Don Cleveland, PhD - US grants

DESCRIPTION: This application is aimed at elucidating the fundamental role of neurofilament subunits (NF-L, NF-M, NF-H) in specifying the axonal volume of neural cells and their role in motor neuron disease. Aim 1 will continue the investigation of the mechanism of neurofilament dependent growth using transgenic mice that over express or are deleted for each subunit. In particular the role played by the phosphorylated tail domains of NF-M and NF-H in specifying the three dimensional array of neurofilaments and how they are linked to other axonal components will be examined. Aim 2 will study the mechanism of neurofilament- dependent motor neuron disease, expanding previous observations that increased neurofilament burden in axons or expression of a mutant NF-L subunit cause selective motor neuron failure in transgenic mice. Point mutations in superoxide dismutase 1 (SOD1) which cause amyotrophic lateral sclerosis (ALS) in human also cause motor neuron disease in transgenic mice and Aim 3 will investigate the mechanism by which defects in this ubiquitously expressed cytoplasmic protein lead to the selective death of motor neurons. Experiments will test directly whether the toxic properties of SOD1 mutations arise through an effect on neurofilament accumulation and transport.

The research interests of the Cleveland group focus on mechanisms of neuronal growth and death using molecular genetics to assess how axons grow and what factors provoke selective death of motor neurons. Unlike most eukaryotic cells, an intrinsic feature of neurons is their extreme asymmetry. for example, in the human peripheral nervous system, single motor neurons extend over a meter in length. Asymmetry is achieved in two phases. The first is when a neurite extends towards its target. After stable synapse formation, a second phase initiates in which the axon grows up to ten fold in diameter. This radial growth phase, concomitant with meylination and essential for establishment of proper conduction velocity, yields an enormous increase in axonal volume and a huge cell, 99.9% of which is in the axon. We have focused part of our effort on using molecular genetics and transgenic mice to test how radial growth of axons is achieved. Already we have shown that neurofilaments, the most abundant structural element in axons, are essential for radial growth. We have now identified a set of neurofilament-associated proteins and, in collaboration with Dr. Burlingame, now wish to use modern protein analysis methods to determine the identities of these associated proteins. Once the corresponding genes are isolated we will then use transgenic and gene disruption methods in mice to determine the corresponding in vivo properties. Beyond this, essentially all human motor neuron disorders are characterized by the maldistribution of neurofilaments, a finding clearly suggesting that they may play an essential role in disease pathogenesis. This is particularly true in the most prominent motor neuron disease, amyotrophic lateral sclerosis, or ALS, which is characterized by selective death of motor neurons. By producing transgenic mice expressing mutations in neurofilaments, we have proven that such mutations can cause ALS in mice and we are now searching for the presence of similar mutations in human patients. The cause of only 1.5% of human ALS is known and this is point mutations in an enzyme superoxide dismutase. By expression of these mutations in mice, we have proven that disease arises from a toxic property of the mutant proteins and we are now looking for what that property is and what is the cascade of events leading to selective motor neuron death. By using high resolution protein analysis methods, we now seek to determine whether neurofilaments are indeed targets for covalent damage arising from ALS-linked SOD1 mutations, and if so, to identify the nature of the aberrant chemistry mediated by these mutant proteins.

embryo /fetus cell /tissue; neoplasm /cancer

mass spectrometry

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Like the skeleton in humans, the cytoskeleton provides the mechanic support for the individual cells. The cytoskeleton is also essential for compartmentation and signal transduction pathways within the cells. There are three types of cytoskeleton: microtubules, microfilaments, and intermediate filaments. These cytoskeletal systems are essentially composed by simple subunits that in turn form polymers to give rise to the final filamentous form. Unlike microtubules and microfilaments, which are formed by cytosolic tubulin and actin, respectively, intermediate filaments (IFs) are formed by a large family of differentially-expressed genes. Typically, IF can take up to 1~5% of total proteins inside the cells. The disorganization and/or aggregation of IF are a hallmark of many human diseases. The outstanding question is how individual IF subunit assemble into filaments and how the filaments disassemble into subunits in vivo. The goal of this project is to identify the factors required for the assembly and disassembly processes and to delineate the in vivo pathways. Finally, we would like to know whether these pathways are altered in the disease settings and whether the molecules identified in this project can be used as therapeutic targets.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Amyotrophic Lateral Sclerosis (ALS) is a late onset neurodegenerative disease leading to paralysis. Mutations in the gene for the ubiquitously expressed Cu/Zn superoxide dismutase (SOD1) are the best-known cause for familial ALS and transgenic mice constitutively expressing mutant SOD1 develop a late-onset, ALS-like disease. It is known that degeneration of upper and lower motor neurons is responsible for paralysis in ALS and that glial cell types expressing mutant SOD1 contribute to disease mechanism. We showed that microglial cells, the immune cells of the CNS, were important players, since diminishing mutant SOD1 specifically in macrophages/ microglial cells extended survival in ALS mice. Microglial cells are activated in any injury of the CNS including sporadic and familial ALS and when activated, they release factors that can be toxic or trophic for neurons. Therefore, identifying those factors could be a key to finding new targets implicated in motor neuron death. As downregulating mutant SOD1 from macrophages/microglial cells slowed disease progression in ALS mice, mutant SOD1 must act directly within microglial cells to generate toxicity toward motor neurons. Therefore, microglial cells expressing mutant or wild-type SOD1 will be screened for the different factors that they can release using Mass Spectrometry. Conditioned medium from cultured microglial cells will be used as a source of released microglial factors and medium from cells expressing mutant SOD1 will be compared to medium from wild-type SOD1 expressing microglial cells. Identification of the differences between mutant SOD1 expressing microglial cells and control microglial cells should help elucidate the intracellular pathways involved and the toxic factors released by microglial cells that contribute to motor neuron death.

