SummaryMotor neurons (MNs) and astrocytes (ACs) are implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), but their interaction and the sequence of molecular events leading to MN death remain unresolved. Here, we optimized directed differentiation of induced pluripotent stem cells (iPSCs) into highly enriched (> 85%) functional populations of spinal cord MNs and ACs. We identify significantly increased cytoplasmic TDP-43 and ER stress as primary pathogenic events in patient-specific valosin-containing protein (VCP)-mutant MNs, with secondary mitochondrial dysfunction and oxidative stress. Cumulatively, these cellular stresses result in synaptic pathology and cell death in VCP-mutant MNs. We additionally identify a cell-autonomous VCP-mutant AC survival phenotype, which is not attributable to the same molecular pathology occurring in VCP-mutant MNs. Finally, through iterative co-culture experiments, we uncover non-cell-autonomous effects of VCP-mutant ACs on both control and mutant MNs. This work elucidates molecular events and cellular interplay that could guide future therapeutic strategies in ALS.

Nerve growth factor (NGF) is essential for the survival of both peripheral ganglion cells and central cholinergic neurons of the basal forebrain. The accelerated loss of central cholinergic neurons during Alzheimer's disease may be a determinant of dementia in these patients and may therefore suggest a therapeutic role for NGF. However, NGF does not significantly penetrate the blood-brain barrier, which makes its clinical utility dependent on invasive neurosurgical procedures. When conjugated to an antibody to the transferrin receptor, however, NGF crossed the blood-brain barrier after peripheral injection. This conjugated NGF increased the survival of both cholinergic and noncholinergic neurons of the medial septal nucleus that had been transplanted into the anterior chamber of the rat eye. This approach may prove useful for the treatment of Alzheimer's disease and other neurological disorders that are amenable to treatment by proteins that do not readily cross the blood-brain barrier.

Obesity-resistant (A/J) and obesity-prone (C57BL/6J) mice were weaned onto low-fat (LF) or high-fat (HF) diets and studied after 2, 10, and 16 wk. Despite consuming the same amount of food, A/J mice on the HF diet deposited less carcass lipid and gained less weight than C57BL/6J mice over the course of the study. Leptin mRNA was increased in white adipose tissue (WAT) in both strains on the HF diet but to significantly higher levels in A/J compared with C57BL/6J mice. Uncoupling protein 1 (UCP1) and UCP2 mRNA were induced by the HF diet in brown adipose tissue (BAT) and WAT of A/J mice, respectively, but not in C57BL/6J mice. UCP1 mRNA was also significantly higher in retroperitoneal WAT of A/J compared with C57BL/6J mice. The ability of A/J mice to resist diet-induced obesity is associated with a strain-specific increase in leptin, UCP1, and UCP2 expression in adipose tissue. The findings indicate that the HF diet does not compromise leptin-dependent regulation of adipocyte gene expression in A/J mice and suggest that maintenance of leptin responsiveness confers resistance to diet-induced obesity.

Various model systems have been used to study the expression of the recently cloned ob gene, leptin. Here we report that freshly isolated rat white adipocytes incubated with insulin release leptin in a rapid and concentration-dependent manner (EC50 of 0.221 +/- .075 nM). Insulin-stimulated leptin release could be detected as early as 30 min and a maximal 2-3 fold effect was produced by 10 nM insulin. The effect of insulin was completely blocked by simultaneous activation of cAMP-dependent protein kinase. Using the activation of lipolysis as an index of cAMP-dependent protein kinase activity, we show that inhibition of leptin release by norepinephrine or the selective beta 3-adrenergic receptor agonist, CL316,243, occurred in parallel to activation of cAMP-dependent protein kinase. In addition, beta 1- and beta 2-adrenergic receptor antagonists did not impair the ability of norepinephrine or CL316,243 to inhibit leptin release from the adipocytes. These findings suggest that the beta 3-adrenergic receptor plays a central role in regulating the release of leptin from the adipocyte.

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BackgroundModelling the blood-CNS barriers of the brain and spinal cord in vitro continues to provide a considerable challenge for research studying the passage of large and small molecules in and out of the central nervous system, both within the context of basic biology and for pharmaceutical drug discovery. Although there has been considerable success over the previous two decades in establishing useful in vitro primary endothelial cell cultures from the blood-CNS barriers, no model fully mimics the high electrical resistance, low paracellular permeability and selective influx/efflux characteristics of the in vivo situation. Furthermore, such primary-derived cultures are typically labour-intensive and generate low yields of cells, limiting scope for experimental work. We thus aimed to establish protocols for the high yield isolation and culture of endothelial cells from both rat brain and spinal cord. Our aim was to optimise in vitro conditions for inducing phenotypic characteristics in these cells that were reminiscent of the in vivo situation, such that they developed into tight endothelial barriers suitable for performing investigative biology and permeability studies.MethodsBrain and spinal cord tissue was taken from the same rats and used to specifically isolate endothelial cells to reconstitute as in vitro blood-CNS barrier models. Isolated endothelial cells were cultured to expand the cellular yield and then passaged onto cell culture inserts for further investigation. Cell culture conditions were optimised using commercially available reagents and the resulting barrier-forming endothelial monolayers were characterised by functional permeability experiments and in vitro phenotyping by immunocytochemistry and western blotting.ResultsUsing a combination of modified handling techniques and cell culture conditions, we have established and optimised a protocol for the in vitro culture of brain and, for the first time in rat, spinal cord endothelial cells. High yields of both CNS endothelial cell types can be obtained, and these can be passaged onto large numbers of cell culture inserts for in vitro permeability studies. The passaged brain and spinal cord endothelial cells are pure and express endothelial markers, tight junction proteins and intracellular transport machinery. Further, both models exhibit tight, functional barrier characteristics that are discriminating against large and small molecules in permeability assays and show functional expression of the pharmaceutically important P-gp efflux transporter.ConclusionsOur techniques allow the provision of high yields of robust sister cultures of endothelial cells that accurately model the blood-CNS barriers in vitro. These models are ideally suited for use in studying the biology of the blood-brain barrier and blood-spinal cord barrier in vitro and for pre-clinical drug discovery.

