Heterozygote galactocerebrosidase (GALC) mutants have reduced remyelination and impaired myelin debris clearance following demyelinating injury.

Genome-wide association studies are identifying multiple genetic risk factors for several diseases, but the functional role of these changes remains mostly unknown. Variants in the galactocerebrosidase (GALC) gene, for example, were identified as a risk factor for Multiple Sclerosis (MS); however, the potential biological relevance of GALC variants to MS remains elusive. We found that heterozygote GALC mutant mice have reduced myelin debris clearance and diminished remyelination after a demyelinating insult. We found no histological or behavioral differences between adult wild-type and GALC +/- animals under normal conditions. Following exposure to the demyelinating agent cuprizone, however, GALC +/- animals had significantly reduced remyelination during recovery. In addition, the microglial phagocytic response and elevation of Trem2, both necessary for clearing damaged myelin, were markedly reduced in GALC +/- animals. These altered responses could be corrected in vitro by treatment with NKH-477, a compound discovered as protective in our previous studies on Krabbe disease, which is caused by mutations in both GALC alleles. Our data are the first to show remyelination defects in individuals with a single mutant GALC allele, suggesting such carriers may have increased vulnerability to myelin damage following injury or disease due to inefficient myelin debris clearance. We thus provide a potential functional link between GALC variants and increased MS susceptibility, particularly due to the failure of remyelination associated with progressive MS. Finally, this work demonstrates that genetic variants identified through genome-wide association studies may contribute significantly to complex diseases, not by driving initial symptoms, but by altering repair mechanisms.

Structural equation models and the quantification of behavior.

Quantifying behavior often involves using variables that contain measurement errors and formulating multiequations to capture the relationship among a set of variables. Structural equation models (SEMs) refer to modeling techniques popular in the social and behavioral sciences that are equipped to handle multiequation models, multiple measures of concepts, and measurement error. This work provides an overview of latent variable SEMs. We present the equations for SEMs and the steps in modeling, and we provide three illustrations of SEMs. We suggest that the general nature of the model is capable of handling a variety of problems in the quantification of behavior, where the researcher has sufficient knowledge to formulate hypotheses.

Are gestational age, birth weight, and birth length indicators of favorable fetal growth conditions? A structural equation analysis of Filipino infants.

The fetal origins hypothesis emphasizes the life-long health impacts of prenatal conditions. Birth weight, birth length, and gestational age are indicators of the fetal environment. However, these variables often have missing data and are subject to random and systematic errors caused by delays in measurement, differences in measurement instruments, and human error. With data from the Cebu (Philippines) Longitudinal Health and Nutrition Survey, we use structural equation models, to explore random and systematic errors in these birth outcome measures, to analyze how maternal characteristics relate to birth outcomes, and to take account of missing data. We assess whether birth weight, birth length, and gestational age are influenced by a single latent variable that we call favorable fetal growth conditions (FFGC) and if so, which variable is most closely related to FFGC. We find that a model with FFGC as a latent variable fits as well as a less parsimonious model that has birth weight, birth length, and gestational age as distinct individual variables. We also demonstrate that birth weight is more reliably measured than is gestational age. FFGCs were significantly influenced by taller maternal stature, better nutritional stores indexed by maternal arm fat and muscle area during pregnancy, higher birth order, avoidance of smoking, and maternal age 20-35 years. Effects of maternal characteristics on newborn weight, length, and gestational age were largely indirect, operating through FFGC.

Acute axon damage and demyelination are mitigated by 4-aminopyridine (4-AP) therapy after experimental traumatic brain injury.

Damage to long axons in white matter tracts is a major pathology in closed head traumatic brain injury (TBI). Acute TBI treatments are needed that protect against axon damage and promote recovery of axon function to prevent long term symptoms and neurodegeneration. Our prior characterization of axon damage and demyelination after TBI led us to examine repurposing of 4-aminopyridine (4-AP), an FDA-approved inhibitor of voltage-gated potassium (Kv) channels. 4-AP is currently indicated to provide symptomatic relief for patients with chronic stage multiple sclerosis, which involves axon damage and demyelination. We tested clinically relevant dosage of 4-AP as an acute treatment for experimental TBI and found multiple benefits in corpus callosum axons. This randomized, controlled pre-clinical study focused on the first week after TBI, when axons are particularly vulnerable. 4-AP treatment initiated one day post-injury dramatically reduced axon damage detected by intra-axonal fluorescence accumulations in Thy1-YFP mice of both sexes. Detailed electron microscopy in C57BL/6 mice showed that 4-AP reduced pathological features of mitochondrial swelling, cytoskeletal disruption, and demyelination at 7 days post-injury. Furthermore, 4-AP improved the molecular organization of axon nodal regions by restoring disrupted paranode domains and reducing Kv1.2 channel dispersion. 4-AP treatment did not resolve deficits in action potential conduction across the corpus callosum, based on ex vivo electrophysiological recordings at 7 days post-TBI. Thus, this first study of 4-AP effects on axon damage in the acute period demonstrates a significant decrease in multiple pathological hallmarks of axon damage after experimental TBI.

