Lack of replication of KIF1B gene in an Italian primary progressive multiple sclerosis cohort

Background: KIF1B gene represents the first non‐inflammatory gene with a putative role on axonal loss and neurodegeneration found to be associated with multiple sclerosis (MS). The objective of this study is to test the association of the rs10492972 C allelic variant of KIF1B gene in a large Italian cohort of patients with primary progressive and progressive relapsing MS (PPMS and PRMS), which represents a subtype of MS mainly driven by neurodegenerative phenomena. Methods: rs10492972 has been genotyped in an outbred sample of 222 primary PPMS and PRMS and 221 healthy controls of unique northern Italian origin using the TaqMan assay. Results: A non‐significant age‐ and sex‐adjusted odds ratio of 0.96 [95% confidence interval (CI) 0.71–1.31] has been found in C carriers, and a non‐significant risk of 0.99 [95% CI 0.77–1.63] in C carriers according to a dominant model. Stratification by sex, age at onset younger than 35 years and symptoms at the onset of the disease did not reveal any significant findings. No influence on disability progression, measured with the multiple sclerosis severity score, was found in C carriers. Conclusions: These results suggest that there is no effect in carrying the rs10492972 C variant on the risk of disease as well as on the rate of disability progression in a cohort of Italian patients with PPMS and patients with PRMS. These data need to be confirmed in an independent sample of patients with progressive MS.     

The role of Kif5B in axonal localization of Kv1 K(+) channels

Here we present evidence that the kinesin, Kif5B, is involved in the transportation and axonal targeting of Kv1 channels. We show that a dominant negative variant of Kif5B specifically blocks localization to the axon of expressed, tagged versions of Kv1.3 in cultured cortical slices. In addition, the dominant negative variant of Kif5B blocks axonal localization of endogenous Kv1.1, Kv1.2, and Kv1.4 in cortical neurons in dissociated cultures. We also found evidence that Kif5B interacts with Kv1 channels. Endogenous Kv1.2 colocalized with Kif5B in cortical neurons and coimmunoprecipitated with Kif5B from brain lysate. The T1 domain of Shaker K(+) channels has been shown to play a critical role in targeting the channel to the axon. We have three pieces of evidence to suggest that the T1 domain also mediates interaction between Kv1 channels and Kif5B: Addition of the T1 domain to a heterologous protein, TfR, is sufficient to cause the resulting fusion protein, TfRT1, to colocalize with Kif5B. Also, the T1 domain is necessary for interaction of Kv1.3 with Kif5B in a coimmunoprecipitation assay. Finally, dominant negative variants of Kif5B block axonal targeting of TfRT1, but have no effect on dendritic localization of TfR. Together these data suggest a model where Kif5B interacts with Kv1 channels either directly or indirectly via the T1 domain, causing the channels to be transported to axons.     

Axonal branch shapes

To distinguish the relative roles of the intrinsic and the extrinsic determinants of axonal branching shapes, a number of key branch parameters were measured under a variety of conditions. Branch shapes of frog and of chick axons were analyzed in tissue culture, and these in vitro patterns were compared with in vivo branch patterns of axons in tadpole tail fins. The shape of a branch junction can be characterized by the sizes of its branch angles. In all cases, branch junctions had only two branches (3 branch angles), and the bifurcation angle between them was usually the smallest. The shape of the branch junction was xonstant in a wide variety of environments, but the exact branch angles, as well as the numbers of branches per axon and the numbers of axons per neuron, could be modulated by changes in the substrate adhesivity.     

Development of the crossed retinocollicular projection in the mouse

Changes in the distribution of axons of the crossed retinal projection within the superior colliculus of the developing mouse were studied by means of normal fiber and Golgi impregnations and by anterograde horseradish peroxidase labelling. Retinal axons advance along the optic tract from gestational days E12 to E14 and first invade the superior colliculus on E15. Over the subsequent days until birth (E19), the retinal axons extend within rostrocaudally oriented fascicles that distribute through the full thickness of the uppermost collicular layer, the stratum superficiale (SS). A dramatic transformation of this fiber stratification pattern into the mature pattern occurs over the first postnatal week. The fiber bundles are progressively cleared from the upper half of SS, identified as the future stratum griseum superficiale (SGS). Concurrently, the fiber bundles in the deep SS, identified as the stratum opticum (SO), give rise to individual, nonfasciculated fibers, which arborize within SGS. The contralateral retinal origin of the transient population of axons in SGS as well as the majority of axons that persist in SO is evident from the observation that they degenerate following neonatal enucleation. The number of fiber bundles lost is estimated to be 40-50% of the total population present in the superficial layers at birth. The combined set of observations indicates that axon elimination plays a major role in shaping the laminar pattern of retinal innervation of the colliculus. Retinal ganglion cell death, and not axon pruning, is proposed as the most probable mechanism by which axon fascicles are eliminated from SGS.     

