Close interaction between oligodendrocytes and axons is essential to initiate myelination and to form specialised domains along myelinated fibres. These domains are characterised by the assembly of protein complexes at the axon-glia interface and key components of these complexes are the Neurofascins. Neurofascins are transmembrane glycoproteins belonging to the L1 subgroup of the Immunoglobulin (Ig) superfamily of cell adhesion molecules. The Neurofascin (Nfasc) gene is subject to extensive alternative splicing. Two of the best characterised isoforms are Nfasc155 and Nfasc186, which are expressed in glia and neurons respectively. In myelinated fibres, Nfasc186 is the predominant isoform expressed at nodes of Ranvier and axon initial segments (AIS) in both the central and peripheral nervous system (CNS and PNS), whereas Nfasc155 resides on the glial side of the paranodal axoglial junction. The Neurofascin gene has been inactivated by homologous recombination and Neurofascin-null mice die within the first week of postnatal life. The main focus of this work was to investigate the role of the Neurofascins in the developing CNS. Similarly to what has been previously observed in the PNS, this study shows that in myelinated fibres of the spinal cord, nodal and paranodal markers are mislocalised and axoglial junctions do not form in the absence of the Neurofascins. In contrast to the PNS, where ensheathment of axons is unaffected, myelin proteins in the CNS are greatly reduced in the mutant. This appears to be due to the reduced ability of oligodendrocyte myelinating processes to extend along axons. This work also shows that the role of Nfasc186 is to maintain the long term stability of the AIS rather than its assembly. In the PNS, Nfasc186 was found to play an essential role in node assembly. However, PNS and CNS nodes are likely to assemble by different mechanisms. To investigate the relative contribution of the Neurofascin isoforms in CNS node assembly, this work made use of transgenic lines in which either neuronal Nfasc186 or glial Nfasc155 was expressed on a Neurofascin null background. Expression of either isoform was found to independently rescue the nodal complex and a model of how the Neurofascins cooperate in the assembly of the CNS node of Ranvier is proposed.

A method for modeling and simulating neural action potential (AP) propagation along the length of an axon containing a number of Ranvier nodes is proposed in this dissertation. A system identification approach is adopted to represent node of Ranvier (NR) response to current pulse stimulus in the form of transfer function representations for NR excitability. Segments of myelinated internodal (IN) and NR regions are cascaded, representing the remaining downstream axon after a site-of-stimulus introduction of an external current pulse. This cascading network is used to simulate "cable" properties and signal propagation along the length of the axon. This work proposes possible solutions to attenuation losses inherited in the classical myelinated cable models and accounts for neuronal AP velocity as well as introducing signal attenuation and transient delays associated with internodal demyelination. This model could aide as a predictive tool for the diagnosis and analysis of axonal signal integrity associated with demyelination pathology. Possible applications could include functional stimulation control methodologies for axon bundles that may exhibit signal fidelity issues associated with demyelination. It is further proposed that this model may serve as an instructive tool for further development and incorporation of other axon dynamic behaviors such as: relative refractory periods of AP generation, NR AP recovery mechanisms and responses to varied current stimulus input. / by George Jason Morales. / Thesis (Ph.D.)--Florida Atlantic University, 2011. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2011. Mode of access: World Wide Web.