4-Aminopyridine Induces Nerve Growth Factor to Improve Skin Wound Healing and Tissue Regeneration.

The discovery of ways to enhance skin wound healing is of great importance due to the frequency of skin lesions. We discovered that 4-aminopyridine (4-AP), a potassium channel blocker approved by the FDA for improving walking ability in multiple sclerosis, greatly enhances skin wound healing. Benefits included faster wound closure, restoration of normal-appearing skin architecture, and reinnervation. Hair follicle neogenesis within the healed wounds was increased, both histologically and by analysis of K15 and K17 expression. 4-AP increased levels of vimentin (fibroblasts) and alpha-smooth muscle actin (α-SMA, collagen-producing myofibroblasts) in the healed dermis. 4-AP also increased neuronal regeneration with increased numbers of axons and S100+ Schwann cells (SCs), and increased expression of SRY-Box Transcription Factor 10 (SOX10). Treatment also increased levels of transforming growth factor-ß (TGF-ß), substance P, and nerve growth factor (NGF), important promoters of wound healing. In vitro studies demonstrated that 4-AP induced nerve growth factor and enhanced proliferation and migration of human keratinocytes. Thus, 4-AP enhanced many of the key attributes of successful wound healing and offers a promising new approach to enhance skin wound healing and tissue regeneration.

Drying Climates and Gendered Suffering: Links Between Drought, Food Insecurity, and Women's HIV in Less-Developed Countries.

HIV/AIDS represents the leading cause of death among women of reproductive age globally, and gender inequalities in the burden of HIV/AIDS are most pronounced in poorer countries. Drawing on ideas from feminist political ecology, we explore linkages between suffering from drought, food insecurity, and women's vulnerability to HIV. Using data from 91 less-developed countries, we construct a structural equation model to analyze the direct and indirect influence of these factors, alongside other socio-economic indicators, on the percentage of the adult population living with HIV that are women. We find that droughts are significant in shaping gender inequalities in the HIV burden indirectly through increased food insecurity. We draw on prior research to argue that due to gendered inequalities, food insecurity increases women's vulnerability to HIV by intensifying biological susceptibilities to the disease, reducing access to social and health resources, and motivating women to engage in risky sexual behaviors, such as transactional sex. Overall, our findings demonstrate that droughts serve as an important underlying factor in promoting HIV transmission among vulnerable women in poor countries, and that food insecurity is a key mechanism in driving this relationship.

Heterozygous carriers of galactocerebrosidase mutations that cause Krabbe disease have impaired microglial function and defective repair of myelin damage.

This review addresses two puzzling findings related to mutations in galactocerebrosidase (GALC) that cause Krabbe disease (KD), a severe lysosomal storage disorder characterized by extensive myelin damage in children with mutations in both GALC alleles. First, heterozygous carriers of KD-causing mutations, which include the biological parents of children with KD, exhibit increased risk for developing other diseases. Second, variants in the GALC locus increase the risk of developing multiple sclerosis (MS), another disease characterized by extensive myelin damage. What explains these correlations? In studies on cuprizone-induced myelin damage in heterozygous (GALC+/-) mice carrying one copy of a mutation that causes KD-like disease, the extent of damage was similar in GALC+/- and wild-type (WT) mice. In contrast, GALC+/- mice had striking defects in repair of cuprizone-induced damage. We further found unexpected microglial defects in myelin debris clearance and in the ability to up-regulate the Trem2 microglial protein critical for debris uptake. These defects were rescued by exposure to a lysosomal re-acidifying drug discovered in our studies on KD, and which provides multiple clinically relevant benefits in the twitcher (GALC+/-) mouse model of KD. Thus, heterozygous GALC mutations cause effects on biological function that may help to understand the increased disease risk in heterozygous carriers of such mutations and to understand why GALC variations increase the risk of MS. Our findings indicate that while some genetic risk factors may contribute to complex diseases by increasing the risk of tissue damage, others may do so by compromising tissue repair.

Pharmacological targeting of p38 MAP-Kinase 6 (MAP2K6) inhibits the growth of esophageal adenocarcinoma.

Drug repurposing with a better understanding of the underlying mechanism has provided new avenues to find treatment for malignancies. Esophageal adenocarcinoma (EAC) is a rapidly increasing cancer with a dismal 5-year survival rate of <15%. Lack of efficient treatment options contributes to the high mortality rate of EAC. To find new therapy against EAC we performed unbiased drug screening of an FDA-approved drug library and identified that the cardiac glycosides including Ouabain, Digoxin and Digitoxin efficiently inhibit the proliferation of EAC cell lines (OE33 and OE19) both in vitro and in vivo. RNA-Sequencing analysis combined with RNAi screening revealed that Ouabain suppresses the proliferation of EAC cells through downregulation of p38 MAP-Kinase 6 (MAP2K6, also known as MKK6). Consistently, shRNA-mediated knockdown of MKK6 reduced the proliferation of EAC cells and tumor growth. Further analysis demonstrated that MKK6 inhibition leads to the reduced levels of the transcription factor SOX9. In line with this finding, deletion of SOX9 with CRISPR/Cas9 resulted in decreased proliferation of EACs in 3D organoid culture and reduced tumor growth. Together these findings establish a druggable axis that can be harnessed for therapeutic gain against EAC.

