Current Research

Our research interests include (1) neuronal communication and maintenance, (2) development of tools to control transcript and protein levels in adult neurons to assess which proteins are required for neuronal function and survival, (3) the creation of genome-wide libraries to manipulate genes in vivo. Our lab uses Drosophila melanogaster as a model system because most biological processes are evolutionarily conserved and studies in fruit flies provide many important clues about the aging process in animals and humans.

The demise of neurons

Introduction

Our interest in synaptic transmission prompted us to study the function of the VapB protein. We discovered that the protein did not play a prominent role in neurotransmission, but rather was required for the development and maintenance of the neuromuscular junction (NMJ). It was then discovered that the loss of its human homolog causes Amyotrophic Lateral Sclerosis. We therefore decided to investigate the biological processes leading to the demise of aging neurons in these and other mutants. Our research strives to (1) determine the key biological processes initiating the demise of neurons in aging flies, (2) elucidate the molecular processes that impair the function of the affected organelles, (3) find ways to improve neuronal function in degenerating neurons, and (4) develop methods in which neurodegenerative processes may be suppressed. We tackle these problems by combining forward genetics, electrophysiological paradigms, transmission electron microscopy, cell biology, biochemistry, pharmacology, and genomic approaches.
Our model neuron, the fly photoreceptor (PR), offers many advantages. Its electrical activity can be gauged upon light exposure via electroretinograms (ERGs). Unlike vertebrate PRs, the fly PRs sense light and directly project their axons into two different layers of the fly brain. The stereotyped arrangements of cell bodies of the photoreceptors in fly ommatidia makes performing light and electron microscopy in the retina simple while stereotyped projections of the photoreceptor terminals in the fly brain allows ultrastructural analyses of defined axons and synapses. The ERGs provide information about the functional properties of the neurons while the morphological studies provide cell biology information, including the types of organelles that have been affected.

Strategies

To define the key processes that initiate the demise of neurons we performed large scale forward unbiased mosaic genetic screens in adult flies. Our strategy was to create random lethal mutations, induced on chromosomes carrying a transgene that promoted recombination between specific chromosome arms (FRT) upon expression of a recombinase (Flipase). This allowed us to produce homozygous mutant clones in the eye of heterozygous adult flies. We then monitored the demise of PR neurons over a period of up to 6 weeks.

A high throughput screening procedure led to the rapid identification of numerous mutations causing a functional loss of PRs. The development of a new mapping strategy, using a BAC transgenic technique, P[acman], permits, in combination with whole genome sequencing, rapid mapping of these mutations.

Once we identify the gene, we define the cells or organelles in which the proteins are expressed, assess when the loss of the gene leads to the first symptoms, and attempt to unravel the molecular processes that lead to the demise of the neurons by studying the presymptomatic stages of neurodegeneration. Finally, we attempt to suppress the neurodegenerative features associated with the deleterious mutations using genetic and pharmacologic approaches.

Results from these screens have caused us to focus on nuclear genes whose products are trafficked to the mitochondria and on genes that are involved in autophagy and endo-lysosomal trafficking. We also study NMNAT and VapB. We isolated the first fly mutations in these genes and have been trying to unravel how they cause the demise of neurons.

Mitochondria

40% of the genes we have identified so far from our unbiased genetic screens encode mitochondrial proteins, indicating that mitochondria play a critical role in numerous forms of neurodegeneration. We have isolated mutations in 34 different genes (Figure 1), 18 of which have human homologs associated with diseases in infants and adults. These diseases, which exhibit both shared and distinct clinical presentations, led us to investigate the phenotypes associated with mutations in these genes in Drosophila. Based on characterization and comparison of these mutants, we have developed common phenotypic profiles and identified common underlying mechanisms. We found that mutations in very different genes and affecting very different processes can cause strikingly similar phenotypes while genes affecting the same pathway or process can lead to very different phenotypes. These observations permit us to classify the genes based on various criteria and develop targeted strategies to suppress the neurodegenerative phenotypes for seemingly unrelated genes and processes.

Figure 1
Figure 1. Through a forward genetic screen, aimed at isolating genes that are required for neuronal maintenance, we identified numerous proteins that are targeted to or associated with mitochondria (bold lined proteins). These proteins function in very diverse pathways, including mitochondrial transcription /translation (green), lipid metabolism (blue), bio-energetics (yellow) and mitochondrial dynamics (red). We are studying these mutants with the aim of delineating their mitochondrial function(s). In addition, we discovered that genes that function in the same pathway, display a wide variety of phenotypes when their function is lost. Similar observations have been made for patients suffering from mitochondrial diseases. We are currently studying the underlying cause(s) of this phenotypic variation in order to find common molecular mechanisms associated with different mitochondrial mutants that cause similar phenotypes.