Cerebral hypoperfusion can occur in normal ageing and is proposed to underlie white matter disturbances observed in the ageing brain. Moreover, cerebral hypoperfusion and white matter attenuation are early events in the progression of Alzheimer’s disease (AD). White matter mostly consists of myelinated axons which have distinct protein architecture, segregated into defined regions; the axon initial segment (AIS), the node of Ranvier, paranode, juxtaparanode, and internode. These sites are essential for action potential initiation and/or propagation and subsequently effective brain function. At the outset of the studies in the thesis there was evidence that the different regions within the myelinated axons are vulnerable to injury and disease. Thus it is hypothesised that in response to normal ageing and/or cerebral hypoperfusion these structures are altered and associated with cognitive impairment and that these effects are exacerbated in a transgenic mouse model (APPSw,Ind, J9 line) which develops age-dependent amyloid-β (Αβ) pathology. The first study aims to investigate the effect of normal ageing and Aβ deposition on myelinated axons and on learning and memory. To address this, the effects of normal ageing on the integrity of the AIS, nodes of Ranvier, myelin, axons, synapses and spatial working memory are examined in young and aged wild-type and TgAPPSw,Ind mice. A significant reduction in the length of nodes of Ranvier is demonstrated in aged wild-type and TgAPPSw,Ind mice. In addition, the length of AIS, is significantly reduced in the aged wild-type animals while the young TgAPPSw,Ind have significantly shorter AIS than the young wild-type mice. These effects are not influenced by the presence of Aβ. Myelin integrity is affected by age but this is more prominent in the wild-type animals whilst axonal integrity is intact. Moreover, there is an age-related decrease of presynaptic boutons only in the TgAPPSw,Ind mice. Contrary to the original hypothesis, working memory performance is not altered with age or influenced by increasing Aβ levels. The second study aims to examine the effects of cerebral hypoperfusion in combination with Αβ pathology and/or ageing on cognitive performance and the structure of myelinated axons. To address this, the effects of surgically induced cerebral hypoperfusion on the integrity of the nodes of Ranvier, paranodes, myelin, axons and spatial working memory performance are investigated in young and aged wild-type and TgAPPSw,Ind mice. A decrease in nodal length is observed in response to hypoperfusion in young and aged animals. This effect is shown to be exacerbated in the young TgAPPSw,Ind animals. Moreover, the disruption of the nodal domain is shown to occur without any gross alterations in myelin and axonal integrity. It is also demonstrated that in response to hypoperfusion, spatial working memory performance is defected in young and aged animals of both genotypes. This deficit is exacerbated in the young TgAPPSw,Ind. The observed changes in the nodal structure are associated with poor working memory performance indicating functional implication for the nodal changes. These data highlight that structures within myelinated axons are vulnerable to ageing and cerebral hypoperfusion. Therefore, the development of strategies that minimize injury or drive repair to these regions is necessary together with therapeutic approaches against the vascular insults that induce hypoperfusion and lead to white matter attenuation and cognitive decline. In the future, it would be interesting to investigate how alterations at the AIS/nodes of Ranvier affect neuronal excitability.

L'agrégation des canaux sodium (Nav) aux nœuds de Ranvier est une étape importante pour la propagation électrique saltatoire rapide le long des axones myélinisés. L'assemblage des nœuds dépend d'interactions neurones-cellules gliales myélinisantes, les oligodendrocytes dans le système nerveux central (SNC) et les cellules de Schwann dans le système nerveux périphérique (SNP). Bien décrits dans le SNP, les mécanismes cellulaires et moléculaires restent à caractériser dans le SNC. Lors de ma thèse, je me suis focalisé sur les étapes précoces d'assemblage des nœuds dans le SNC. Ce travail montre que des agrégats de protéines nodales (ou pré-nœuds) sont formés le long des axones de neurones GABAergiques avant la myélinisation dans des cultures neurones-glies d'hippocampe et également au cours du développement chez les rongeurs. La formation de pré-nœuds dépend de protéines sécrétées par les oligodendrocytes et de la protéine axonale d'échafaudage, ankyrineG. En outre, la transition des isoformes de Nav le long des axones est régulée par la présence des cellules gliales. Enfin, les pré-nœuds permettent d'accélérer la vitesse de conduction de l'influx nerveux par un facteur 1,5, indépendamment de la myélinisation et du calibre axonal. Globalement, ces résultats renforcent notre connaissance des mécanismes d'assemblage des nœuds de Ranvier dans le SNC et suggèrent une fonction développementale de l'agrégation nodale avant le début de la myélinisation. Si la vitesse de conduction a été décrite comme liée aux propriétés isolantes de la gaine de myéline, les résultats de cette thèse apportent un concept novateur de régulation de la conduction axonale en l'absence de myéline. / The clustering of sodium channels (Nav) at the nodes of Ranvier is an important step in permitting rapid saltatory conduction along myelinated axons. Nodal assembly is neuron-glia dependent, mediated by myelinating oligodendrocytes of the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). While the mechanisms of nodal assembly are currently best characterized in the PNS, cellular and molecular mechanisms underlying their assembly in the CNS are only partially understood. In the core of my PhD dissertation, I focused on the early developmental steps of nodal protein clustering in the CNS and show that clusters of nodal proteins, called prenodes, are detected before myelination along GABAergic axons in hippocampal neuron-glia cultures and also in the developing rodent hippocampus. Prenodal clustering requires extrinsic oligodendroglial secreted proteinaceous factors, and also the intrinsic axonal cytoskeletal scaffolding protein ankyrinG. Furthermore, the transition of sodium channels isoforms is tightly regulated along GABAergic axons during development, but this transition is lost in the absence of the physical presence of glial cells. Lastly, prenodes increase axonal conduction by a factor of 1.5x, independently of myelination and axonal caliber. Taken together, these results further our understanding of CNS nodes of Ranvier assembly mechanisms and the developmental function of nodal clustering prior to myelin ensheathment. While conduction velocity along axons has long been thought to mostly rely on the insulating properties of myelin, these results may shed light on a new concept of axonal conduction in the absence of myelination.