Nerve regeneration through holey silicone tubes

Recent studies focus on regeneration where nerve stumps are placed in a silicone tube. Since the tube is impermeable, the fluid and cells that collect from the stumps bath the axons. This is presumably beneficial. Making the tube permeable by making holes in its walls should change the patterns of regeneration. If this is done, the major cytologic change is an increase in the fascicular perineurium. There are more individual fascicles, more cells line each fascicle and the lining cells are coated by more prominent external laminae than after similar regeneration in a regular silicone tube or in the normal untransected nerve. For axonal numbers, there are more myelinated and unmyelinated axons in the gap and more unmyelinated axons in the distal stump than after regeneration in a regular silicone tube. The numbers in the holey tube regenerate are statistically different from normal but they are closer to normal than after similar regeneration in a regular silicone tube. There are significantly fewer myelinated and unmyelinated axons than in the normal sural nerve after regeneration through a holey tube, but there are more than after regeneration through a regular tube. The numbers of axons in the nerve to the medial gastrocnemius muscle are not significantly different from normal or from the other regeneration paradigms. These data allow the suggestion that regeneration through a silicone tube with macroscopic holes in its walls may be superior in certain respects to regeneration through a regular impermeable silicone tube.     

Sorting and convergence of primary olfactory axons are independent of the olfactory bulb

Primary olfactory axons expressing the same odorant receptor gene sort out and converge to fixed sites in the olfactory bulb. We examined the guidance of axons expressing the P2 odorant receptor when they were challenged with different cellular environments in vivo. In the mutant extratoes mouse, the olfactory bulb is lacking and is replaced by a fibrocellular mass. In these animals, primary olfactory axons form glomerular-like loci despite the absence of normal postsynaptic targets. P2 axons are able to sort out from other axons in this fibrocellular mass and converge to form loci of like axons. The sites of these loci along mediolateral and ventrodorsal axes were highly variable. Similar convergence was observed for larger subpopulations of axons expressing the same cell surface carbohydrates. The sorting out and convergence of like axons also occurred during regeneration following bulbectomy. Olfactory axon behaviour in these models demonstrates that sorting and convergence of axons are independent of the target, which instead provides distinct topographic cues for guidance.     

A re-evaluation of the question of ascending fibers in the pyramidal tract

In 1952 we published a study in the cat with the Glees method, demonstrating the occurrence of degenerating fibers in the pyramidal tract rostral to transections of the tract in the spinal cord. These fibers were interpreted as spinocortical fibers, which have also been described in man. However, other authors have disputed the existence of such fibers. In an attempt to provide more information about this subject, multiple injections of horseradish peroxidase (free and lectin-labeled) were made in the sensorimotor cortex of 4 cats. No retrogradely labeled cells were found in the spinal cord in these cases. Our present and previously reported findings are discussed in the light of other studies of pathological changes in fiber tracts within the central nervous system. Although the present experiments were negative, the degenerating axons previously observed by us in silver sections from the pyramid, pons and internal capsule after lesions of the pyramidal tract in the spinal cord, can not be satisfactorily explained as evidence of retrograde, indirect Wallerian, degeneration of corticospinal fibers.     

Isolated satellite cells of a peripheral nerve direct the growth of regenerating frog axons

Frog motor axons regenerate and grow back to reinnervate their targets, the original motor end plates, after a lesion. When the cutaneous pectoris muscle is cut away and a segment of peripheral nerve is placed in the vicinity of regenerating axons they turn and grow toward it. This is in marked contrast to the random pattern of axonal outgrowth seen in the absence of a target. The influence on the direction of axonal growth of motor neurons can be produced by a 1-mm segment of nerve satellite cells over a distance of more than 8 mm. The nerve satellite cells have no influence on the direction of growth of the regenerating axons after all the cells in the nerve segment have been killed, leaving only the Schwann cell basal lamina tubes intact. These results show that the cells in the segment of the nerve trunk contain cues that actively direct the growth of motor neurons. Two possible explanations for this effect might be that the cells act indirectly by influencing the organization of the substructure over which axons regenerate or that the nerve satellite cells release a diffusible substance that acts directly on the regenerating axons.     

