Alfredo Giménez-Cassina

BAD-Dependent Regulation of Fuel Metabolism and KATP Channel Activity Confers Resistance to Epileptic Seizures

Neuronal excitation can be substantially modulated by alterations in metabolism, as evident from the anticonvulsant effect of diets that reduce glucose utilization and promote ketone body metabolism. We provide genetic evidence that BAD, a protein with dual functions in apoptosis and glucose metabolism, imparts reciprocal effects on metabolism of glucose and ketone bodies in brain cells. These effects involve phosphoregulation of BAD and are independent of its apoptotic function. BAD modifications that reduce glucose metabolism produce a marked increase in the activity of metabolically sensitive K(ATP) channels in neurons, as well as resistance to behavioral and electrographic seizures in vivo. Seizure resistance is reversed by genetic ablation of the K(ATP) channel, implicating the BAD-K(ATP) axis in metabolic control of neuronal excitation and seizure responses.

Infectious Delivery and Expression of a 135 kb Human FRDA Genomic DNA Locus Complements Friedreich's Ataxia Deficiency in Human Cells

Friedreichs ataxia (FA) is the most common recessive ataxia, affecting 1-2 in 50,000 Caucasians, and there is currently no effective cure or treatment. FA results from a deficiency of the mitochondrial protein frataxin brought about by a repeat expansion in intron 1 of the FRDA gene. The main areas affected are the central nervous system (particularly the spinocerebellar system) and cardiac tissue. Therapies aimed at alleviating the neurological degeneration have proved unsuccessful to date. Here, we describe the construction and delivery of high capacity herpes simplex virus type 1 (HSV-1) amplicon vectors expressing the entire 80 kb FRDA genomic locus, driven by the endogenous FRDA promoter and including all introns and flanking regulatory sequences within a 135 kb genomic DNA insert. FA patient primary fibroblasts deficient in frataxin protein and exhibiting sensitivity to oxidative stress were transduced at high efficiency by FRDA genomic locus vectors. Following vector transduction, expression of FRDA protein by immunofluorescence was shown. Finally, functional complementation studies demonstrated restoration of the wild-type cellular phenotype in response to oxidative stress in transduced FA patient cells. These results suggest the potential of the infectious bacterial artificial chromosome-FRDA vectors for gene therapy of FA.

Mitochondrial Hexokinase II Promotes Neuronal Survival and Acts Downstream of Glycogen Synthase Kinase-3

Mitochondrial alterations are detected in most neurodegenerative disorders and may contribute to the dysfunction and demise of neuronal cells. Because glycogen synthase kinase-3 (GSK-3) is considered to be a critical factor in regulating neuronal cell survival and death, we studied the effects of modulating GSK-3 activity in cultured neurons treated with the mitochondrial inhibitor, rotenone. Interestingly, chronic inhibition of GSK-3 protects against rotenone-induced apoptosis in cultured neuronal cells. In an attempt to elucidate the molecular mechanisms underlying this neuroprotection, we demonstrated that chronic inhibition of GSK-3 reprograms the metabolism of neuronal cells, leading to an enhancement of glycolysis. This effect was accompanied by the induction and accumulation of hexokinase II (HKII) in the mitochondria. Interfering with either the mitochondrial binding of HKII or HKII expression significantly diminished the neuroprotection evoked by GSK-3 inhibition, and importantly, HKII overexpression is sufficient to protect against rotenone-induced cell death. Thus, mitochondrial HKII is a promoter of neuronal survival under the regulation of GSK-3. Furthermore, the neuroprotective effect of HKII may be relevant to neurodegenerative diseases in which glucose hypometabolism and mitochondrial dysfunction are prominent features.

Homeostatic functions of BCL-2 proteins beyond apoptosis.