DESCRIPTION (provided by applicant): Spinal Muscular Atrophy (SMA) and Amyotrophic Lateral Sclerosis (ALS) are fatal motor neuron disorders for which no significant treatments currently exist. Delivery of a therapeutic agent across the blood brain barrier (BBB) to the central nervous system is a significant problem that prevents the effective development of therapies to treat neurodegenerative diseases such as SMA and ALS. Here we propose to develop a simple vascular delivery to transduce genes across the BBB and have an impact on treatment of neurological disorders. We have discovered the unique capacity for the adeno-associated virus (serotype 9) to traverse the BBB and to efficiently target motor neurons and astrocytes within the brain and spinal cord. Here we wish to expand on these studies in mice and to translate them into the non-human primate in order to develop promising therapies for motor neuron disease. Here we propose (1) to optimize the correction of SMA and treatment of ALS in mouse models, (2) to develop a vascular delivery route for motor neuron and astrocytes targeting in the non-human primate and (3) to determine if ALS targets identified in mutant SOD1 mouse models function in human sporadic and familial SOD1 ALS models. We have assembled a team of investigators with all the critical expertise for the study of both SMA and ALS. This proposal includes studies that will not only further the understanding of the biological mechanism of motor neuron disease, but will also lead to the development of a technique for vascular delivery of therapeutics that will have widespread impact for many neurological disorders. PUBLIC HEALTH RELEVANCE: This delivery system will revolutionize therapies for all neurological disorders. We will specifically focus on the two major motor neuron disorders, Spinal Muscular Atrophy (SMA) and Amyotrophic Lateral Sclerosis (ALS). The delivery of SMN in SMA will have a major benefit.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Progression through the cell cycle is regulated at multiple checkpoints. For example, the spindle checkpoint monitors the fidelity of chromosome segregation. This checkpoint inhibits cells from progressing into anaphase until all sister chromatids are properly attached to the mitotic spindle and aligned at the metaphase plate. A number of proteins have been shown to be important in this process including MAD1, MAD2, TTK, Bub1, BubR1 and BubR2. To further understand the molecular mechanism by which these protein regulated the spindle checkpoint a mass spectrometry analysis was performed to identify phosphorylation sites on Mad1 by the TTK kinase.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Progression through the cell cycle is regulated at multiple checkpoints. For example, the spindle checkpoint monitors the fidelity of chromosome segregation. This checkpoint inhibits cells from progressing into anaphase until all sister chromatids are properly attached to the mitotic spindle and aligned at the metaphase plate. A number of proteins have been shown to be important in this process including MAD1, MAD2, TTK, Bub1, BubR1 and BubR2. Previous studies have shown that the kinesin-related motor protein CENP-E interacts with BubR1 and TTK. To further understand the importance of CENP-E to the spindle checkpoint mass spectrometry will be used to interaction partners of CENP-E and it post-translational modifications.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Accurate control of the number of centrosomes, the major microtubule- organizing centers of animal cells, is critical for the maintenance of genomic integrity. Abnormalities in centrosome number can promote errors in spindle formation that lead to subsequent chromosome missegregation and extra centrosomes are found in many cancers. Centrosomes are comprised of a pair of centrioles surrounded by amorphous pericentriolar material and centrosome duplication is controlled by centriole replication. Polo-like kinase 4 (Plk4) plays a key role in initiating centriole duplication and overexpression of Plk4 promotes centriole overduplication and the formation of extra centrosomes. Recently, we showed that kinase active Plk4 is inherently unstable and targeted for degradation. Using mass spectrometry we demonstrated that Plk4 multiply self-phosphorylates within a 24 amino-acid ?phosphodegron" and that phosphorylation of multiple sites within this region is required for Plk4 instability. We continue to investigate how Plk4 auto-regulated instability acts to self-limits Plk4 activity so as to prevent centrosome amplification and genomic instability.

This project addresses the challenge of increasing diversity in the biological science academic workforce as well as amongst the recipients of grants from national agencies. The Faculty Research and Education Development Program to be implemented in this project focuses on promoting the competitive grant-writing skills of junior faculty at Minority Serving Institutions by matching junior faculty with experienced mentors who are successful senior faculty at research-intensive institutions. The year-long structured mentoring process will focus on the preparation and submission of a grant proposal. In addition to regular (at least monthly) communication between junior faculty and mentors, the program will include 1) Career Development Workshops for junior faculty and mentors, to be held each year 2) reciprocal visits by junior faculty and their mentors to each other's institution, at which they will present a seminar 3) meetings for junior faculty and mentors at the Annual Conference in December of the American Society for Cell Biology. A curated publicly accessible and searchable website will be developed to disseminate results of this program and to serve as a resource for junior faculty and mentors throughout the US. Summative and formative evaluation will be performed by a professional evaluation consultant. Broader Impacts: The goal of this project is to promote the skills in research and education of junior faculty at Minority Serving Institutions, and thereby to improve the educational environment for the undergraduates whom they teach and mentor. Web-based dissemination of the results of this program will make this model accessible to others, and will provide a resource for young scientists to assist them in their professional advancement.