Peter W. Baas, BSc, MS, PhD - US grants

Project Summary/Abstract Neurons are terminally post-mitotic cells that use their microtubule arrays not for cell division but rather as architectural elements required for the elaboration of elongated axons and dendrites. In addition to acting as compression-bearing struts that provide for the shape of the neuron, microtubules also act as directional railways for organelle transport. Microtubules in the axon are nearly uniformly oriented, with the plus ends of the microtubules directed away from the cell body (?plus-ends-out?). Preservation of this microtubule pattern is crucial for the normal functioning of an axon throughout the life of the neuron. This microtubule polarity pattern is also important for distinguishing features of the axon from the dendrite, as dendrites of vertebrate neurons have a mixed pattern of microtubule polarity orientation. Long-standing questions in cellular neuroscience are how the plus-end-out polarity pattern of microtubules arises in the axon, how axons maintain this pattern during the life of the neuron and how flaws arising from plastic events in the life of the neuron are repaired. This competing renewal builds on a mechanism called ?polarity sorting,? in which microtubules are organized according to their polarity orientation via their transport by molecular motor proteins. Specifically, the investigators hypothesize that in the case of the healthy axon, microtubules are transported with plus-ends leading, so that plus-end-out microtubules move forward down the axon while minus-end-out microtubules are transported back to the cell body to clear them from the axon. If not for this clearing mechanism, minus-end- out microtubules would accumulate in the axon and corrupt its polarity pattern. The proposed experiments will first test the polarity-sorting hypothesis. Experiments will then be conducted to test the hypothesis that two minus-end-directed molecular motor proteins, namely cytoplasmic dynein and KIFC1, share the responsibility of polarity sorting microtubules in the axon. Finally, studies will be conducted to identify the structures against which the molecular motors generate forces to transport microtubules in the axon. Mobile microtubules are generally quite short, and are hypothesized to move against either long microtubules or actin bundles, depending on the particular motor protein. Computational modeling will add additional rigor to the project, especially in terms of explaining why the axon?s microtubule polarity pattern is corrupted when various molecular players are manipulated. The experiments will utilize contemporary live-cell imaging techniques with unprecedented documentation of the polarity orientation of mobile microtubules in the axon, together with methods for acutely inhibiting molecular motor proteins. The work has relevance to potential treatments for diseases of the nervous system that may corrupt the microtubule polarity pattern of the axon.

DESCRIPTION: The proposed research will examine the mechanisms by which the axonal microtubule array is elaborated during axon extension. The ability of neurons to extend axons over long distances is dependent upon the elaboration of highly organized arrays of microtubules. In the proposed experiments, the role of microtublule transport during axon growth will be addressed in living cells. There are 3 specific aims: (1) In the first aim, microtubule behaviors at the neuronal centrosome will be observed within living neurons to determine whether centrosomal microtubules actually detach and move into the axon. (2) In the second aim, fluorescent tubulin will be introduced into neurons in the form of stable microtubule fragments, assembly-incompetent tubulin subunits, or potential intermediate tubulin aggregates, and it will be determined whether tubulin in any of these forms is actively transported down the axon. (3) In the third aim, microtubule behaviors in the axon that underlie the formation of collateral branches will be investigated. Direct observation as well as pharmacologic tools will be used to determine whether microtubules arise within newly-forming branches via transport from the parent axon. Collectively, these efforts will resolve the contribution of microtubule transport to critical features of axon growth.

animal tissue; microscopy; nervous system; growth factor; biomedical resource;

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] This application is the competing renewal for the ongoing postdoctoral training program at Drexel University College of Medicine focused on the biological bases of nervous system disorders. The objective of the program is to provide trainees with the perspectives and skills needed to prepare them for independent careers in neurobiological research. We request support for four fellows, who will have either the Ph.D. or M.D. degree. The program will provide opportunities for multidisciplinary research projects in the laboratories of both basic and clinical scientists and exposure to a wide variety of experimental and clinical issues. The program is broadly-based, and consists of four focal groups that are highly interactive with one another. The first group focuses on cellular and developmental neurobiology; the second group on systems and behavioral neurobiology, the third group on spinal cord and regeneration; and the fourth group on neuroengineering. There are a total of 10 senior level faculty members who will act as the primary training faculty. There are an additional 8 junior level faculty members who will also act as trainers, under the supervision of the senior faculty. The entire participating faculty, both senior and junior, is funded by the NIH, well published, and active in both research and training. In addition, there is an abundance of clinical researchers at three different hospitals within our university system who will participate in the training of the fellows. In addition to the primary relationship between a trainee and the mentor, there are a number of activities that ensure a broad exposure to the issues and techniques of modern neurobiology. These include courses developed around the four focal groups, journal clubs and seminars, research conferences and shared laboratory facilities. The progress of each trainee will be formally reviewed regularly and monitored constantly, through research meetings and seminars. This process assures full access to the various disciplines and perspectives available in the program. [unreadable] [unreadable]