Bilayer embedded proteins are affected by stress. This general affirmation is, in this thesis, embodied by two types of proteins: membrane spandex and voltage-gated sodium channels. In this work, we essentially explore, using methods from physics, the theoretical consequences of ideas drawn from experimental biology. Membrane spandex was postulated to exist and we study the theoretical implications and possible benefits for a cell to have such proteins embedded in its bilayer. There are no specific membrane spandex proteins, rather any protein with a transition involving a large enough area change between two non-conducting states could act as spandex. Bacterial cells have osmovalve channels which open at near-lytic tensions to protect themselves against rupture. Spandex expanding at tensions just below the osmovalves’ opening tension could relieve tension enough as to avoid costly accidental osmovalve opening due to transient bilayer tension excursions. Another possible role for spandex is a tension-damper: spandex could be used to maintain bilayer tension at a fixed level. This would be useful as many bilayer embedded channels are known to be modulated by tension. The Stress/shear experienced in traumatic brain injury cause an immediate (< 2 min) and irreversible TTX-sensitive rise in axonal calcium. In situ, this underlies an untreatable condition, diffuse axonal injury. TTX sensitivity indicates that leaky voltage-gated sodium (Nav) channels mediate the calcium increase. Wang et al. showed that the mammalian adult CNS Nav isoform, Nav1.6, expressed in Xenopus oocytes becomes “leaky” when subjected to bleb-inducing pipette aspiration. This “leaky” condition is caused by a hyperpolarized-shift (left-shift or towards lower potentials, typically 20 mV) of the kinetically coupled processes of activation and inactivation thus effectively degrading a well-confined window conductance into a TTX-sensitive Na leak. We propose experimental protocols to determine whether this left-shift is the result of an all-or-none or graded process and whether persistent Na currents are also left-shifted by trauma. We also use modeling to assess whether left-shifted Nav channel kinetics could lead to Na+ (and hence Ca2+ ) loading of axons and to study saltatory propagation after traumatizing a single node of Ranvier.

Bilayer embedded proteins are affected by stress. This general affirmation is, in this thesis, embodied by two types of proteins: membrane spandex and voltage-gated sodium channels. In this work, we essentially explore, using methods from physics, the theoretical consequences of ideas drawn from experimental biology. Membrane spandex was postulated to exist and we study the theoretical implications and possible benefits for a cell to have such proteins embedded in its bilayer. There are no specific membrane spandex proteins, rather any protein with a transition involving a large enough area change between two non-conducting states could act as spandex. Bacterial cells have osmovalve channels which open at near-lytic tensions to protect themselves against rupture. Spandex expanding at tensions just below the osmovalves’ opening tension could relieve tension enough as to avoid costly accidental osmovalve opening due to transient bilayer tension excursions. Another possible role for spandex is a tension-damper: spandex could be used to maintain bilayer tension at a fixed level. This would be useful as many bilayer embedded channels are known to be modulated by tension. The Stress/shear experienced in traumatic brain injury cause an immediate (< 2 min) and irreversible TTX-sensitive rise in axonal calcium. In situ, this underlies an untreatable condition, diffuse axonal injury. TTX sensitivity indicates that leaky voltage-gated sodium (Nav) channels mediate the calcium increase. Wang et al. showed that the mammalian adult CNS Nav isoform, Nav1.6, expressed in Xenopus oocytes becomes “leaky” when subjected to bleb-inducing pipette aspiration. This “leaky” condition is caused by a hyperpolarized-shift (left-shift or towards lower potentials, typically 20 mV) of the kinetically coupled processes of activation and inactivation thus effectively degrading a well-confined window conductance into a TTX-sensitive Na leak. We propose experimental protocols to determine whether this left-shift is the result of an all-or-none or graded process and whether persistent Na currents are also left-shifted by trauma. We also use modeling to assess whether left-shifted Nav channel kinetics could lead to Na+ (and hence Ca2+ ) loading of axons and to study saltatory propagation after traumatizing a single node of Ranvier.

