Fractones and basement membranes: matrix for patterning, growth and development

Growth factors, cytokines and chemokines are proteins that influence cell proliferation, differentiation, migration, apoptosis and other biological determinants that determine cell fate and cell functions, ultimately the phenomena of generation, regeneration, and degeneration throughout life. Most of these molecules diffuse throughout biological fluids and bind heparin. In vivo they require heparan sulfate proteoglycans (HSPG) located in the extracellular matrix (ECM) to target their cell‐surface receptors and ultimately induce specific biological effects. We have characterized by immunohistochemistry and electron microscopy a new structure found in some parts of the extracellular space as part of the ECM that we named fractones. Fractones are chemically similar to basement membranes and consist of laminin isoforms, collagen‐IV, nidogen and HSPG. However, fractones and basement membranes differ by their location. Fractones reside in the parenchyma of tissues and organs and are visualized as particles by light microscopy whereas they often display a fractal ultrastructure (a reference to their name). In contrast, basement membranes are located as sheaths at the connective tissue/parenchyma interface and in between the layers of the vascular wall. We have shown that fractone‐associated heparan sulfates bind, concentrate, and activate the growth factors FGF‐2, BMP‐4 and BMP‐7 to regulate stem cell proliferation in the neurogenic zone of the adult brain. We have also found fractones are abundantly expressed throughout mammalian development and distributed in a manner that reflects morphogenesis. Fractones were found in all animals that we investigated including vertebrates (human, mouse and rats) and invertebrates (cnidarians, sponges, copepods, decapodes, insects). HSPG are highly dynamic molecules formed by post‐translational processes in both the Golgi apparatus, and in the ECM where extracellular enzymes are also secreted. Our results strongly suggest that fractones are essential chemicals for constructing and maintaining biological structures. We are currently elaborating the model of tissue explants, i.e. miniature fragments of organs to study growth factors/fractone interactions that will ultimately provide insights into the mechanisms that control cell fate in a near‐physiological context. We anticipate that the model explants originating from physiological animals and from animal disease models will revolutionize our understanding of molecular interactions between proteoglycans and their ligands through tissues and organs and throughout life. This research will provide the basics for new paradigms that orchestrate biological structures and functions in health and disease. We are interested in establishing methods to identify specific carbohydrate sequences in the ECM structures that bind signaling proteins. This may allow elucidation of a signaling code used by tissues for growth and maintenance. Establishment of such a code would allow for discovery of disease.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Figure S1. Attachment and growth of clonal-density primary human melanocytes on ECM. Figure S2. Presence and requirement for laminin-332. Figure S3. Clonal survival of UV-irradiated primary human melanocytes after UVB exposure. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Biological scaffolds mimicking basement membranes are valuable tools to study biological processes like cellular differentiation and phenotypic maintenance in vitro. Such artificial basement membranes also hold great promise for engineering tissues and organs and promoting regenerative processes. Basement membrane‐like sheets were fabricated by electrospinning of nanofibers with fiber size, geometry, and the porosity of authentic basement membranes. Nanofibers were electrospun from pure murine laminin 111, murine reconstituted basement membrane (containing laminin, type IV collagen and perlecan) and human cardiac muscle basement membrane enriched in laminins 411 and 511). These proteins and complex extracts were blended with polymers such as polycaprolactone to create hybrid nanofibers with mechanical properties determined by the degradable polymer and biological properties derived from laminin. Importantly, the protein and hybrid nanofibers, unlike others reported, required no chemical crosslinking, which often disrupts biological activity, to maintain structure. These biological scaffolds were compared to films of pure laminin 111 and the mouse and human basement membrane extracts for their ability to maintain the pluripotent state of induced pluripotent stem cells and embryonic stem cells in defined media. All cell lines examined attached more rapidly and to a greater extent on the nanofibrous basement membrane scaffolds, and antibody and enzymatic staining for a variety of markers of pluripotency showed they resisted the tendency to differentiate in vitro for weeks when maintained on these substrates. Next we examined the ability of the scaffolds to promote attachment and differentiation of mesenchymal stem and PC12 cells to a neuron‐like phenotype. The basement membrane like scaffolds promoted neuronal precursor attachment and differentiation to neuron‐like cells in serum free media without the addition of cAMP or nerve growth factor, suggesting a possible biomimetic effect on differentiation from the scaffold. Together these initial studies illustrate methods to create substrates that mimic native basement membranes in structure and function‐‐these scaffolds may take our in vitro models a bit closer to the in vivo situation.Support or Funding InformationResearch generously supported by: Virginia Biosciences Health Research Corporation Grant, Nanofabrication of Tissue ScaffoldsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

