the intestinal epithelium. These gastrointestinal sensors of food and bacteria have been difficult to study because they are dispersed among other epithelial cells at a ratio of 1:1,000. Recently, transgenic reporter mice have been generated to identify enteroendocrine cells by means of fluorescence. One of those is the peptide YY-GFP mouse. Using this mouse, we developed a method to correlate confocal and serial block-face scanning electron microscopy. We named the method cocem3D and applied it to identify a specific enteroendocrine cell in tissue and unveil the cell's ultrastructure in 3D. The resolution of cocem3D is sufficient to identify organelles as small as secretory vesicles and to distinguish cell membranes for volume rendering. Cocem3D can be easily adapted to study the 3D ultrastructure of other specific cell types in their native tissue.

The Anti-Aging Protein Klotho Enhances Remyelination Following Cuprizone-Induced Demyelination.

The current study examined whether overexpression of Klotho (KL) in transgenic mice can enhance remyelination following cuprizone-induced demyelination and improves the clinical outcome in experimental autoimmune encephalomyelitis (EAE). Demyelination was achieved by feeding transgenic mice overexpressing the transmembrane form of Klotho (KL-OE) and wild-type (WT) littermates cuprizone-containing chow for 6 weeks. The animals were then allowed to remyelinate for 3 weeks. Paraphenylenediamine staining and platelets-derived growth factor receptor α (PDGFRα) and glutathione S-transferase pi (GSTpi) immunohistochemistry were performed on corpus callosum (CC) sections for quantification of myelin and progenitor and mature oligodendrocytes, respectively. The EAE model was induced with the MOG35-55 peptide. The animals were scored daily for clinical symptoms for 30 days. Following 6 weeks of demyelination, both KL-OE mice and WT littermates demonstrated almost complete and comparable demyelination of the CC. However, the level of spontaneous remyelination was increased approximately two-fold in KL-OE mice, although no significant differences in the numbers of PDGFRα and GSTpi-positive cells were observed. Following EAE induction, Klotho overexpression did not affect the clinical scores, likely due to the different roles Klotho plays in the brain and spinal cord. Thus, increasing Klotho expression should be considered as a therapy for enhancing remyelination in the brains of individuals with multiple sclerosis.

An enteroendocrine cell-enteric glia connection revealed by 3D electron microscopy.

The enteroendocrine cell is the cornerstone of gastrointestinal chemosensation. In the intestine and colon, this cell is stimulated by nutrients, tastants that elicit the perception of flavor, and bacterial by-products; and in response, the cell secretes hormones like cholecystokinin and peptide YY--both potent regulators of appetite. The development of transgenic mice with enteroendocrine cells expressing green fluorescent protein has allowed for the elucidation of the apical nutrient sensing mechanisms of the cell. However, the basal secretory aspects of the enteroendocrine cell remain largely unexplored, particularly because a complete account of the enteroendocrine cell ultrastructure does not exist. Today, the fine ultrastructure of a specific cell can be revealed in the third dimension thanks to the invention of serial block face scanning electron microscopy (SBEM). Here, we bridged confocal microscopy with SBEM to identify the enteroendocrine cell of the mouse and study its ultrastructure in the third dimension. The results demonstrated that 73.5% of the peptide-secreting vesicles in the enteroendocrine cell are contained within an axon-like basal process. We called this process a neuropod. This neuropod contains neurofilaments, which are typical structural proteins of axons. Surprisingly, the SBEM data also demonstrated that the enteroendocrine cell neuropod is escorted by enteric glia--the cells that nurture enteric neurons. We extended these structural findings into an in vitro intestinal organoid system, in which the addition of glial derived neurotrophic factors enhanced the development of neuropods in enteroendocrine cells. These findings open a new avenue of exploration in gastrointestinal chemosensation by unveiling an unforeseen physical relationship between enteric glia and enteroendocrine cells.

Percutaneous adventitial delivery of allogeneic bone marrow-derived stem cells via infarct-related artery improves long-term ventricular function in acute myocardial infarction.