Bilayer embedded proteins are affected by stress. This general affirmation is, in this thesis, embodied by two types of proteins: membrane spandex and voltage-gated sodium channels. In this work, we essentially explore, using methods from physics, the theoretical consequences of ideas drawn from experimental biology. Membrane spandex was postulated to exist and we study the theoretical implications and possible benefits for a cell to have such proteins embedded in its bilayer. There are no specific membrane spandex proteins, rather any protein with a transition involving a large enough area change between two non-conducting states could act as spandex. Bacterial cells have osmovalve channels which open at near-lytic tensions to protect themselves against rupture. Spandex expanding at tensions just below the osmovalves’ opening tension could relieve tension enough as to avoid costly accidental osmovalve opening due to transient bilayer tension excursions. Another possible role for spandex is a tension-damper: spandex could be used to maintain bilayer tension at a fixed level. This would be useful as many bilayer embedded channels are known to be modulated by tension. The Stress/shear experienced in traumatic brain injury cause an immediate (< 2 min) and irreversible TTX-sensitive rise in axonal calcium. In situ, this underlies an untreatable condition, diffuse axonal injury. TTX sensitivity indicates that leaky voltage-gated sodium (Nav) channels mediate the calcium increase. Wang et al. showed that the mammalian adult CNS Nav isoform, Nav1.6, expressed in Xenopus oocytes becomes “leaky” when subjected to bleb-inducing pipette aspiration. This “leaky” condition is caused by a hyperpolarized-shift (left-shift or towards lower potentials, typically 20 mV) of the kinetically coupled processes of activation and inactivation thus effectively degrading a well-confined window conductance into a TTX-sensitive Na leak. We propose experimental protocols to determine whether this left-shift is the result of an all-or-none or graded process and whether persistent Na currents are also left-shifted by trauma. We also use modeling to assess whether left-shifted Nav channel kinetics could lead to Na+ (and hence Ca2+ ) loading of axons and to study saltatory propagation after traumatizing a single node of Ranvier.

Bilayer embedded proteins are affected by stress. This general affirmation is, in this thesis, embodied by two types of proteins: membrane spandex and voltage-gated sodium channels. In this work, we essentially explore, using methods from physics, the theoretical consequences of ideas drawn from experimental biology. Membrane spandex was postulated to exist and we study the theoretical implications and possible benefits for a cell to have such proteins embedded in its bilayer. There are no specific membrane spandex proteins, rather any protein with a transition involving a large enough area change between two non-conducting states could act as spandex. Bacterial cells have osmovalve channels which open at near-lytic tensions to protect themselves against rupture. Spandex expanding at tensions just below the osmovalves’ opening tension could relieve tension enough as to avoid costly accidental osmovalve opening due to transient bilayer tension excursions. Another possible role for spandex is a tension-damper: spandex could be used to maintain bilayer tension at a fixed level. This would be useful as many bilayer embedded channels are known to be modulated by tension. The Stress/shear experienced in traumatic brain injury cause an immediate (< 2 min) and irreversible TTX-sensitive rise in axonal calcium. In situ, this underlies an untreatable condition, diffuse axonal injury. TTX sensitivity indicates that leaky voltage-gated sodium (Nav) channels mediate the calcium increase. Wang et al. showed that the mammalian adult CNS Nav isoform, Nav1.6, expressed in Xenopus oocytes becomes “leaky” when subjected to bleb-inducing pipette aspiration. This “leaky” condition is caused by a hyperpolarized-shift (left-shift or towards lower potentials, typically 20 mV) of the kinetically coupled processes of activation and inactivation thus effectively degrading a well-confined window conductance into a TTX-sensitive Na leak. We propose experimental protocols to determine whether this left-shift is the result of an all-or-none or graded process and whether persistent Na currents are also left-shifted by trauma. We also use modeling to assess whether left-shifted Nav channel kinetics could lead to Na+ (and hence Ca2+ ) loading of axons and to study saltatory propagation after traumatizing a single node of Ranvier.