dystrophin-glycoprotein complex The sarcolemma (muscle plasma membrane) plays a central role in skeletal muscle structure and function (1Engel A.G. Franzini-Armstrong C. Myology. 2nd Ed. McGraw-Hill, New York1994Google Scholar). In addition to the housekeeping functions of a cell plasma membrane, the sarcolemma is directly involved in synaptic transmission, action potential propagation, and excitation-contraction coupling (1Engel A.G. Franzini-Armstrong C. Myology. 2nd Ed. McGraw-Hill, New York1994Google Scholar). Besides these well established physiological functions, the sarcolemma, subsarcolemma cytoskeleton, and surrounding basement membrane (extracellular matrix) play an essential structural role in skeletal muscle (1Engel A.G. Franzini-Armstrong C. Myology. 2nd Ed. McGraw-Hill, New York1994Google Scholar, 2Clark K.A. McElhinny A.S. Beckerle M.C. Gregorio C.C. Annu. Rev. Cell Biol. 2002; 18: 637-706Crossref Scopus (474) Google Scholar, 3Colognato H. Yurchenco P.D. Dev. Dyn. 2000; 218: 213-234Crossref PubMed Scopus (1052) Google Scholar). The biological importance of the basement membrane-sarcolemma-cytoskeleton in skeletal muscle is underscored by the number of inherited muscle diseases caused by mutations in components of the basement membrane, or cytoskeleton, or the sarcolemma protein complexes that link the basement membrane to the cytoskelton (4Cohn R.D. Campbell K.P. Muscle Nerve. 2000; 23: 1456-1471Crossref PubMed Scopus (426) Google Scholar, 5Blake D.F. Weir A. Newey S.E. Davies K.E. Physiol. Rev. 2002; 82: 291-329Crossref PubMed Scopus (925) Google Scholar, 6Spence H.J. Chen Y.J. Winder S.J. Bioessays. 2002; 24: 542-552Crossref PubMed Scopus (57) Google Scholar, 7Brown S.C. Muntoni F. Sewer C.A. Brain Pathol. 2002; 11: 193-205Crossref Scopus (17) Google Scholar, 8Durbeej M. Campbell K.P. Curr. Opin. Genet. Dev. 2002; 9: 2019-2027Google Scholar). The minireview in this and the following issues updates our understanding of the structure and function of the basement membrane, cytoskeletal costameres, and the major trans-sarcolemma links (integrins and dystroglycan) in skeletal muscle. The basement membrane surrounds skeletal muscle fibers and is now known to be critical in muscle fiber structure and function. The skeletal muscle basement membrane is composed of the basal lamina and the reticular lamina. Basal lamina is directly linked to the sarcolemma. Genetic studies of muscular dystrophy patients and animal models of muscular dystrophy have demonstrated the importance of the basement membrane in maintenance of muscle integrity. In addition to maintenance of muscle integrity, the basement membrane is essential in the promotion of myogenesis and muscle development. Muscle regeneration is also a process that depends on the skeletal muscle basement membrane. Satellite cells (endogenous stem cells of skeletal muscle) reside between the muscle fiber and the basal lamina. Following injury, new muscle fibers regenerate within a basement membrane tube, which is believed to act as a mechanical barrier to limit migration of satellite cells, and a scaffold to orient myotube regeneration. Finally, the basement membrane is structurally and functionally specialized in areas of neuromuscular and myotendinous junctions and is required for the assembly of these structures. In the first minireview of this series entitled "The Basement Membrane/Basal Lamina of Skeletal Muscle," Joshua R. Sanes reviews the structure and function of the skeletal muscle basement membrane. In particular, he focuses on recent molecular studies that have led to a better understanding of the function of the basement membrane in skeletal muscle physiology and pathophysiology (9Sanes J.R. J. Biol. Chem. 2003; 278: 12601-12604Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). A major cytoskeletal structure in muscle that has the unique role of connecting the sarcomere to the basement membrane is the costamere. Costameres were originally described as subsarcolemma protein complexes that align in register with the Z-disk and are physically coupled to the sarcomeres. Costameres may be equivalent to focal adhesions that are expressed in non-muscle cells and are believed to be involved in the lateral transmission of contractile forces from sarcomeres across the sarcolemma to the basement membrane. In the second minireview, entitled "Costameres: the Achilles Heel of Herculean Muscle," James M. Ervasti reviews the structure and function of the striated muscle costamere (10Ervasti J.M. J. Biol. Chem. 2003; 278: 13591-13594Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). As with the basement membrane, the importance of the costamere for normal muscle function has been revealed by genetic studies of muscular dystrophies and dilated cardiomyopathies. Dystrophin is known to be enriched in costameres, but dystrophin is not required for costamere assembly. In the absence of dystrophin, there is a disorganization of the costameric lattice, as well as disruption of the sarcolemma integrity. Disruption of the costameric lattice correlates with functional studies that show reduction of contractile force in muscles lacking dystrophin. Ervasti's minireview provides insights into the growing costameric protein network and illustrates how these