Regulation of the actin cytoskeleton in the neuronal growth cone

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2005.Includes bibliographical references (leaves 94-111).During the development of the nervous system, axons and dendrites are guided to their targets throughout the brain and body through the detection of diffusible and surface-bound guidance cues. The growth cone, a specialized structure found at the tips of axons and dendrites, detects and interprets these cues. Activation of downstream intracellular singling pathways leads to changes in the cytoskeleton of the growth cone that affect its motility. Although a number of the key players in this system have been identified, the means by which the growth cone is able to directly rearrange its cytoskeleton in response to guidance cues is not known. In the following experiments, I have examined the roles of several known actin- binding proteins in the remodeling of the growth cone cytoskeleton. The Arp2/3 protein complex binds to an existing actin filament and nucleates formation of a daughter branch. Arp2/3 activity generates a dense actin network in fibroblast cells that drives membrane protrusions. However, the role of this activity in neuronal growth cones has not been established. We have used an inhibitory strategy to explore the requirement for Arp2/3 in growth cone morphology and function. Arp2/3 is not required for growth cone morphology or the generation of protrusive structures; however, it may be required for proper motility in response to axon guidance cues. Activity the EnaNASP family of proteins promotes the formation of long, unbranched filaments in fibroblast systems. Ena/VASP has also been genetically linked to axon guidance pathways in Drosophila, C. elegans, and mice.(cont.) To examine the role of Ena/VASP proteins in growth cones, we used a strategy to simultaneously inhibit all three mammalian family members in neurons. Ena/VASP proteins were found to have a key role in filopodia formation in growth cones. During the course of my graduate work I was involved in several collaborations that examined the roles of several other proteins involved in the regulation of the actin cytoskeleton. Relevant excerpts from these experiments are presented.by Geraldine A. Strasser.Ph.D

Integrators of the Cytoskeleton that Stabilize Microtubules

AbstractSensory neurodegeneration occurs in mice defective in BPAG1, a gene encoding cytoskeletal linker proteins capable of anchoring neuronal intermediate filaments to actin cytoskeleton. While BPAG1 null mice fail to anchor neurofilaments (NFs), BPAG1/NF null mice still degenerate in the absence of NFs. We report a novel neural splice form that lacks the actin-binding domain and instead binds and stabilizes microtubules. This interaction is functionally important; in mice and in vitro, neurons lacking BPAG1 display short, disorganized, and unstable microtubules defective in axonal transport. Ironically, BPAG1 neural isoforms represent microtubule-associated proteins that when absent lead to devastating consequences. Moreover, BPAG1 can functionally account for the extraordinary stability of axonal microtubules necessary for transport over long distances. Its isoforms interconnect all three cytoskeletal networks, a feature apparently central to neuronal survival

Recommendations for the design of therapeutic trials for neonatal seizures

Although seizures have a higher incidence in neonates than any other age group and are associated with significant mortality and neurodevelopmental disability, treatment is largely guided by physician preference and tradition, due to a lack of data from welldesigned clinical trials. There is increasing interest in conducting trials of novel drugs to treat neonatal seizures, but the unique characteristics of this disorder and patient population require special consideration with regard to trial design. The Critical Path Institute formed a global working group of experts and key stakeholders from academia, the pharmaceutical industry, regulatory agencies, neonatal nurse associations, and patient advocacy groups to develop consensus recommendations for design of clinical trials to treat neonatal seizures. The broad expertise and perspectives of this group were invaluable in developing recommendations addressing: (1) use of neonate-specific adaptive trial designs, (2) inclusion/exclusion criteria, (3) stratification and randomization, (4) statistical analysis, (5) safety monitoring, and (6) definitions of important outcomes. The guidelines are based on available literature and expert consensus, pharmacokinetic analyses, ethical considerations, and parental concerns. These recommendations will ultimately facilitate development of a Master Protocol and design of efficient and successful drug trials to improve the treatment and outcome for this highly vulnerable population

Arp2/3 Is a Negative Regulator of Growth Cone Translocation

Arp2/3 is an actin binding complex that is enriched in the peripheral lamellipodia of fibroblasts, where it forms a network of short, branched actin filaments, generating the protrusive force that extends lamellipodia and drives fibroblast motility. Although it has been assumed that Arp2/3 would play a similar role in growth cones, our studies indicate that Arp2/3 is enriched in the central, not the peripheral, region of growth cones and that the growth cone periphery contains few branched actin filaments. Arp2/3 inhibition in fibroblasts severely disrupts actin organization and membrane protrusion. In contrast, Arp2/3 inhibition in growth cones minimally affects actin organization and does not inhibit lamellipodia protrusion or de novo filopodia formation. Surprisingly, Arp2/3 inhibition significantly enhances axon elongation and causes defects in growth cone guidance. These results indicate that Arp2/3 is a negative regulator of growth cone translocation.National Institutes of Health (U.S.) (Grant 6895154)W. M. Keck Foundation (Distinguished Young Scholar Award

The Murine Nck SH2/SH3 Adaptors Are Important for the Development of Mesoderm-Derived Embryonic Structures and for Regulating the Cellular Actin Network

