Peter W. Grabham

Cytoplasmic dynein and LIS1 are required for microtubule advance during growth cone remodeling and fast axonal outgrowth

Recent evidence has implicated dynein and its regulatory factors dynactin and LIS1 in neuronal and non-neuronal cell migration. In the current study we sought to test whether effects on neuronal cell motility might reflect, in part, a role for these proteins in the growth cone. In chick sensory neurons subjected to acute laminin treatment dynein, dynactin, and LIS1 were mobilized strikingly and rapidly to the leading edge of the growth cone, where they were seen to be associated with microtubules converging into the laminin-induced axonal outgrowths. To interfere acutely with LIS1 and dynein function and to minimize secondary phenotypic effects, we injected antibodies to these proteins just before axon initiation. Antibody to both proteins produced an almost complete block of laminin-induced growth cone remodeling and the underlying reorganization of microtubules. Penetration of microtubules into the peripheral zone of differentiating axonal growth cones was decreased dramatically by antibody injection, as judged by live analysis of enhanced green fluorescent protein-tubulin and the microtubule tip-associated EB3 (end-binding protein 3). Dynein and LIS1 inhibition had no detectable effect on microtubule assembly but reduced the ability of microtubules to resist retrograde actin flow. In hippocampal neurons dynein, dynactin, and LIS1 were enriched in axonal growth cones at stage 3, and both growth cone organization and axon elongation were altered by LIS1 RNA interference. Together, our data indicate that dynein and LIS1 play a surprisingly prominent role in microtubule advance during growth cone remodeling associated with axonogenesis. These data may explain, in part, the role of these proteins in brain developmental disease and support an important role in diverse aspects of neuronal differentiation and nervous system development.

Effects of ionizing radiation on three-dimensional human vessel models: differential effects according to radiation quality and cellular development.

Abstract Little is known about the effects of space radiation on the human body. There are a number of potential chronic and acute effects, and one major target for noncarcinogenic effects is the human vasculature. Cellular stress, inflammatory response, and other radiation effects on endothelial cells may affect vascular function. This study was aimed at understanding the effects of space ionizing radiation on the formation and maintenance of capillary-like blood vessels. We used a 3D human vessel model created with human endothelial cells in a gel matrix to assess the effects of low-LET protons and high-LET iron ions. Iron ions were more damaging and caused significant reduction in the length of intact vessels in both developing and mature vessels at a dose of 80 cGy. Protons had no effect on mature vessels up to a dose of 3.2 Gy but did inhibit vessel formation at 80 cGy. Comparison with γ radiation showed that photons had even less effect, although, as with protons, developing vessels were more sensitive. Apoptosis assays showed that inhibition of vessel development or deterioration of mature vessels was not due to cell death by apoptosis even in the case of iron ions. These are the first data to show the effects of radiation with varying linear energy transfer on a human vessel model.

Microtubule and Rac 1-dependent F-actin in growth cones

Extracellular cues control the rate and direction of growth of neuronal processes in large part by regulating the cytoskeleton of the growth cone. The actin filament network of the peripheral region is thought to be the primary target for these cues, with consequences for the advance and organization of microtubules. Binding of laminin to integrin receptors is a cue that accelerates the growth of processes from many types of neurons. It was applied acutely to sympathetic neurons in culture to study its effects on the cytoskeleton of the growth cone. Microtubules advance to the edge of the growth cone and bundle in response to laminin, and it was found that small veils of membrane appear near the ends of some of those microtubules. To examine more clearly the relationship between the microtubules and the appearance of actin-rich structures at the periphery, a low dose of cytochalasin D was used to deplete the peripheral region of the growth cone of pre-existing F-actin. The subsequent addition of laminin resulted in the bundling of ends of dynamic (tyrosinated) microtubules at the distal edge of the growth cone, most of which were associated with foci of F-actin. Observations of labeled actin within living growth cones confirmed that these foci formed in response to laminin. Suppression of microtubule dynamics with drugs eliminated the actin foci; washout of drug restored them. Rac 1 did not co-concentrate with F-actin in the peripheral region of the growth cone in the absence of laminin, but did co-concentrate with the foci of F-actin that formed in response to laminin. Inhibition of Rac 1 functioning prevented the formation of the foci and also inhibited laminin-induced neurite growth with or without cytochalasin. These results indicate that extracellular cues can affect actin in the growth cone via microtubules, as well as affect microtubules via actin. They also point to the mediation of microtubule-dependent accumulation of F-actin at the front of the growth cone as a role of Rac 1 in neurite growth.

Recruitment of the Arp2/3 complex and mena for the stimulation of actin polymerization in growth cones by nerve growth factor.