Objective-It has been generally believed that adipocytes are derived from mesenchymal stem cells via fibroblasts. We recently reported that fibroblasts/myofibroblasts in a number of tissues and organs are derived from hematopoietic stem cells (HSCs).Methods-In the present study, we tested the hypothesis that HSCs also give rise to adipocytes using transplantation of single EGFP + HSCs and primary culture.Results-Adipose tissues from clonally engrafted mice showed EGFP + adipocytes that stained positive for leptin, perilipin and fatty acid binding protein 4. A diet containing rosiglitazone, a peroxisome proliferator-activated receptor-gamma agonist, significantly enhanced the number of the EGFP + adipocytes. When EGFP + bone marrow cells from clonally engrafted mice were cultured under adipogenic conditions, all of the cultured cells stained positive with oil red O and sudan black B and exhibited the presence of abundant mRNA for adipocyte markers. Finally, clonal culture and sorting based studies of Mac-1 expression of hematopoietic progenitors suggested that adipocytes are derived from HSCs via progenitors for monocytes/macrophages. Conclusion-Together, these studies not only clarify the current controversy regarding the ability of HSCs to give rise to adipocytes, but do so based on the analysis of single hematopoietic stem cells in vivo and in vitro. Furthermore, our primary culture method that generates adipocytes from uncommitted hematopoietic cells should contribute to the studies of the mechanisms of early adipocytic differentiation and may lead to development of therapeutic solutions for many general obesity issues.

The three known subtypes of ␤-adrenoreceptors (␤ 1 -AR, ␤ 2 -AR, and ␤ 3 -AR) are differentially expressed in brown and white adipose tissue and mediate peripheral responses to central modulation of sympathetic outflow by leptin. To assess the relative roles of the ␤-AR subtypes in mediating leptin's effects on adipocyte gene expression, mice with a targeted disruption of the ␤ 3 -adrenoreceptor gene (␤ 3 -AR KO) were treated with vehicle or the ␤ 1 /␤ 2 -AR selective antagonist, propranolol (20 g/g body weight/day) prior to intracerebroventricular (ICV) injections of leptin (0.1 g/g body weight/day). Leptin produced a 3-fold increase in UCP1 mRNA in brown adipose tissue of wild type (FVB/NJ) and ␤ 3 -AR KO mice. The response was unaltered by propranolol in wild type mice, but was completely blocked by this antagonist in ␤ 3 -AR KO mice. In contrast, ICV leptin had no effect on leptin mRNA in either epididymal or retroperitoneal white adipose tissue (WAT) from ␤ 3 -AR KOs. Moreover, propranolol did not block the ability of exogenous leptin to reduce leptin mRNA in either WAT depot site of wild type mice. These results demonstrate that the ␤ 3 -AR is required for leptin-mediated regulation of ob mRNA expression in WAT, but is interchangeable with the ␤ 1 /␤ 2 -ARs in mediating leptin's effect on UCP1 mRNA expression in brown adipose tissue.In mice the absence of leptin (ob/ob) or its functional receptor (db/db) produces a complex metabolic syndrome characterized by hyperphagia, endocrine abnormalities, and morbid obesity (1). Deposition of excess body fat occurs even when food intake is controlled, suggesting that an important function of leptin is to regulate energy balance through modulation of metabolic efficiency. This view is supported by studies in ob/ob mice showing that leptin-injected animals lose more weight than pair-fed vehicle-injected littermates (2, 3). Of particular interest is the observation that leptin-induced weight loss occurs specifically in adipose tissue with little effect in other tissues (3, 4). The loss of adipose tissue is associated with an increase in fat oxidation, and the associated shift in fuel selection can be measured as a decrease in the respiratory quotient during leptin repletion (5, 6). Thus, adipose tissue is an important target of leptin action and the primary effect is a shift from fat storage to fat mobilization and oxidation.This leptin-mediated shift in adipocyte function involves a coordinated change in gene expression. Two mechanisms have been postulated and include both centrally mediated effects and direct effects through functional leptin receptors (Ob-Rb) on the adipocyte (7-9). It should be noted, however, that although supraphysiologic levels of leptin are capable of producing significant direct effects on adipose tissue (10, 11), increments of plasma leptin in the physiological range are thought to act primarily through receptors in the hypothalamus (10). Occupancy of hypothalamic leptin receptors promotes activation of the sympathetic nervous system (12-15),...