Mammalian Nck1 and Nck2 are closely related adaptor proteins that possess three SH3 domains, followed by an SH2 domain, and are implicated in coupling phosphotyrosine signals to polypeptides that regulate the actin cytoskeleton. However, the in vivo functions of Nck1 and Nck2 have not been defined. We have mutated the murine Nck1 and Nck2 genes and incorporated β-galactosidase reporters into the mutant loci. In mouse embryos, the two Nck genes have broad and overlapping expression patterns. They are functionally redundant in the sense that mice deficient for either Nck1 or Nck2 are viable, whereas inactivation of both Nck1 and Nck2 results in profound defects in mesoderm-derived notochord and embryonic lethality at embryonic day 9.5. Fibroblast cell lines derived from Nck1(−/−) Nck2(−/−) embryos have defects in cell motility and in the organization of the lamellipodial actin network. These data suggest that the Nck SH2/SH3 adaptors have important functions in the development of mesodermal structures during embryogenesis, potentially linked to a role in cell movement and cytoskeletal organization

Combined absence of TRP53 target genes ZMAT3, PUMA and p21 cause a high incidence of cancer in mice

Transcriptional activation of target genes is essential for TP53-mediated tumour suppression, though the roles of the diverse TP53-activated target genes in tumour suppression remains poorly understood. Knockdown of ZMAT3, an RNA-binding zinc-finger protein involved in regulating alternative splicing, in haematopoietic cells by shRNA caused leukaemia only with the concomitant absence of the PUMA and p21, the critical effectors of TRP53-mediated apoptosis and cell cycle arrest respectively. We were interested to further investigate the role of ZMAT3 in tumour suppression beyond the haematopoietic system. Therefore, we generated Zmat3 knockout and compound gene knockout mice, lacking Zmat3 and p21, Zmat3 and Puma or all three genes. Puma-/-p21-/-Zmat3-/- triple knockout mice developed tumours at a significantly higher frequency compared to wild-type, Puma-/-Zmat3-/- or p21-/-Zmat3-/-deficient mice. Interestingly, we observed that the triple knockout and Puma-/-Zmat3-/- double deficient animals succumbed to lymphoma, while p21-/-Zmat3-/- animals developed mainly solid cancers. This analysis suggests that in addition to ZMAT3 loss, additional TRP53-regulated processes must be disabled simultaneously for TRP53-mediated tumour suppression to fail. Our findings reveal that the absence of different TRP53 regulated tumour suppressive processes changes the tumour spectrum, indicating that different TRP53 tumour suppressive pathways are more critical in different tissues.This work was supported by grants and fellowships from the Australian Phenomics Network (APN), the Australian National Health and Medical Research Council (NHMRC) to MJH and AS (1143105), Programme Grant to AS (1016701), Investigator grant to AS (2007887), Investigator Grant to MJH (2017971); the Leukaemia and Lymphoma Society of America to AS and MJH (LLS SCOR 7015-18); the Cancer Council of Victoria Project grant to AS (1052309) and Venture Grant to MJH and AS; support to AS from the estate of Anthony (Toni) Redstone OAM; support to AJ from Spanish Ministry of Economy and Development Grant (PID2021-127710OB-I00) and Programa Captació de Talent Investigador ‘La Caixa’ foundation (51110009). AJ is supported by Ramon y Cajal Research Fellowship (RYC2018-025244-I), AS and MJH are supported by NHMRC Fellowships (1020363 and 1156095), MSB is supported by Cancer Council Victoria Postdoctoral Fellowship and Swedish Cancer Society (21 0355 PT). This work was made possible through the Victorian Government Operational Infrastructure Support and Australian Government, the ‘Unidad de Excelencia María de Maeztu’ funded by the Spanish Government

A dysfunctional desmin mutation in a patient with severe generalized myopathy

Mice lacking desmin produce muscle fibers with Z disks and normal sarcomeric organization. However, the muscles are mechanically fragile and degenerate upon repeated contractions. We report here a human patient with severe generalized myopathy and aberrant intrasarcoplasmic accumulation of desmin intermediate filaments. Muscle tissue from this patient lacks the wild-type desmin allele and has a desmin gene mutation encoding a 7-aa deletion within the coiled-coil segment of the protein. We show that recombinant desmin harboring this deletion cannot form proper desmin intermediate filament networks in cultured cells, nor is it able to assemble into 10-nm filaments in vitro. These findings provide direct evidence that a mutation in desmin can cause human myopathies

VEGF mediates commissural axon chemoattraction through its receptor Flk1

Growing axons are guided to their targets by attractive and repulsive cues. In the developing spinal cord, Netrin-1 and Shh guide commissural axons toward the midline. However, the combined inhibition of their activity in commissural axon turning assays does not completely abrogate turning toward floor plate tissue, suggesting that additional guidance cues are present. Here we show that the prototypic angiogenic factor VEGF is secreted by the floor plate and is a chemoattractant for commissural axons in vitro and in vivo. Inactivation of Vegf in the floor plate or of its receptor Flk1 in commissural neurons causes axon guidance defects, whereas Flk1 blockade inhibits turning of axons to VEGF in vitro. Similar to Shh and Netrin-1, VEGF-mediated commissural axon guidance requires the activity of Src family kinases. Our results identify VEGF and Flk1 as a novel ligand/receptor pair controlling connmissural axon guidance