The growth of axons and dendrites during development and regeneration is regulated by cues in the environment. Many of these cues regulate the actin cytoskeleton of the protrusive structures (like filopodia) of the growth cone that are essential for detecting and responding to cues. Nerve growth factor, which promotes the formation of protrusive structures, stimulated actin polymerization in rat sympathetic growth cones, resulting within 1–2 min in accumulations of F‐actin at the distal edge and in splotches of F‐actin farther back. We examined the potential involvement of a protein machinery important in at least certain types of actin polymerization in non‐neuronal cells. Members of the Arp2/3 complex, p34‐Arc and p21‐Arc, heavily concentrated in the early accumulations of F‐actin, as did one member of the Ena/VASP family (Mena) but not another (VASP). Retention of Arc proteins at preferred sites of actin polymerization did not require polymerization itself. Growth cones of differentiated PC12 cells were similar to sympathetic growth cones in their response to NGF. Introduction into these cells of a peptide that should block the binding of Ena/VASP family proteins to the protein complex at sites of actin polymerization reduced the formation of splotches and filopodia in response to NGF. These results point to the early involvement of the Arp2/3 complex and the Ena/VASP family in growth factor‐stimulated actin polymerization that gives rise to protrusive structures at the growth cone. J. Neurosci. Res. 4:458–467, 2000

Braking news: calcium in the growth cone.

The critical unknown was whether signaling through Ca1 in the growth cone is physiologically relevant. It now seems likely that it is. One line of evidence is its Daniel J. Goldberg* and Peter W. Grabham Department of Pharmacology and Center for Neurobiology and Behavior apparent involvement in transducing responses in the Columbia University growth cone to certain cues that are likely to function New York, New York 10032 in vivo. NI-35 is a growth-inhibitory protein expressed on oligodendrocytes in the CNS (see Bandtlow et al., 1993, for references). Regeneration of injured axons Growing axons are guided by cues in the environment: within the spinal cord is promoted by antibodies that local or released from a distance, positive and negative neutralize NI-35, attesting to its importance in vivo. NI(Goodman, 1996). The motile growth cone at the tip of 35 induces growth cones in culture to collapse. The the axon senses and interprets these cues, changing its collapse is associated with a large rise in [Ca]i, at least behavior to alter the direction or speed of growth. Many partially due to release from the smooth endoplasmic cues have been identified and their in vivo importance reticulum (SER) (Bandtlow et al., 1993). Blockage of Ca1 has been verified, but how their binding is transduced release prevents collapse. into changes in growth cone behavior remains poorly Certain positive cues that are likely to function in vivo understood. Given the plethora of cues, it would simplify may also work through Ca1. NCAM and L1 are growththings if common elements of transduction pathways promoting members of the immunoglobulin superfamily emerged: for example, if stopping of a growth cone at (Walsh and Doherty, 1997). Disruption of the functioning a choice point were always (or, at least, often) caused of either in vivo leads to what appear to be abnormalities by elevation of messenger X within the growth cone, in axonal growth (Cohen et al., 1998; Seki and Rutiswhatever the external cue. Recent work points to such hauser, 1998). The promotion of growth by either in vitro key transduction molecules. Moreover, a new paper in depends on influx of Ca1 through channels in the Nature literally and figuratively illuminates the inner plasma membrane, though it is merely assumed these workings of the growth cone by fluorescently imaging are in the growth cone (Walsh and Doherty, 1997). LamiCa1 in neurons growing within the developing spinal nin is a prominent constituent of the extracellular matrix cord, providing evidence for its importance in vivo in and is expressed by Schwann cells in peripheral nerve, regulating axonal elongation (Gomez and Spitzer, 1999). where it probably promotes growth. It elicits rises in Considering that the response of growth cones to cues [Ca]i in growth cones in culture that may be important is, for technical reasons, typically studied in the reducin affecting both the rate and direction of growth (Kuhn tionist world of the culture dish, this is a noteworthy et al., 1998; but see below). accomplishment. The aforementioned environmental cues all act as Calcium Is a Physiologically Important Transduction substrate-bound molecules. Molecules released from Molecule in the Growth Cone afar can also influence growth, either attracting or repelIt has long been known that the growth cone is sensitive ling neurites. Here, Ca1 may also be involved. Netrin-1 to its [Ca]i. Large increases caused by neurotransmitis produced by the floor plate of the developing spinal ters or depolarization cause growth cones in culture to cord, from where it emanates to attract certain axons collapse, stopping neuritic elongation (Kater and Mills, to, and repel others from, the midline (see Ming et al., 1991). Decreases caused by removing Ca1 from the 1997, for references). This Janus-like behavior can be bathing medium can have similar effects. In some cases, displayed in culture in a particularly intriguing way. A growth cone activity and neuritic elongation can be prospinal neurite that turns and grows toward a micropimoted by elevations of [Ca]i over resting levels; focal pette emitting netrin-1 turns away from that micropipette changes within the growth cone can produce focal prowhen the activity of cAMP-dependent protein kinase in trusive activity appropriate for turning, for example (Davthe neurite is pharmacologically suppressed (Ming et enport and Kater, 1992). These seemingly discordant al., 1997). Neither behavior is seen when [Ca]o is drastiresults were accommodated by Kater’s set-point model, cally reduced, though straight-ahead growth proceeds in which substantial deviations in either direction from apace. Similar results are seen with brain-derived neuroan optimal [Ca]i inhibit motile activity of the growth trophic factor (BDNF) and acetylcholine. A complemencone and slow growth (Kater and Mills, 1991). This is tary situation is seen with myelin-associated glycoproso reasonable as to be unsurprising; very high or low tein (MAG), another inhibitory protein expressed by levels of [Ca]i are cytotoxic, presumably one reason oligodendrocytes (see Song et al., 1998, for references). that [Ca]i is normally tightly buffered. However, there Neurites that are repelled by a distant source of MAG were indications that the deviations from normal did are instead attracted when cAMP is artificially elevated not have to be great to inhibit growth (Lankford and (Song et al., 1998). Both the repulsion and the attraction Letourneau, 1991). Microfilaments, so important to the disappear in low [Ca1] medium. motile activity of the growth cone, are particularly sensiAlso pointing to a role for Ca1 in regulating neuritic tive to [Ca]i. growth was the discovery that certain growth cones advancing in culture display periodic brief rises in [Ca]i (termed waves) that seem to affect the speed of neuritic * To whom correspondence should be addressed (e-mail: dlg5@

