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The overall goals of our research program are to understand the mechanisms of degeneration of motor neurons and to develop therapeutic candidates for motor neuron diseases. We use a combination of biochemical, genetic, cell biological, anatomic, pharmacological and behavioral methods to study neurodegeneration and neuroprotection.

Our primary focus is on childhood onset motor neuron diseases such as spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS) and distal hereditary motor neuronopathies (HMNs). However, the approaches we use can also be applied to other neurodegenerative diseases as well as to neuronal injury.

Main Interests

• Developing neuroprotective therapeutic strategies
• Establishing cell-culture models
• Ribonucleoprotein complexes

Current Projects

• Determination of the mechanism(s) of the neuroprotective effect of butyrate compounds on motor neuron diseases
• Identification of compounds that increase SMN2 promoter activity and inclusion of exon 7
• Generation of a SMN2 promoter and exon 7 inclusion dual reporter transgenic mouse
• Characterization of SMN-containing macromolecular complexes in motor neuron processes
• Characterization of embryonic stem cell models for SMA
• Establishment of a pediatric motor neuron diseases cell collection
• Identification of neuroprotective agents against motor neuron excitotoxicity

Spinal muscular atrophy (SMA) is a leading genetic cause of infant death in the world. SMA is the result of reduced expression of SMN protein. In humans there are two nearly identical copies of the SMN gene (SMN1 and SMN2). There is a C-T transition in exon 7 of SMN2 that alters a splicing regulatory element which leads to reduced inclusion of exon 7 in SMN2-derived transcripts and, hence, reduced levels of SMN protein. In SMA, SMN1 (which produces full-length SMN protein) is lost while SMN2 is retained. SMN gene replacement using gene therapy vectors and modulation of SMN expression by drug compounds have been shown to improve the survival and phenotypes of various mouse models of SMA.

Studies using transgenic mice have shown that increasing SMN2 copy number improves the survival and phenotype of SMA mouse models. Likewise, in humans, patients with a higher the SMN2 copy number generally have the milder the SMA phenotype. These observations collectively suggest that SMN2 may be a therapeutic target for SMA. In this project, we will generate novel methods to monitor SMN2 induction and exon 7 inclusion in the spinal cord of living SMA mice. This information will be extremely useful in understanding the effectiveness of SMN-inducing drugs in treating SMA and will lead to the design of newer drugs with better protective properties.

Butyrate-based compounds such as phenylbutyrate have been suggested to be potential drug compounds for treating SMA patients. These compounds improve survival of a mouse model of SMA but do not increase SMN in the spinal cord. Work from others has shown that administration of 4-phenylbutyrate to SOD1(G93A) amyotrophic lateral sclerosis (ALS) transgenic mice improves their survival by ~21%. In this project, we propose to test butyrate-based compounds in transgenic mouse models for motor neuron diseases such as SMA, ALS and SMA with respiratory distress 1 (SMARD1). We will also determine the mechanism(s) by which these compounds exert their neuroprotective effects. These compounds, therefore, will be more potent therapeutics for motor neuron diseases which can be moved forward into clinical trials.

The identification of therapeutic agents for early-onset motor neuron diseases (MNDs) like SMA is driven by the use of skin fibroblasts as an in cellulo model. Unfortunately, therapeutics identification in this manner has met with limited success because some compounds which increase SMN protein — the defective protein in SMA — in fibroblasts do not increase SMN expression in vivo in motor neurons. It is, therefore, desirable to have a cellular model of SMA motor neurons for dissecting the mechanisms of motor neuron loss in SMA as well as for testing the efficacies of novel therapeutics. Somatic cells such as fibroblasts can be transformed into pluripotent cells (creating induced pluripotent stem (iPS) cells) that are capable of being differentiated into many types of cells — including motor neurons.

In this project, we propose to generate iPS cell lines from our collection of fibroblasts from MND patients with varying phenotypes. These iPS cell lines will then be differentiated into motor neurons and compared against motor neurons from normal iPS cell lines. With a collection of MND iPS cell lines, we will ultimately be able to, in future studies, investigate the mechanisms of disease in affected motor neurons, identify genes and transcripts that modulate disease severity and test therapeutic agents for efficacy in affected motor neurons. Once we establish the procedure for the conversion of fibroblasts into iPS cells, we will be able to apply this technique toward other genetic disorders for which fibroblasts are available.

Deficiency of SMN in motor neurons is known to cause SMA, however, the underlying biochemical reason that motor neurons degenerate is not known. SMN is required for the assembly of small nuclear ribonucleoprotein (snRNP) complexes which function as the machinery for splicing. It has been suggested that there is a greater sensitivity of motor neurons to reduced snRNP assembly; however, this may not be the case as recent data suggests that some SMN mutations from severe SMA patients are capable of normal snRNP biogenesis but cannot rescue abnormal motor neuron axon pathfinding seen in zebrafish embryos with reduced SMN levels. SMN macromolecular complexes within motor neuron axons are critical for the normal functioning of neurons; however, this function is not yet known nor is the composition of these axonal SMN complexes. It is critical that we understand the composition of axonal SMN complexes. The other components of these axonal SMN macromolecular complexes, however, need to first be identified. Subcellular fractionation, coimmunoprecipitation and confocal microscopy will be used to identify the protein composition of these complexes. Once the components of the axonal SMN macromolecular complex have been identified, we will determine which components, if any, are altered in SMA by using various mouse models of SMA.

There are other motor neuron diseases aside from SMA that involve perturbation of some aspect of RNA metabolism. Examples of such diseases include SMA with respiratory distress (SMARD1; IGHMBP2), various familial forms of ALS (FUS/TSL and TDP-43) and lethal congenital contracture syndromes (like LCCS1; GLE1). The gene products for these familial motor neuron diseases are ubiquitously expressed but only certain cell types (i.e. motor neurons) are affected. One possible explanation for this observation is that these proteins may be involved in some aspect of RNA metabolism that is unique to neuronal processes such as axons and dendrites. To begin to address this hypothesis, we will characterize axonal RNP complexes containing IGHMBP2, FUS/TSL and TDP-43 using biochemical and cell biological approaches.