Role of EphA/ephrin‐A signaling in the development of topographic maps in mouse corticothalamic projections

Corticothalamic (CT) feedback outnumbers thalamocortical projections and regulates sensory information processing at the level of the thalamus. It is well established that EphA7, a member of EphA receptor family, is involved in the topographic mapping of CT projections. The aim of the present study was to dissect the precise impact of EphA7 on each step of CT growth. We used in utero electroporation‐mediated EphA7 overexpression in developing somatosensory CT axons to dissect EphA7/ephrin‐A‐dependent mechanisms involved in regulating both initial targeting and postnatal growth of the CT projections. Our data revealed that topographic maps of cortical afferents in the ventrobasal complex and medial part of the posterior complex in the thalamus become discernible shortly after birth and are fully established by the second postnatal week. This process starts with the direct ingrowth of the CT axons to the designated areas within target thalamic nuclei and by progressive increase of axonal processes in the terminal zones. Large‐scale overproduction and elimination of exuberant widespread axonal branches outside the target zone was not observed. Each developmental event was coordinated by spatially and temporally different responsiveness of CT axons to the ephrin‐A gradient in thalamic nuclei, as well as by the matching levels of EphA7 in CT axons and ephrin‐As in thalamic nuclei. These results support the concept that the topographic connections between the maps in the cerebral cortex and corresponding thalamic nuclei are genetically prespecified to a large extent, and established by precise spatiotemporal molecular mechanisms that involve the Eph family of genes. J. Comp. Neurol. 521:626–637, 2013.     

Axon reflex flare and quantitative sudomotor axon reflex contribute in the diagnosis of small fiber neuropathy

Introduction: An international clinical trial enrolled 174 ambulatory males ≥5 years old with nonsense mutation Duchenne muscular dystrophy (nmDMD). Pretreatment data provide insight into reliability, concurrent validity, and minimal clinically important differences (MCIDs) of the 6‐minute walk test (6MWT) and other endpoints. Methods: Screening and baseline evaluations included the 6‐minute walk distance (6MWD), timed function tests (TFTs), quantitative strength by myometry, the PedsQL, heart rate–determined energy expenditure index, and other exploratory endpoints. Results: The 6MWT proved feasible and reliable in a multicenter context. Concurrent validity with other endpoints was excellent. The MCID for 6MWD was 28.5 and 31.7 meters based on 2 statistical distribution methods. Conclusions: The ratio of MCID to baseline mean is lower for 6MWD than for other endpoints. The 6MWD is an optimal primary endpoint for Duchenne muscular dystrophy (DMD) clinical trials that are focused therapeutically on preservation of ambulation and slowing of disease progression. Muscle Nerve 48 : 357–368, 2013     

Differential expression of molecular motors in the motor cortex of sporadic ALS

The molecular mechanisms underlying the selective neurodegeneration of motor neurons in amyotrophic lateral sclerosis (ALS) are inadequately understood. Recent breakthroughs have implicated impaired axonal transport, mediated by molecular motors, as a key element for disease onset and progression. The current work identifies the expression of 15 kinesin-like motors in healthy human motor cortex, including three novel isoforms. Our comprehensive quantitative mRNA analysis in control and sporadic ALS (SALS) motor cortex specimens detects SALS-specific down-regulation of KIF1Bbeta and novel KIF3Abeta, two isoforms we show to be enriched in the brain, and also of SOD1, a key enzyme linked to familial ALS. This is accompanied by a marked reduction of KIF3Abeta protein levels. In the motor cortex KIF3Abeta localizes in cholinergic neurons, including upper motor neurons. No mutations causing splicing defects or altering protein-coding sequences were identified in the genes of the three proteins. The present study implicates two motor proteins as possible candidates in SALS pathology.     

Remodeling of the proximal segment of crayfish motor nerves following transection