Two distinct types of the inhibition of vasculogenesis by different species of charged particles

BackgroundCharged particle radiation is known to be more biologically effective than photon radiation. One example of this is the inhibition of the formation of human blood vessels. This effect is an important factor influencing human health and is relevant to space travel as well as to cancer radiotherapy. We have previously shown that ion particles with a high energy deposition, or linear energy transfer (LET) are more than four times more effective at disrupting mature vessel tissue models than particles with a lower LET. For vasculogenesis however, the relative biological effectiveness between particles is the same. This unexpected result prompted us to investigate whether the inhibition of vasculogenesis was occurring by distinct mechanisms.MethodsUsing 3-Dimensional human vessel models, we developed assays that determine at what stage angiogenesis is inhibited. Vessel morphology, the presence of motile tip structures, and changes in the matrix architecture were assessed. To confirm that the mechanisms are distinct, stimulation of Protein Kinase C (PKC) with phorbol ester (PMA) was employed to selectively restore vessel formation in cultures where early motile tip activity was inhibited.ResultsEndothelial cells in 3-D culture exposed to low LET protons failed to make connections with other cells but eventually developed a central lumen. Conversely, cells exposed to high LET Fe charged particles extended cellular processes and made connections to other cells but did not develop a central lumen. The microtubule and actin cytoskeletons indicated that motility at the extending tips of endothelial cells is inhibited by low LET but not high LET particles. Actin-rich protrusive structures that contain bundled microtubules showed a 65% decrease when exposed to low LET particles but not high LET particles, with commensurate changes in the matrix architecture. Stimulation of PKC with PMA restored tip motility and capillary formation in low but not high LET particle treated cultures.ConclusionLow LET charged particles inhibit the early stages of vasculogenesis when tip cells have motile protrusive structures and are creating pioneer guidance tunnels through the matrix. High LET charged particles do not affect the early stages of vasculogenesis but they do affect the later stages when the endothelial cells migrate to form tubes.

The effects of radiation on angiogenesis

The average human body contains tens of thousands of miles of vessels that permeate every tissue down to the microscopic level. This makes the human vasculature a prime target for an agent like radiation that originates from a source and passes through the body. Exposure to radiation released during nuclear accidents and explosions, or during cancer radiotherapy, is well known to cause vascular pathologies because of the ionizing effects of electromagnetic radiations (photons) such as gamma rays. There is however, another type of less well-known radiation – charged ion particles, and these atoms stripped of electrons, have different physical properties to the photons of electromagnetic radiation. They are either found in space or created on earth by particle collider facilities, and are of significant recent interest due to their enhanced effectiveness and increasing use in cancer radiotherapy, as well as a health risk to the growing number of people spending time in the space environment. Although there is to date, relatively few studies on the effects of charged particles on the vascular system, a very different picture of the biological effects of these particles compared to photons is beginning to emerge. These under researched biological effects of ion particles have a large impact on the health consequences of exposure. In this short review, we will discuss the effects of charged particles on an important biological process of the vascular system, angiogenesis, which creates and maintains the vasculature and is highly important in tumor vasculogenesis.