The proposed research is important for understanding how nerves grow and branch. In particular, the project focuses on microtubules, which are elongated polymers that provide architecture for the nerve and also act as railways to move proteins and structures down the nerve. For the nerve to grow and branch, there must be mobility within the microtubule array itself. Notably, it has been discovered in recent years that only the shortest microtubules in the nerves are mobile. To generate more mobility within the microtubule array, nerves use special proteins that cut long microtubules into short ones. The purpose of this project is to study how these microtubule "cutters" work, and how they are controlled to get their job done properly. The cutting of microtubules is also important for many other types of cells, so the studies will be of interest and importance across various fields of biology. The general approach will be to make cultures of rat neurons, and then use reliable methods to alter the microtubule cutting proteins, and then see what the effects are on microtubules and the shape of the nerves. The results are expected to show what other molecules and mechanisms the neuron uses to control the cutting proteins so they cut microtubules at the right time and place in the nerves. Broad impact will be achieved through strong efforts to recruit minority students into the graduate program at Drexel University. Even broader impact will be achieved by an outreach to scientists and students in the developing nations of Africa. The Principal Investigator will work with African partners to develop strategies for bringing African students to the USA for part of their training so that the students can prosper, upon their return to their home countries.

PROJECT SUMMARY / ABSTRACT Hereditary Spastic Paraplegias (HSP) are heritable neurodegenerative diseases in which progressive degeneration of corticospinal axonal tracts results in limb weakness, spasticity and gait deficiencies. These symptoms result from a dying back pattern of degeneration of corticospinal axons, which also display prominent swellings of unclear pathological significance. The commonest form of HSP, termed SPG4-HSP, is caused by mutations in the SPAST gene, which codes for a microtubule-severing protein called spastin. To date, the prevailing mechanistic hypothesis for the etiology of SPG4-HSP is haploinsufficiency, meaning that the corticospinal tracts degenerate because of insufficient functional spastin. However, several major disease features are not readily explained by this etiology, and it is not clear how reduced microtubule severing would promote corticospinal axonal degeneration. Providing novel information that may fill a major gap in our knowledge of SPG4-HSP pathogenesis, recent work of the Principal Investigators revealed toxic properties of mutant spastin proteins, suggesting that a gain-of-function mechanism operates in SPG4-HSP. Curiously, both mechanisms negatively affect fast axonal transport (FAT), a cellular process fueled by molecular motor proteins that allows bidirectional movement of vesicular cargoes along axons. Based on a strong experimental premise, it is hypothesized in this multi-PI grant proposal that abnormalities in microtubule organization associated with reduced spastin levels cause FAT deficits and axonal swellings (loss-of-function). On the other hand, toxic effects of mutant spastin protein cause different FAT deficits that are mediated by casein kinase 2 (CK2), and these deficits promote corticospinal axon degeneration (gain-of-function). The former makes the axon more vulnerable, but it is the latter that suffices for corticospinal axon degeneration. The proposed work seeks to test these hypotheses by directly comparing a mouse model with a single SPAST allele (SPAST +/-) with a transgenic mouse model with both endogenous mouse SPAST alleles intact that additionally expresses human spastin bearing a pathogenic mutation associated with SPG4-HSP (spastin-C448Y mice). In Aim 1, these models will be individually crossed with mice that selectively express eGFP in corticospinal motor neurons (CSMN), so that loss-of and gain-of-function contributions to the disease can be investigated. In Aim 2, FAT deficits will be studied in neurons cultured from these animals, and specific hypotheses for the etiology of the deficits will be tested. In Aim 3, studies are proposed using transgenic spastin-C448Y mice in which autophagy is experimentally enhanced or CK2 levels are experimentally reduced, to test the hypothesis that these manipulations will prevent or reduce corticospinal axon degeneration and associated behavioral deficits. The overall significance of this project is to establish mechanisms underlying SPG4-HSP and forge a path toward effective therapies for patients.