proteins can interact with many components of both the sarcolemma and cytoskeleton. In addition, the newly identified proteins suggest a role for costameric proteins in converting mechanical stimuli to alterations in cell signaling and gene expression. Finally, Ervasti discusses non-sarcolemmal mechanical defects associated with the loss of costameric proteins. It is well recognized that the function and maintenance of skeletal muscle cell integrity is dependent upon interactions of the muscle cell with the surrounding basement membrane and underlying cytoskeleton. Trans-sarcolemma receptors are known to be involved in providing critical mechanical links between the basement membrane and the cytoskeleton. In addition, recent data suggest that these receptors transmit signals from the basement membrane into the muscle cell. Over the past 10 years there has been great progress in the identification and characterization of the two sarcolemma protein complexes that connect cytoskeleton to the basement membrane in skeletal muscle: integrins and the dystrophin-glycoprotein complex (DGC).1 Integrins form a large family of cell surface receptors that mediate cell-extracellular matrix interactions. In the third minireview, entitled "Integrins, Redundant or Important Players in Skeletal Muscle?," Ulrike Mayer focuses on the role of integrins in skeletal muscle (11Mayer U. J. Biol. Chem. 2003; 278: 14587-14590Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Of the current members of this family, a subset is expressed in skeletal muscle, in particular, at the sarcolemma, neuromuscular junction, myotendinous junction, and costameres. Expression of the integrin family is regulated during skeletal muscle development. Integrins play a major role in muscle differentiation, and α7β1 integrin is a critical receptor for myoblast migration. In adult skeletal muscle, integrins are concentrated at the neuromuscular junction and the myotendinous junction and provide an important link to the basement membrane in these specialized regions of muscle. α7β1 integrin is the major form found in adult skeletal muscle. Integrins have also been implicated in skeletal muscle diseases, and the absence of α7 integrin leads to a mild muscular dystrophy in both mice and humans. Myotendinous junctions were severely disturbed in mouse models with α7integrin deficiency. Both mouse and human studies suggest that muscle weakness arises from destruction of the myotendinous junction, rather than from compromised sarcolemma integrity. Finally, α7β1 integrin is reduced in several muscular dystrophies, and α7β1 integrin overexpression may provide a possible therapeutic approach for Duchenne muscular dystrophy. In the final minireview, entitled "Dystrophin-Glycoprotein Complex: Post-translational Processing and Dystroglycan Function," Daniel E. Michele and Kevin P. Campbell focus on the major sarcolemma membrane complex in adult skeletal muscle that links the cytoskeleton to the basement membrane (12Michele D.E. Campbell K.P. J. Biol. Chem. 2003; (January 29, 10.1074/jbc.R200031200)Google Scholar). DGC is a large oligomeric complex of proteins in the sarcolemma of skeletal muscle. The DGC is composed of both integral and peripheral membrane proteins and provides a structural connection between the basement membrane and the actin cytoskeleton and has been hypothesized to protect the sarcolemma from mechanical damage during muscle contraction. Several forms of muscular dystrophy arise from primary mutations in genes encoding components of the DGC. The DGC is grouped into three subcomplexes, dystroglycan (α- and β-dystroglycan), the sarcoglycan-sarcospan subcomplex, and the cytoskeletal components dystrophin, syntrophyin, and dystrobrevin. Michele and Campbell review the current status of our understanding of the DGC, and in particular focus on the structure and post-translational processing of dystroglycan. Interestingly, recent genetic data have demonstrated that proteins with homology similar to glycosyltransferases are linked to muscular dystrophy and appear to preferentially or exclusively modify α-dystroglycan. The role of glycosylation in the function of dystroglycan is discussed, and the mechanisms whereby the loss of functional dystroglycan leads to clinical symptoms, including muscular dystrophy, and abnormal central nervous system development and function are presented. Finally, new insights into dystroglycan function revealed from the studies of mouse models and patients with incomplete glycosylation (dystroglycanopathies) are reviewed. Much progress has been made in our understanding of the role of basement membrane-sarcolemma-cytoskeleton interactions in the development, structure, and function of striated muscle. The field continues to be a dynamic arena for future research, in which new functional molecules, their interactions, and their functional significance, continue to be identified from molecular genetics of human diseases, biochemistry, cell biology, and gene targeting in the mouse. Given the essential role of these molecules in human disease, understanding the interactions of the skeletal muscle basement membrane-sarcolemma-cytoskeleton will hopefully lead to a better understanding of disease pathogenesis and therapeutic opportunity.