Lysosomes

So far, we have identified 5 genes that affect lysosome function. Mutations in subunits of the retromer complex (Vps26 and Vps35) reveal that Rhodopsin1 (Rh1), a light sensing protein in PRs, is recycled from endosomes upon activation and endocytosis. The loss of retromer-dependent recycling leads to constitutive Rh1 lysosomal degradation, burdening the endo-lysosomal pathway, with subsequent photoreceptor dysfunction. Loss of Crag, the first Guanylate Exchange Factor (GEF) for Rab11 identified, leads to increased Rh1 lysosomal degradation and photoreceptor degeneration.

We also found that loss of subunits of the voltage gated Ca2+ channel (VGCC), a complex that has only been studied in the context of synaptic transmission, causes an age-dependent accumulation of autophagic vacuoles (AVs) in PR terminals. Loss of the mouse VGCC homologs leads to similar autophagic defects and degeneration of neurons, indicating that VGCC plays a previously uncharacterized evolutionarily conserved role in autophagy and lysosomal degradation.

NMNAT

Mutations in nmnat (NAD synthase nicotinamide mononucleotide adenylyltransferase) cause the quickest and most severe PR loss of all genes identified so far. Importantly, overexpression of NMNAT provides strong protective effects against light induced PR degeneration in flies and protects against Spinocerebellar ataxia 1 (SCA1)-induced neurodegeneration, suggesting a broad neuroprotective function. NMNAT protects neuronal function and survival partly through a proteasome-mediated pathway, independent of its enzymatic activity. NMNAT displays strong chaperone activity both in biochemical assays and cultured cells, and shares significant structural similarity with a known yeast chaperone.

We are unraveling the precise mechanism by which this protein acts as a potent neuroprotective agent by studying genetic and protein interactors of NMNAT. In order to exploit NMNAT’s neuroprotective functions in human diseases, especially Alzheimer’s disease, we are collaborating with Hui-Chen Lu and Huda Zoghbi at BCM and scientists at MD Anderson to develop potential drugs.

VapB and Amyotrophic Lateral Sclerosis

Mutations in the human gene VapB were shown to cause Amyotrophic Lateral Sclerosis 8 (ALS8), a severe neuromuscular disease in which motor neurons stop functioning. In Drosophila, we unraveled a new signaling pathway whereby the human VapB protein, which is an ER associated protein, is cleaved, secreted and circulated in the blood (Figure 2). We showed in collaboration with Michael Miller at University of Alabama and Hiroshi Tsuda at McGill University that its aminoterminal MSP domain binds to growth cone guidance receptors on muscles and regulates glutamate receptor clustering as well as mitochondrial dynamics in a cell non-autonomous manner. A point mutation associated with ALS8 blocks the secretion of VapB. Other familial forms of ALS, as well as sporadic cases, also exhibit diminished levels of VapB, indicating that defects in this novel signaling pathway may underlie multiple forms of this disease. We are now pursuing experiments to determine if VapB can be used as a diagnostic tool and a therapeutic molecule for ALS.

Figure 2
Figure 2. A schematic model of the VapB signaling pathway. VapB (blue) is associated with the ER and regulates protein folding and ceramide transport from the ER to the Golgi together with CERT. The full length VAP protein is cleaved, releasing the MSP domain from the neuron. MSP binds the growth cone guidance receptors in the muscle, including Eph, Lar, and Robo. MSP binding to Eph leads to unclustering of Glutamate receptors, and binding to Lar and Robo is required for proper mitochondrial morphology, localization, and function through regulation of the actin and tubulin cytoskeleton.

Genome wide tools to manipulate flies

We have been developing technology and reagents for the fly community for the past 25 years. These resources are popular to manipulate the fly genome. We helped develop enhancer detector transposable elements that permitted the cloning of hundreds of genes and also lead to the development of the UAS-GAL4 system by Brand and Perrimon, the most widely used Drosophila technology for expressing a gene/protein/RNAi of interest in a tissue specific manner. In collaboration with Allan Spradling at Carnegie and Roger Hoskins at Lawrence Berkeley National Laboratory, we are generating a library of transgenic flies with tagged genes using transposable elements, permitting the mutational analysis of nearly 10,000 genes in vivo. We also developed a new transformation vector named P[acman] that allows the insertion of transgenes up to 250 kb and the integration of the transgene at defined sites to avoid positional effects. This vector also allows elegant manipulations of the DNA in bacteria using recombineering. The vector also permitted the creation of a library of over 100,000 transformation ready P[acman] clones covering the whole fly genome. Recently, we developed a new transposable element collection of MiMIC insertions that permits genomic tagging of thousands of proteins as well as the temporal and spatial knockdown of genes and proteins in a reversible manner in specific cells at all stages of development and in adult flies.

References