PROJECT SUMMARY / ABSTRACT Tau, a fibrous microtubule-associated protein concentrated in the axon, binds to microtubules, thereby influencing its properties. For decades, the prevailing theory has been that tau stabilizes microtubules in the axon. This is based on test tube studies showing that excess tau can indeed stabilize microtubules, as well as overexpression studies showing this to be the case in non-neuronal cells as well. Disease researchers are so invested in the idea of tau as a microtubule stabilizer that work is underway to use microtubule-stabilizing drugs to treat diseases such as Alzheimer?s, in which tau loses its association with microtubules. Recent published work of the Principal Investigator has challenged this dogma, building on evidence that tau is enriched on the labile domains of microtubules in the axon than on the stable domains. Moreover, in that work, when tau was depleted from the neuron with RNA interference, there was a loss of microtubule mass but not due to destabilization of the stable domains of the microtubules. Rather, there was a preferential loss of the labile domains, with the remaining portion of the labile domains actually becoming more stable rather than less stable. Additional studies suggested that tau prevents the labile domain from becoming stable by outcompeting genuine microtubule-stabilizers such as MAP6, and also promotes the assembly of the labile domain. This is a strikingly different scenario from the one that has become so established in the scientific literature, and potentially transformational to both the basic sciences and medical sciences of tau. However, all of that work was done on developing rodent neurons in culture, with the very real possibility that the situation is not the same in adult brain. In this proposal, the Principal Investigator seeks to test whether the findings hold true in adult mouse brain, using viral-driven RNA interference to lower tau levels. From there, studies are proposed to delve into the proposed competition between tau and genuine microtubule stabilizers such as MAP6 by ectopically expressing fluorescently tagged versions of them in non-neuronal cells in which binding of these proteins to microtubules can be readily visualized. Collectively, these studies will contribute significantly to understanding of tau, a crucial protein for both normal functioning of the axon and for disease, and will open the door toward mechanism-based therapies to rectify the ill effects of tau loss-of-function in disease.

PROJECT SUMMARY/ABSTRACT Spinal cord injury (SCI) is a devastating condition that affects about 250,000 Americans with 17,700 new cases annually costing upwards of $2 million each. There is very little that can be done to treat these patients that improves their prospects for even partial recovery or the amelioration of symptoms that negatively impact their quality of life. SCI research continues to advance, but bolder and more innovative breakthroughs are needed, if the efforts are to translate into therapies that improve symptoms, alleviate pain, and restore functionality. As highlighted by the NIH-hosted SCI-2020 meeting, a new generation of SCI researchers is desperately needed, one that is thoroughly educated in all of the needed background and history of the field while also equipped to bring new technologies and insights into the field through strong collaborative interactions with scientists from other fields. Drexel University College of Medicine is home to the Marion Murray Spinal Cord Research Center, which has over three decades of history advancing the SCI field while simultaneously educating and training doctoral students to take the field forward. Within the Center is the Drexel SCI Training Program, which consists of well-funded and vibrant investigators who study SCI from many different perspectives, and mentor trainees in many different approaches. These include engineering, electrophysiology, cell transplantation, rehabilitation and others. The center has extensive multidisciplinary collaborations with Drexel investigators from other fields, many of whom are involved in the SCI Training Program. The Center is based within the Department of Neurobiology and Anatomy, with the students affiliated with the Neuroscience Graduate Program. SCI students are exposed to a multitude of training experiences, including but not limited to what the Neuroscience Graduate Program offers, with flexibility that welcomes students from other graduate programs to transfer into the program in order to conduct SCI-relevant research. The program has existed for over thirty years, with consistent success of the students securing F31 funding to fund their senior years, and then achieving success in their careers after completing the program. T32 funding for the program will enable expansion of the research and the number of trainees, thus improving the prospects of SCI patients, for whom treatments have remained elusive for too long.