How cell numbers are determined is not understood. Hedgehog-Gli activity is involved in precursor cell proliferation and stem cell self-renewal, and its deregulation sustains the growth of many human tumours. However, it is not known whether GLI1, the final mediator of Hh signals, controls stem cell numbers, and how its activity is restricted to curtail tumourigenesis. Here we have altered the levels of GLI1 and p53, the major tumour suppressor, in multiple systems. We show that GLI1 expression in Nestin+ neural progenitors increases precursor and clonogenic stem cell numbers in vivo and in vitro. In contrast, p53 inhibits GLI1-driven neural stem cell self-renewal, tumour growth and proliferation. Mechanistically, p53 inhibits the activity, nuclear localisation and levels of GLI1 and in turn, GLI1 represses p53, establishing an inhibitory loop. We also find that p53 regulates the phosphorylation of a novel N' truncated putative activator isoform of GLI1 in human cells. The balance of GLI1 and p53 functions, thus, determines cell numbers, and prevalence of p53 restricts GLI1-driven stem cell expansion and tumourigenesis.

Donor corneas are processed in eye banks and used for transplantation as a standard routine. The maximum time limit post-mortem for harvesting donor tissue varies greatly between eye banks. This study aimed to examine the corneal epithelium for structural changes post-mortem. A total of 51 corneas harvested between 14 and 163 hours post-mortem were examined using scanning electron and light microscopy. Cell loss occurred through desquamation of flat superficial cells during the first days. In corneas with a post-mortem time of more than 2-3 days, large superficial cell sheets and deeper cells detached, starting centrally. Deep peripheral cells remained. The loss of the superficial cells revealed the 3-dimensional structure of the epithelium and the membrane characteristics of deeper cells. The longer the time post-mortem, the greater the epithelial cell loss. However, a rim of peripheral cells remained, even after 7 days. The superficial cell layer showed signs of strong lateral attachment and broke up in a sheet-like fashion. The intercellular adhesion between deeper cells and adhesion between the basal cells and the basement membrane appeared to be weak post-mortem. The cell membrane structures of the remaining cells were surprisingly well retained. The clinical implication of the study is discussed.

Microassay for the assessment of low levels of hydroxyproline.