Transected crustacean motor axons consist of a soma-endowed proximal segment that regenerates and a soma-less distal segment that survives for up to a year. We report on the anatomical remodeling of the proximal segment of phasic motor nerves innervating the deep flexor muscles in the abdomen of adult crayfish following transection. The intact nerve with 10 phasic axons and its two branches with subsets of 6 and 7 of these 10 axons undergo several remodeling changes. First, the transected nerve displays many more and smaller axon profiles than the 6 and 7 axons of the intact nerve, approximately 100 and 300 profiles in the two branches of a preparation transected 8 weeks previously. Serial images of the transected nerve denote that the proliferation of profiles is due to several orders of axon sprouting primary, secondary, and tertiary branches. The greater proliferation of axon sprouts, their smaller size, and the absence of intervening glia in the one nerve branch compared with the other branch denote that sprouting is more advanced in this branch. Second, the axon sprouts are regionally differentiated; thus, although in most regions the sprouts are basically axon-like, with a cytoskeleton of microtubules and peripheral mitochondria, in some regions they appear nerve terminal-like and are characterized by numerous clear synaptic vesicles, a few dense-core vesicles, and dispersed mitochondria. Both regions possess active zone dense bars with clustered synaptic vesicles found opposite other sprouts, glia, hemocytes, and connective tissue, but because the opposing membranes are not differentiated into a synaptic contact, the active zones are extrasynaptic. Third, some of the transected axons display a glial cell nucleus denoting assimilation of an adaxonal glial cell by the transected axons. Fourth, within the nerve trunk are a few myocytes and muscle fibers. These most likely originate from adjoining and intimately connected hemocytes, because such transformation occurs during muscle repair. In a crustacean nerve, however, where muscle is clearly misplaced, its presence implies an instructive role for motor nerves in muscle formation.     

Noradrenergic systems in human cerebellum

Thin, beaded axons, immunostained with antisera to human β-hydroxylase (DBH), were present in all layers of the anterior vermis of human cerebellum. This plexus appears similar to that described in rodents and provides information complementary to receptor autoradiographic studies that show significant noradrenergic innervation of mammalian cerebellum. Moreover, in two aged controls, we demonstrated abnormal, swollen, tortuous axons not visualized in young controls.     

Myelinated axon physiology and regulation of neural circuit function

The study of structural and functional plasticity in the central nervous system (CNS) to date has focused primarily on that of neurons and synapses. However, more recent studies implicate glial cells as key regulators of neural circuit function. Among these, the myelinating glia of the CNS, oligodendrocytes, have been shown to be responsive to extrinsic signals including neuronal activity, and in turn, tune neurophysiological function. Due to the fact that myelin fundamentally alters the conduction properties of axons, much attention has focused on how dynamic regulation of myelination might represent a form of functional plasticity. Here, we highlight recent research that indicates that it is not only myelin, but essentially all the function-regulating components of the myelinated axon that are responsive to neuronal activity. For example, the axon initial segment, nodes of Ranvier, heminodes, axonal termini, and the morphology of the axon itself all exhibit the potential to respond to neuronal activity, and in so doing might underpin specific functional outputs. We also highlight emerging evidence that the myelin sheath itself has a rich physiology capable of influencing axonal physiology. We suggest that to fully understand nervous system plasticity we need to consider the fact that myelinated axon is an integrated functional unit and adaptations that influence the entire functional unit are likely to underpin modifications to neural circuit function.     

Axons of callosal neurons bifurcate transiently at the white matter before consolidating an interhemispheric projection

The main alternative output routes of adult cortical axons are the internal capsule and the corpus callosum. How do callosal axons choose their trajectories? We hypothesized that bifurcation followed by elimination of one branch is a developmental strategy for accomplishing this aim. Using embryonic and postnatal mice, we labelled cortical projecting neurons and quantified their axonal bifurcations in correlation with the mediolateral position of their somata. Bifurcating axons were numerous in the younger brains but declined during further development. Most bifurcating axons pertained to neurons located in the dorsolateral cortex. Moreover, callosal neurons bifurcate more often than subcortically projecting cells. We then quantified bifurcations formed by dissociated green fluorescent cells plated onto cortical slices. Cells grown over dorsolateral cortex bifurcated more often than those grown over medial cortex, irrespective of their positional origin in the donor. Removal of intermediate targets from the slices prevented bifurcation. We concluded that transient bifurcation and elimination of the lateral branch is a strategy employed by developing callosal axons in search of their targets. As cell body position and intermediate targets determine axon behaviour, we suggest that bifurcations are regulated by cues expressed in the environment.     

Effect of temperature on long-term survival of anucleate giant axons in crayfish and goldfish

The effect of temperature on the electrophysiology and morphology of anucleate axons was examined following severance of crayfish medial giant axons and goldfish Mauthner axons from their respective cell bodies. Although anucleate segments of each giant axon exhibited long-term survival for weeks to months at 5-25 degrees C in crayfish and 10-30 degrees C in goldfish, the two axons differed in their survival characteristics. All measures of long-term survival in crayfish medial giant axons were independent of animal holding temperature, whereas all measures in Mauthner axons were dependent on holding temperature. Medial giant axons survived for at least 90 days in crayfish maintained at 5-25 degrees C in this and previous studies. Mauthner axons survived for over 5 months in goldfish maintained at 10 degrees C but survived for 1 month at 30 degrees C. Postoperative time had different effects on many single measures of long-term survival (axonal diameter, amplitude of action or resting potentials) in medial giant axons compared to Mauthner axons. For example, resting and action potentials in crayfish medial giant axons remained remarkably constant at all holding temperatures for 0-90 postoperative days. In contrast, resting and action potentials in goldfish Mauthner axons declined abruptly in the first 10-20 postoperative days followed by a slower decline at each holding temperature. We suggest that the mechanism of long-term survival is not necessarily the same in all anucleate axons.     

