Ravits Laboratory
The Neurogenomics Laboratory at Benaroya Research Center is devoted to amyotrophic lateral sclerosis (ALS), commonly known as Lou Gehrig's disease. ALS is a progressive disease causing paralysis of muscles from degeneration of motor neurons in the central nervous system. About 10% of cases are genetic and 90% are sporadic. The cause and mechanisms are unknown. Our research focuses on understanding the molecular mechanisms of motor neuron degeneration, especially the anatomical propagation or spread of disease. In 2007, we analyzed the clinical onset of ALS and postulated that degeneration is a focal process that spreads contiguously outward over time, a phenomenon seen most clearly in early disease before it becomes complex by temporal-spatial summation (1). As a consequence of this focality and the way ALS marches anatomically into the respiratory system, death in effect arrests the disease at a point in time when motor neuron loss is arranged relative to the site of onset. In a companion analysis to our clinical one, we showed that indeed this was usually true, that “stage” of degeneration can be defined on the basis of the degree of neuronal loss relative to the site of onset, an idea that had not previously been in the neuropathological literature (2). These observations of ALS focality and spread suggest key properties of ALS neurodegeneration (3) including that what is critically important is not just cell death but propagation of disease—that ALS motor neuron degeneration is a 3-dimensional disease: neuronal death over time and space. Spatial propagation seem to involve two types of mechanisms—those that are trans-synaptic and those that are local (such as controlled by so called paracrine factors). Interestingly, since genetically-caused ALS is phenotypically identical to sporadic ALS, it seems reasonable to presume that a combination of local and generalized abnormalities together produce motor neuron degeneration and its propagation—that a generalized abnormality is locally primed.
Our research exploits this unique focality for direct study in ALS nervous systems to identify candidate pathobiology: focality creates regions in that are early to intermediate in their stage of degeneration and these regions present research opportunity. Among the difficulties for investigation of ALS are the discreteness of the affected neuronal population; the diminishing nature of the neuronal targets of investigation; and the low ratio of pathogenic signal to noise. By applying newly available microchip and laser genomic technologies, it becomes possible to bridge pathology and molecular biology to key directly and comprehensively on specific cellular compartments in these regions. These technologies include laser microdissection, RNA amplification, microelectrophoresis, microarray, and recently RNAseq. In the last 6 years that we have been working in this area, there has been rapid advance of these technologies. Together, they allow relatively comprehensive cell-specific exploration of the human genome. In order for these studies to be valid, there must be optimal tissue acquisition for simultaneous preservation of molecular quality and structural integrity and we first spent several years working out and validating the details of this and establishing a repository of nervous systems to serve as the basis of our work. In order for genomic signals to be detected, there are major challenges for data analysis of the genome-scale data and we work with and are supported by a collaboration with investigators at the Microsoft Research (Microsoft’s research division, http://research.microsoft.com/en-us/ ).
Our latest studies have profiled exon splicing in SALS motor neurons and are especially interesting in light of the growing appreciation of the complexity of RNA biology in general and the growing recognition of the importance of RNA processing in ALS in particular. We have recently reported three main observations from our transcriptional studies (4):
• key biological signals are compartment-specific, strongest in the pool of motor neurons isolated by laser microdissection and weak in the surrounding anterior horn microenvironment;
• abnormalities of exon splicing are more significant than abnormalities of differential gene expression and are more up-regulated than down-regulated (consistent with an active energy-dependent pathological process);
• aberrantly spliced genes are highly enriched in cell-matrix adhesion biology, suggesting the attachment and communication of the motor neuron to its microenvironment plays an important role in molecular pathogenesis (and interesting in view of our beliefs about pathological spread).
The perturbed extracellular matrix adhesion that we have identified in SALS motor neurons is very similar to that recently identified in a mouse model of spinal muscular atrophy (SMA) motor neuron degeneration. What is particularly interesting is that the common denominator in SALS and SMA motor neuron degeneration is not that genes are aberrantly spliced but that in the two types of motor neuron degeneration there are very different and non-overlapping cohorts of aberrantly spliced genes that have the same biological function, altered extracellular matrix adhesion biology.
We are now pursing a variety of experiments in transgenic mice of ALS1 (SOD1) and in 2- and 3-dimensional cell culture of mouse and human motor neuron-like cells transfected with wild-type and mutant TDP-43 to help clarify the significance of this and study cell-cell interactions for models of propagation.
Dr. John Ravits is a clinical neurologist at the Virginia Mason Medical Center, Research Associate Member of the Benaroya Research Institute and a Clinical Professor of Neurology at the University of Washington. His clinical work has concentrated on neuromuscular diseases and clinical neurophysiology especially ALS over the last 20 years and this been the driving force behind the establishment of the neurogenomics laboratory. He now spends 50% of his time in patient care and 50% in genomics research outlined above.
Ravits Laboratory Members
Stuart Rabin, PhD, Staff Scientist
Hugo Kim
Ryan Libby
Michael Baughn
Aaron Bodansky
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