Neurogenesis involves generation of new neurons through finely tuned multistep processes, such as neural stem cell (NSC) proliferation, migration, differentiation, and integration into existing neuronal circuitry in the dentate gyrus of the hippocampus and subventricular zone. Adult hippocampal neurogenesis is involved in cognitive functions and altered in various neurodegenerative disorders, including Alzheimer disease (AD). Ethosuximide (ETH), an anticonvulsant drug is used for the treatment of epileptic seizures. However, the effects of ETH on adult hippocampal neurogenesis and the underlying cellular and molecular mechanism(s) are yet unexplored. Herein, we studied the effects of ETH on rat multipotent NSC proliferation and neuronal differentiation and adult hippocampal neurogenesis in an amyloid β (Aβ) toxin-induced rat model of AD-like phenotypes. ETH potently induced NSC proliferation and neuronal differentiation in the hippocampus-derived NSC in vitro. ETH enhanced NSC proliferation and neuronal differentiation and reduced Aβ toxin-mediated toxicity and neurodegeneration, leading to behavioral recovery in the rat AD model. ETH inhibited Aβ-mediated suppression of neurogenic and Akt/Wnt/β-catenin pathway gene expression in the hippocampus. ETH activated the PI3K·Akt and Wnt·β-catenin transduction pathways that are known to be involved in the regulation of neurogenesis. Inhibition of the PI3K·Akt and Wnt·β-catenin pathways effectively blocked the mitogenic and neurogenic effects of ETH. In silico molecular target prediction docking studies suggest that ETH interacts with Akt, Dkk-1, and GSK-3β. Our findings suggest that ETH stimulates NSC proliferation and differentiation in vitro and adult hippocampal neurogenesis via the PI3K·Akt and Wnt·β-catenin signaling.

The basement membrane of skeletal muscle fibers is believed to persist unchanged during myofiber degeneration and act as a tubular structure within which the regeneration of new myofibers occurs. In the present study we describe macromolecular changes in the basement membrane zone during muscle degeneration and regeneration, as monitored by immunofluorescence using specific antibodies against types IV and V collagen, laminin, and heparan sulfate proteoglycan and by the binding of concanavalin A (Con A). Skeletal muscle regeneration was induced by autotransplantation of the extensor digitorum longus muscle in rats. After this procedure, the myofibers degenerate; this is followed by myosatellite cell activation, proliferation, and fusion, resulting in the formation of new myotubes that mature into myofibers. In normal muscle, the distribution of types IV and V collagen, laminin, heparan sulfate proteoglycan, and Con A binding was seen in the pericellular basement membrane region. In autotransplanted muscle, the various components of the basement membrane zone disappeared, leaving behind some unidentifiable component that still bound Con A. Around the regenerated myotubes a new basement membrane (zone) reappeared, which persisted during maturation of the regenerating muscle. The distribution of various basement membrane components in the regenerated myofibers was similar to that seen in the normal muscle. Based on our present and previous study (Gulati, A.K., A.H. Reddi, and A.A. Zalewski, 1982, Anat. Rec. 204:175-183), it appears that some of the original basement membrane zone components disappear during myofiber degeneration and initial regeneration. As a new basement membrane develops, its components reappear and persist in the mature myofibers. We conclude that skeletal muscle fiber basement membrane (zone) is not a static structure as previously thought, but rather that its components change quite rapidly during myofiber degeneration and regeneration.

Transplant quality human kidneys were sliced (approximately equal to 2 mm thick) and cortical regions further minced to approximately 1 mm3. These samples were treated successively with EDTA and detergents to solubilize cellular materials but leave the extracellular matrix (ECM), including basement membranes (BMs) intact. Resultant acellular cortices bear a remarkable resemblance to their in vivo counterparts. By light microscopy, patent, expanded tubular BM (TBM), peritubular capillary BM (PTCBM) and Bowman's capsule BM (BCBM) are surrounded by a swollen ECM which is occasionally fibrillar. Glomerular BM (GBM) lacks supporting interstitium but nevertheless remains convoluted and does not collapse. At the level of transmission electron microscopy, the diverse morphological characteristics of each BM type are evident. Random thickness measurements (lamina densa only) show that BCBMs are thickest (approximately equal to 2,400 nm) followed by TBMs (approximately equal to 750 nm), GBMs (approximately equal to 335 nm) and PTCBMs (approximately equal to 125 nm). Power transformations to normalize right-sided skew of thickness distribution curves reduce their arithmetic means 3-16%. SEM studies confirm LM and TEM observations that isolated GBMs maintain their free-standing spheroidal shapes and indicate that they may be intrinsically rigid. Moreover, their surface topographical details are preserved. We conclude that the acellular cortex offers a clarified view of renal interstitial morphology and demonstrates structural diversity among major renal BM types. Moreover, it provides a baseline of morphological information on which to assess pathological conditions of renal cortical extracellular matrix.