Acute myocardial infarction (AMI) results in ischemic damage and death of cardiomyocytes and loss of vasculature. Stem cell therapy has emerged as a potentially promising strategy for maximizing cardiac function following ischemic injury. Issues of cell source, delivery, and quantification of response have challenged development of clinically viable strategies. In this study we investigate the effects of a well-defined bone marrow-derived allogeneic cell product delivered by catheter directly to the myocardium via the infarct-related vessel on global and regional measures of left ventricular (LV) function in a porcine model of anterior wall myocardial infarction. Multipotent adult progenitor cells (MAPCs) were derived and expanded from the bone marrow of a donor Yorkshire pig. Anterior wall myocardial infarction (AMI) was induced by 90 min of mid-LAD occlusion using a balloon catheter. Two days after AMI was induced, either vehicle (Plasma Lyte-A, n = 7), low-dose (20 million, n = 6), or high-dose (200 million, n = 6) MAPCs were delivered directly to the myocardium via the infarct-related vessel using a transarterial microsyringe catheter-based delivery system. Echocardiography was used to measure LV function as a function of time after AMI. Animals that received low-dose cell treatment showed significant improvement in regional and global LV function and remodeling compared to the high-dose or control animals. Direct myocardial delivery of allogeneic MAPCs 2 days following AMI through the vessel wall of the infarct-related vessel is safe and results in delivery of cells throughout the infarct zone and improved cardiac function despite lack of long-term cell survival. These data further support the hypothesis of cell-based myocardial tissue repair by a paracrine mechanism and suggest a clinically translatable strategy for delivering cells at any time after AMI to modulate cardiac remodeling and function.

Human umbilical cord matrix stem cells: preliminary characterization and effect of transplantation in a rodent model of Parkinson's disease.

The umbilical cord contains an inexhaustible, noncontroversial source of stem cells for therapy. In the U.S., stem cells found in the umbilical cord are routinely placed into bio-hazardous waste after birth. Here, stem cells derived from human umbilical cord Wharton's Jelly, called umbilical cord matrix stem (UCMS) cells, are characterized. UCMS cells have several properties that make them of interest as a source of cells for therapeutic use. For example, they 1) can be isolated in large numbers, 2) are negative for CD34 and CD45, 3) grow robustly and can be frozen/thawed, 4) can be clonally expanded, and 5) can easily be engineered to express exogenous proteins. UCMS cells have genetic and surface markers of mesenchymal stem cells (positive for CD10, CD13, CD29, CD44, and CD90 and negative for CD14, CD33, CD56, CD31, CD34, CD45, and HLA-DR) and appear to be stable in terms of their surface marker expression in early passage (passages 4-8). Unlike traditional mesenchymal stem cells derived from adult bone marrow stromal cells, small populations of UCMS cells express endoglin (SH2, CD105) and CD49e at passage 8. UCMS cells express growth factors and angiogenic factors, suggesting that they may be used to treat neurodegenerative disease. To test the therapeutic value of UCMS cells, undifferentiated human UCMS cells were transplanted into the brains of hemiparkinsonian rats that were not immune-suppressed. UCMS cells ameliorated apomorphine-induced rotations in the pilot test. UCMS cells transplanted into normal rats did not produce brain tumors, rotational behavior, or a frank host immune rejection response. In summary, the umbilical cord matrix appears to be a rich, noncontroversial, and inexhaustible source of primitive mesenchymal stem cells.

Matrix cells from Wharton's jelly form neurons and glia.

We have identified an easily attainable source of primitive, potentially multipotent stem cells from Wharton's jelly, the matrix of umbilical cord. Wharton's jelly cells have been propagated in culture for more than 80 population doublings. Several markers for stem cells, including c-kit (CD117), and telomerase activity are expressed in these cells. Treatment with basic fibroblast growth factor overnight and low-serum media plus butylated hydroxyanisole and dimethylsulfoxide induced Wharton's jelly cells to express a neural phenotype. Within several hours of this treatment, Wharton's jelly cells developed rounded cell bodies with multiple neurite-like extensions, similar to the morphology of neural stem cells. Neuron-specific enolase (NSE), a neural stem cell marker, was expressed in these cells, as shown by immunocytochemistry. Immunoblot analysis showed similar levels of NSE expression in both untreated and induced Wharton's jelly cells. After 3 days, the induced Wharton's jelly cells resembled bipolar or multipolar neurons, with processes that formed networks reminiscent of primary cultures of neurons. The neuron-like cells in these cultures stained positively for several neuronal proteins, including neuron-specific class III beta-tubulin, neurofilament M, an axonal growth-cone-associated protein, and tyrosine hydroxylase. Immunoblot analysis showed increasing levels of protein markers for mature neurons over time post induction. Markers for oligodendrocytes and astrocytes were also detected in Wharton's jelly cells. These exciting findings show that cells from the matrix of umbilical cord have properties of stem cells and may, thus, be a rich source of primitive cells. This study shows their capacity to differentiate into a neural phenotype in vitro.