Since its introduction in 1930 by physiologist Walter Bradford Cannon, the concept of homeostasis remains the cardinal tenet of biologic regulation. Cells have evolved a highly integrated network of control mechanisms, including positive and negative feedback loops, to safeguard homeostasis in face of a wide range of stimuli. Such control mechanisms ultimately orchestrate cell death, division and repair in a manner concordant with cellular energy and ionic balance to achieve proper biologic fitness. The interdependence of these homeostatic pathways is also evidenced by shared control points that decode intra- and extracellular cues into defined effector responses. As critical control points of the intrinsic apoptotic pathway, the BCL-2 family of cell death regulators plays an important role in cellular homeostasis. The different anti- and pro-apoptotic members of this family form a highly selective network of functional interactions that ultimately governs the permeabilization of the mitochondrial outer membrane and subsequent release of apoptogenic factors such as cytochrome c. The advent of loss- and gain-of-function genetic models for the various BCL-2 family proteins has not only provided important insights into apoptosis mechanisms but also uncovered unanticipated roles for these proteins in other physiologic pathways beyond apoptosis (Fig. 1). Here, we turn our attention to these alternative cellular functions for BCL-2 proteins. We begin with a brief introduction of the cast of characters originally known for their capacity to regulate apoptosis and continue to highlight recent advances that have shaped and reshaped our views on their physiologic relevance in integration of apoptosis with other homeostatic pathways.

Polysome Profiling in Liver Identifies Dynamic Regulation of Endoplasmic Reticulum Translatome by Obesity and Fasting

Obesity-associated metabolic complications are generally considered to emerge from abnormalities in carbohydrate and lipid metabolism, whereas the status of protein metabolism is not well studied. Here, we performed comparative polysome and associated transcriptional profiling analyses to study the dynamics and functional implications of endoplasmic reticulum (ER)–associated protein synthesis in the mouse liver under conditions of obesity and nutrient deprivation. We discovered that ER from livers of obese mice exhibits a general reduction in protein synthesis, and comprehensive analysis of polysome-bound transcripts revealed extensive down-regulation of protein synthesis machinery, mitochondrial components, and bile acid metabolism in the obese translatome. Nutrient availability also plays an important but distinct role in remodeling the hepatic ER translatome in lean and obese mice. Fasting in obese mice partially reversed the overall translatomic differences between lean and obese nonfasted controls, whereas fasting of the lean mice mimicked many of the translatomic changes induced by the development of obesity. The strongest examples of such regulations were the reduction in Cyp7b1 and Slco1a1, molecules involved in bile acid metabolism. Exogenous expression of either gene significantly lowered plasma glucose levels, improved hepatic steatosis, but also caused cholestasis, indicating the fine balance bile acids play in regulating metabolism and health. Together, our work defines dynamic regulation of the liver translatome by obesity and nutrient availability, and it identifies a novel role for bile acid metabolism in the pathogenesis of metabolic abnormalities associated with obesity.

Changing appetites: the adaptive advantages of fuel choice

Cells are capable of metabolizing a variety of carbon substrates, including glucose, fatty acids, ketone bodies, and amino acids. Cellular fuel choice not only fulfills specific biosynthetic needs, but also enables programmatic adaptations to stress conditions beyond compensating for changes in nutrient availability. Emerging evidence indicates that specific switches from utilization of one substrate to another can have protective or permissive roles in disease pathogenesis. Understanding the molecular determinants of cellular fuel preference may provide insights into the homeostatic control of stress responses, and unveil therapeutic targets. Here, we highlight overarching themes encompassing cellular fuel choice; its link to cell fate and function; its advantages in stress protection; and its contribution to metabolic dependencies and maladaptations in pathological conditions.

Regulation of mitochondrial nutrient and energy metabolism by BCL-2 family proteins

Cells have evolved a highly integrated network of mechanisms to coordinate cellular survival/death, proliferation, differentiation, and repair with metabolic states. It is therefore not surprising that proteins with canonical roles in cell death/survival also modulate nutrient and energy metabolism and vice versa. The finding that many BCL-2 (B cell lymphoma 2) proteins reside at mitochondria or can translocate to this organelle has long motivated investigation into their involvement in normal mitochondrial physiology and metabolism. These endeavors have led to the discovery of homeostatic roles for BCL-2 proteins beyond apoptosis. We predominantly focus on recent findings that link select BCL-2 proteins to carbon substrate utilization at the level of mitochondrial fuel choice, electron transport, and metabolite import independent of their cell death regulatory function.