Short term effects of gamma radiation on endothelial barrier function: Uncoupling of PECAM-1

A limiting factor in the treatment of cancer with radiotherapy is the damage to surrounding normal tissue, particularly the vasculature. Vessel pathologies are a major feature of the side effects of radiotherapy and little is known about early events that could initiate subsequent diseases. We tested the hypothesis that gamma radiation has early damaging effects on the human endothelial barrier. Two models were used; Human Brain Microcapillary Endothelial Cells (HBMEC), and Human Umbilical Vein Endothelial Cells (HUVEC). Endpoints included Trans-Endothelial Electrical Resistance (TEER), barrier permeability to 10 kDa and 70 kDa tracer molecules, and the localization of F-actin, and junction proteins and the Platelet Endothelial Cell Adhesion Molecule (PECAM-1). Radiation induced a rapid and transient decrease in TEER at 3 h, with effects also seen at the radiotherapy doses. This dip in resistance correlated to the transient loss of PECAM-1 in discrete areas where cells often detached from the monolayer leaving gaps. Redistribution of PECAM-1 was also seen in 3-D human tissue models. By 6 h, the remaining cells had migrated to reseal the barrier, coincident with TEER returning to control levels. Resealed monolayers contained fewer cells per unit area and their barrier function was weakened as evidenced by an increased permeability over 24 h. This is the first demonstration of a transient and rapid effect of gamma radiation on human endothelial barriers that involves cell detachment and the loss of PECAM-1. Considering the association of cell adhesion molecules with vasculopathies, such an effect has the potential to be clinically relevant to the longer-term effects of radiotherapy.

Intrachromosomal Changes and Genomic Instability in Site-Specific Microbeam-Irradiated and Bystander Human-Hamster Hybrid Cells

Exposure to ionizing radiation may induce a heritable genomic instability phenotype that results in a persisting and enhanced genetic and functional change among the progeny of irradiated cells. Since radiation-induced bystander effects have been demonstrated with a variety of biological end points under both in vitro and in vivo conditions, this raises the question whether cytoplasmic irradiation or the radiation-induced bystander effect can also lead to delayed genomic instability. In the present study, we used the Radiological Research Accelerator Facility charged-particle microbeam for precise nuclear or cytoplasmic irradiation. The progeny of irradiated and the bystander human hamster hybrid (AL) cells were analyzed using multicolor banding (mBAND) to examine persistent chromosomal changes. Our results showed that the numbers of metaphase cells involving changes of human chromosome 11 (including rearrangement, deletion and duplication) were significantly higher than that of the control in the progeny of both nuclear and cytoplasmic targeted cells. These chromosomal changes could also be detected among the progeny of bystander cells. mBAND analyses of clonal isolates from nuclear and cytoplasm irradiations as well as the bystander cell group showed that chromosomal unstable clones were generated. Analyses of clonal stability after long-term culture indicated no significant change in the number of unstable clones for the duration of culture in each irradiated group. These results suggest that genomic instability that is manifested after ionizing radiation exposure is not dependent on direct damage to the cell nucleus.

DNA damage foci formation and decline in two-dimensional monolayers and in three-dimensional human vessel models: Differential effects according to radiation quality

Purpose: To analyze the effect of different radiation qualities on the kinetics of p53 Binding Protein 1 (53BP1) formation and decline in human three-dimensional (3-D) vessel models. Material and methods: Two-dimensional (2-D) and 3-D cultures of human umbilical vein cells were exposed to 80 cGy of Gamma radiation and high-energy protons and Fe ions. 53BP1 antibodies were used for foci visualization via immunocytochemistry. Computer analysis was used to determine the number and the size of foci up to 48 hours after irradiation. Results: DNA foci kinetics in 2-D and 3-D human vessel cultures show that foci formation and removal were the same in each type of culture. After 48 h, the number of foci induced by high-energy protons and gamma rays reduced to almost control levels while high linear energy transfer (LET) Fe particles produced more persistent damage. Conclusion: The kinetics of radiation-induced 53BP1 foci in 3-D vessel models is essentially the same as in 2-D monolayers. Since the basal level of spontaneous foci is low in these differentiated non-proliferating cultures, the persistence of radiation-induced 53BP1 foci is detected longer than previously noted. Furthermore, analysis of foci sizes revealed that abnormal radiation-induced foci can persist even when foci frequencies are close to basal levels. The detection of these latent abnormalities could be useful for a more sensitive dosimetry.