Delayed transplantation of precursor cell-derived astrocytes provides multiple benefits in a rat model of Parkinsons.

In addition to dopaminergic neuron loss, it is clear that Parkinson disease includes other pathological changes, including loss of additional neuronal populations. As a means of addressing multiple pathological changes with a single therapeutically-relevant approach, we employed delayed transplantation of a unique class of astrocytes, GDAs(BMP), that are generated in vitro by directed differentiation of glial precursors. GDAs(BMP) produce multiple agents of interest as treatments for PD and other neurodegenerative disorders, including BDNF, GDNF, neurturin and IGF1. GDAs(BMP) also exhibit increased levels of antioxidant pathway components, including levels of NADPH and glutathione. Delayed GDA(BMP) transplantation into the 6-hydroxydopamine lesioned rat striatum restored tyrosine hydroxylase expression and promoted behavioral recovery. GDA(BMP) transplantation also rescued pathological changes not prevented in other studies, such as the rescue of parvalbumin(+) GABAergic interneurons. Consistent with expression of the synaptic modulatory proteins thrombospondin-1 and 2 by GDAs(BMP), increased expression of the synaptic protein synaptophysin was also observed. Thus, GDAs(BMP) offer a multimodal support cell therapy that provides multiple benefits without requiring prior genetic manipulation.

Glial restricted precursor cell transplant with cyclic adenosine monophosphate improved some autonomic functions but resulted in a reduced graft size after spinal cord contusion injury in rats.

Transplantation of glial restricted precursor (GRP) cells has been shown to reduce glial scarring after spinal cord injury (SCI) and, in combination with neuronal restricted precursor (NRP) cells or enhanced expression of neurotrophins, to improve recovery of function after SCI. We hypothesized that combining GRP transplants with rolipram and cAMP would improve functional recovery, similar to that seen after combining Schwann cell transplants with increasing cAMP. A short term study, (1) uninjured control, (2) SCI+vehicle, and (3) SCI+cAMP, showed that spinal cord [cAMP] was increased 14days after SCI. We used 51 male rats subjected to a thoracic SCI for a 12-week survival study: (1) SCI+vehicle, (2) SCI+GRP, (3) SCI+cAMP, (4) SCI+GRP+cAMP, and (5) uninjured endpoint age-matched control (AM). Rolipram was administered for 2weeks after SCI. At 9days after SCI, GRP transplantation and injection of dibutyryl-cAMP into the spinal cord were performed. GRP cells survived, differentiated, and formed extensive transplants that were well integrated with host tissue. Presence of GRP cells increased the amount of tissue in the lesion; however, cAMP reduced the graft size. White matter sparing at the lesion epicenter was not affected. Serotonergic input to the lumbosacral spinal cord was not affected by treatment, but the amount of serotonin immediately caudal to the lesion was reduced in the cAMP groups. Using telemetric monitoring of corpus spongiosum penis pressure we show that the cAMP groups regained the same number of micturitions per 24hours when compared to the AM group, however, the frequency of peak pressures was increased in these groups compared to the AM group. In contrast, the GRP groups had similar frequency of peak pressures compared to baseline and the AM group. Animals that received GRP cells regained the same number of erectile events per 24hours compared to baseline and the AM group. Since cAMP reduced the GRP transplant graft, and some modest positive effects were seen that could be attributable to both GRP or cAMP, future research is required to determine how cAMP affects survival, proliferation, and/or function of progenitor cells and how this is related to function. cAMP may not always be a desirable addition to a progenitor cell transplantation strategy after SCI.

Directed nerve outgrowth is enhanced by engineered glial substrates.

In the present study, the influence of astrocyte alignment on the direction and length of regenerating neurites was examined in vitro. Astrocytes were experimentally manipulated by different approaches to create longitudinally aligned monolayers. When cultured on the aligned monolayers, dorsal root ganglion neurites grew parallel to the long axis of the aligned astrocytes and were significantly longer than controls. Engineered monolayers expressed linear arrays of fibronectin, laminin, neural cell adhesion molecule, and chondroitin sulfate proteoglycan that were organized parallel to one another, suggesting that a particular spatial arrangement of these molecules on the astrocyte surface may be necessary to direct nerve regeneration in vivo. In contrast, no bias in directional outgrowth was observed for neurites growing on unorganized monolayers. The results suggest that altering the organization of astrocytes and their scar-associated matrix at the lesion site may be used to influence the direction and the length of adjacent regenerating axons in the damaged brain and spinal cord.