Differentiation of a human neuroblastoma into neuron-like cells increases their susceptibility to transduction by herpesviral vectors

Gene transfer is a powerful tool for functional gene analysis in human cells. In this respect, there is a need to develop experimental models that involve homogeneous cultures of human neuron‐like cells susceptible to gene transduction and that are easy to handle. Here we describe an optimized and reproducible procedure to differentiate human SH‐SY5Y neuroblastoma cells into a homogeneous population of neuron‐like cells. The fully differentiated cells are postmitotic and resemble primary cultured neurons in terms of their cytoskeletal polarity. Notably, differentiated SH‐SY5Y cells are far more susceptible to transduction by herpes simplex virus (HSV‐1)‐based vectors than proliferating SH‐SY5Y cells. This increase in transduction efficiency after neuronal differentiation may be due to the up‐regulation of cell surface receptors for herpesvirus entry. In summary, we propose that fully differentiated human neuron‐like cells obtained from the SH‐SY5Y neuroblastoma may constitute an excellent and versatile experimental tool for gene transfer and functional genomic studies with HSV‐1 vectors.

Hexokinase II gene transfer protects against neurodegeneration in the rotenone and MPTP mouse models of Parkinson's disease.

A typical feature of Parkinsons disease is the progressive loss of dopaminergic neurons in the substantia nigra, in which inhibition of mitochondrial complex I activity may play an important role. Rotenone or 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) inhibit the mitochondrial complex I and they cause the death of substantia nigra dopaminergic neurons, thereby providing acute murine models of Parkinsons disease. We have found that increasing mitochondrial hexokinase II activity can prevent cell death in neuronal cultures treated with rotenone. As a result, we have studied the effects of hexokinase II gene transfer in vivo using a herpes simplex virus type 1 (HSV‐1) amplicon vector. The placHK2 amplicon vector was injected into substantia nigra of mice that were subsequently administered rotenone or MPTP. Overexpression of hexokinase II prevented both rotenone and MPTP‐induced dopaminergic neuronal cell death, as well as reducing the associated motor defects. Our results provide the first proof‐of‐principle that hexokinase II protects against dopaminergic neurodegeneration in vivo, emphasizing the role of this enzyme in promoting neuronal survival. Thus, the increase of hexokinase II expression by gene transfer or other means represents a promising approach to treat Parkinsons and other neurodegenerative diseases.

Peroxisome Proliferator-activated Receptor γ Coactivator-1 α Isoforms Selectively Regulate Multiple Splicing Events on Target Genes.

Endurance and resistance exercise training induces specific and profound changes in the skeletal muscle transcriptome. Peroxisome proliferator-activated receptor γ coactivator-1 α (PGC-1α) coactivators are not only among the genes differentially induced by distinct training methods, but they also participate in the ensuing signaling cascades that allow skeletal muscle to adapt to each type of exercise. Although endurance training preferentially induces PGC-1α1 expression, resistance exercise activates the expression of PGC-1α2, -α3, and -α4. These three alternative PGC-1α isoforms lack the arginine/serine-rich (RS) and RNA recognition motifs characteristic of PGC-1α1. Discrete functions for PGC-1α1 and -α4 have been described, but the biological role of PGC-1α2 and -α3 remains elusive. Here we show that different PGC-1α variants can affect target gene splicing through diverse mechanisms, including alternative promoter usage. By analyzing the exon structure of the target transcripts for each PGC-1α isoform, we were able to identify a large number of previously unknown PGC-1α2 and -α3 target genes and pathways in skeletal muscle. In particular, PGC-1α2 seems to mediate a decrease in the levels of cholesterol synthesis genes. Our results suggest that the conservation of the N-terminal activation and repression domains (and not the RS/RNA recognition motif) is what determines the gene programs and splicing options modulated by each PGC-1α isoform. By using skeletal muscle-specific transgenic mice for PGC-1α1 and -α4, we could validate, in vivo, splicing events observed in in vitro studies. These results show that alternative PGC-1α variants can affect target gene expression both quantitatively and qualitatively and identify novel biological pathways under the control of this system of coactivators.