In many recent studies, the inhibitory transmitter gamma-aminobutyric acid has been shown to modulate the proliferation, differentiation and survival of neural stem cells. Most general anesthetics are partial or allosteric gamma-aminobutyric acid A receptor agonists, suggesting that general anesthetics could alter the behavior of neural stem cells. The neuroprotective efficacy of general anesthetics has been recognized for decades, but their effects on the proliferation of neural stem cells have received little attention. This study investigated the potential effect of midazolam, an extensively used general anesthetic and allosteric gamma-aminobutyric acid A receptor agonist, on the proliferation of neural stem cells in vitro and preliminarily explored the underlying mechanism. The proliferation of neural stem cells was tested using both Cell Counting Kit 8 and bromodeoxyuridine incorporation experiments. Cell distribution analysis was performed to describe changes in the cell cycle distribution in response to midazolam. Calcium imaging was employed to explore the molecular signaling pathways activated by midazolam. Midazolam (30–90 μM) decreased the proliferation of neural stem cells in vitro. Pretreatment with the gamma-aminobutyric acid A receptor antagonist bicuculline or Na-K-2Cl cotransport inhibitor furosemide partially rescued this inhibition. In addition, midazolam triggered a calcium influx into neural stem cells. The suppressive effect of midazolam on the proliferation of neural stem cells can be partly attributed to the activation of gamma-aminobutyric acid A receptor. The calcium influx triggered by midazolam may be a trigger factor leading to further downstream events.

The extracellular matrix (ECM), a highly organized network of structural and nonstructural proteins, plays a pivotal role in cellular and tissue homeostasis. Changes in the ECM are critical for normal tissue repair, whereas dysregulation contributes to aberrant tissue remodeling. Pulmonary arterial hypertension is a severe disorder of the pulmonary vasculature characterized by pathologic remodeling of the pulmonary vasculature and right ventricle, increased production and deposition of structural and nonstructural proteins, and altered expression of ECM growth factors and proteases. Furthermore, ECM remodeling plays a significant role in disease progression, as several dynamic changes in its composition, quantity, and organization are documented in both humans and animal models of disease. These ECM changes impact vascular cell biology and affect proliferation of resident cells. Furthermore, ECM components determine the tissue architecture of the pulmonary and myocardial vasculature as well as the myocardium itself and provide mechanical stability crucial for tissue homeostasis. However, little is known about the basement membrane (BM), a specialized, self-assembled conglomerate of ECM proteins, during remodeling. In the vasculature, the BM is in close physical association with the vascular endothelium and smooth muscle cells. While in the myocardium, each cardiomyocyte is enclosed by a BM that serves as the interface between cardiomyocytes and the surrounding interstitial matrix. In this review, we provide a brief overview on the current state of knowledge of the BM and its ECM composition and their impact on pulmonary vascular remodeling and right ventricle dysfunction and failure in pulmonary arterial hypertension.

Microcephaly-associated mutations disrupt microtubule-dependent WDR62 translocation from the Golgi complex to the mitotic spindle poles, impair mitotic progression, and alter neurogenic trajectories in patient induced Pluripotent Stem Cell-derived 2D and 3D models of human neurodevelopment.

After injury of the central and peripheral nervous systems, functional recovery is impaired by axon regeneration failure. Various approaches for promoting axon growth have been attempted but their low efficacy has prevented them from being clinically applicable. It is possible that in spite of all the research that has been performed regarding axon growth, we are still missing key aspects of axon growth biology that are essential in order to design effective treatments for axon regeneration. Most of what we know about how an axon grows has been discovered by using tools that delete, add, block, or activate macromolecules in the whole cell and by looking at its corresponding effect on axon growth. Less is known about the spatiotemporal actions of macromolecules and organelles at the growth cone and its relation with axon growth. Macromolecules and organelles are heterogeneously distributed in the cell and their resulting function depends heavily on when and where they exert their actions. We need tools that allow us to manipulate these macromolecules and organelles in a temporal window during which axon growth events are taking place. The development of such tools will provide enhanced knowledge of how an axon grows and lead to design more effective therapies to promote axon growth.

Eculizumab and Complement Activation in Anti−glomerular Basement Membrane Disease