Chemically and morphologically identifiable glomeruli in the rat olfactory bulb.

Primary olfactory neurons that express the same odorant receptor are distributed mosaically throughout the olfactory neuroepithelium lining the nasal cavity, yet their axons converge and form discrete glomeruli in the olfactory bulb. We previously proposed that cell surface carbohydrates mediate the sorting out and selective fasciculation of primary olfactory axons en route to glomeruli. If this were the case, then axons that terminate in the same glomerulus would express the same complement of cell surface carbohydrates. In this study, we examined the expression of a novel carbohydrate (NOC-3) on neural cell adhesion molecule in the adult rat olfactory system. NOC-3 was expressed by a subset of neurons distributed throughout the olfactory neuroepithelium. The axons of these neurons entered the nerve fiber layer and terminated in a subset of glomeruli. It is interesting to note that we identified three unusually large glomeruli in the lateral, ventrolateral, and ventromedial olfactory bulb that were innervated by axons expressing NOC-3. NOC-3-expressing axons sorted out and fasciculated into discrete fascicles prior to entering these glomeruli. Each of these glomeruli was in a topographically fixed position in the olfactory bulbs of the same animal as well as in different animals, and their lengths were approximately 10% of the total length of the bulb. They could be identified reliably by both their topographical position and their unique morphology. These results reveal that axons expressing the same cell surface carbohydrates consistently target the same topographically fixed glomeruli, which supports a role for these molecules in axon navigation in the primary olfactory nerve pathway.     

Inhibition of tyrosine kinase receptor type B synthesis blocks axogenic effect of estradiol on rat hypothalamic neurones in vitro

17-beta-estradiol (E2) increases axonal growth and tyrosine kinase receptor (Trk)B levels of male-derived hypothalamic neurones in vitro. To investigate whether the axogenic response depends on the upregulation of TrkB, we analysed neuritic growth and neuronal polarization in cultures treated with an antisense oligonucleotide against TrkB mRNA. In cultures without E2, treatment with 7.5 or 10 micro m antisense reduced TrkB levels and the percentage of neurones showing an identifiable axon; the number and length of minor processes were increased. In cultures treated with 5 micro m antisense, morphometric parameters were normal although total TrkB levels were reduced. The same dose prevented the E2-dependent increase of TrkB levels and suppressed the axogenic effect of E2. These results indicate that TrkB is necessary for normal neuronal growth and maturation and further suggest that an increase in TrkB is necessary for E2 to exert its axogenic effect in male-derived neurones.     

Morphology of physiologically identified mitral cells in the carp olfactory bulb: a light microscopic study after intracellular staining with horseradish peroxidase

Physiologically identified mitral cells in the carp olfactory bulb were stained by intracellular injection of horseradish peroxidase in order to study the morphology in detail. The somata were fusiform, elongated, oval, triangular, or irregular. The mean diameters of the somata were 30 microns X 14 microns. Two to five thick dendrites arose from the somata and frequently gave off branches to form glomerular tufts. The dendrites extended less than 400 microns; the dendritic field of single mitral cells in the medial or lateral part of the olfactory bulb was confined within the respective part of the bulb. The axons arose from either the somata or the dendrites and had a conical initial portion, usually with a smooth contour. Some cells had poorly developed intrabulbar axon collaterals. No difference between the mitral cells in the medial part of the olfactory bulb and those in the lateral part was found in the soma diameter, the dendritic diameter at the base, or the number of first-order dendrites. However, there was a difference in the site of the origin of the axon between them: most of the axons of the mitral cells in the medial part arose from the dendrites, while most of the axons of the mitral cells in the lateral part arose from the somata. The morphology of physiologically identified mitral cells is basically consistent with that reported in the Golgi studies of teleosts. The limited dendritic fields of mitral cells may underlie the previously reported functional separation of the olfactory bulb into medial and lateral parts. The results also indicate that the two parts of the teleost olfactory bulb are differentiated not only functionally but also morphologically.