Neuregulin 1 confers neuroprotection in SOD1-linked amyotrophic lateral sclerosis mice via restoration of C-boutons of spinal motor neurons.

INTRODUCTION: Increasing evidence implicates the role of the cell types surrounding motor neurons, such as interneurons and glial cells, in non-cell autonomous neurodegeneration of amyotrophic lateral sclerosis (ALS). C-boutons, the large cholinergic synapses that innervate spinal α-motor neurons to control their excitability, are progressively lost from motor neurons in both human ALS and mutant Cu/Zn superoxide dismutase 1 (SOD1)-ALS mice. Neuregulin-1 (NRG1), a trophic factor implicated in neural development, transmission, and synaptic plasticity, has been reported to localize in the synapse of C-boutons. However, the roles of NRG1 in maintenance of motor neuron health and activity, as well as the functional consequences of its alteration in motor neuron disease, are not fully understood. RESULTS: NRG1 was localized to the post-synaptic face of C-boutons and its expression was significantly lost in SOD1-ALS mice and human ALS patients. Losses of NRG1 expression and C-boutons occurred almost contemporaneously in SOD1-ALS mice. In addition, expressions of ErbB3 and ErbB4, receptors for NRG1, were reduced in the motor neurons of SOD1-ALS mice. Furthermore, viral-mediated delivery of type III-NRG1 to the spinal cord restored the number of C-boutons and extended the survival time of SOD1-ALS mice. CONCLUSIONS: These results suggest that maintenance of NRG1-ErbB4/3 axis by supplementation of NRG1 confers neuroprotection in motor neuron disease, partly through the maintenance of C-boutons of spinal motor neurons.

Topography of striate-extrastriate connections in neonatally enucleated rats.

It is known that retinal input is necessary for the normal development of striate cortex and its corticocortical connections, but there is little information on the role that retinal input plays in the development of retinotopically organized connections between V1 and surrounding visual areas. In nearly all lateral extrastriate areas, the anatomical and physiological representation of the nasotemporal axis of the visual field mirrors the representation of this axis in V1. To determine whether the mediolateral topography of striate-extrastriate projections is preserved in neonatally enucleated rats, we analyzed the patterns of projections resulting from tracer injections placed at different sites along the mediolateral axis of V1. We found that the correlation between the distance from injection sites to the lateral border of V1 and the distance of the labeling patterns in area 18a was strong in controls and much weaker in enucleates. Data from pairs of injections in the same animal revealed that the separation of area 18a projection fields for a given separation of injection sites was more variable in enucleated than in control rats. Our analysis of single and double tracer injections suggests that neonatal bilateral enucleation weakens, but not completely abolishes, the mediolateral topography in area 18a.

Axonal thinning and extensive remyelination without chronic demyelination in spinal injured rats.

Remyelination following spinal cord injury (SCI) is thought to be incomplete; demyelination is reported to persist chronically and is proposed as a compelling therapeutic target. Yet most reports do not distinguish between the myelin status of intact axons and injury-severed axons whose proximal stumps persist but provide no meaningful function. We previously found full remyelination of spared, intact rubrospinal axons caudal to the lesion in chronic mouse SCI. However, the clinical concept of chronically demyelinated spared axons remains controversial. Since mouse models may have limitations in clinical translation, we asked whether the capacity for full remyelination is conserved in clinically relevant chronic rat SCI. We determined myelin status by examining paranodal protein distribution on anterogradely labeled, intact corticospinal and rubrospinal axons throughout the extent of the lesion. Demyelination was evident on proximal stumps of severed axons, but not on intact axons. For the first time, we demonstrate that a majority of intact axons exhibit remyelination (at least one abnormally short internode, <100 µm). Remarkably, shortened internodes were significantly concentrated at the lesion epicenter and individual axons were thinned by 23% compared with their rostral and caudal zones. Mathematical modeling predicted a 25% decrease in conduction velocity at the lesion epicenter due to short internodes and axonal thinning. In conclusion, we do not find a large chronically demyelinated population to target with remyelination therapies. Interventions may be better focused on correcting structural or molecular abnormalities of regenerated myelin.

Increased re-entry into cell cycle mitigates age-related neurogenic decline in the murine subventricular zone.

Although new neurons are produced in the subventricular zone (SVZ) of the adult mammalian brain, fewer functional neurons are produced with increasing age. The age-related decline in neurogenesis has been attributed to a decreased pool of neural progenitor cells (NPCs), an increased rate of cell death, and an inability to undergo neuronal differentiation and develop functional synapses. The time between mitotic events has also been hypothesized to increase with age, but this has not been directly investigated. Studying primary-cultured NPCs from the young adult and aged mouse forebrain, we observe that fewer aged cells are dividing at a given time; however, the mitotic cells in aged cultures divide more frequently than mitotic cells in young cultures during a 48-hour period of live-cell time-lapse imaging. Double-thymidine-analog labeling also demonstrates that fewer aged cells are dividing at a given time, but those that do divide are significantly more likely to re-enter the cell cycle within a day, both in vitro and in vivo. Meanwhile, we observed that cellular survival is impaired in aged cultures. Using our live-cell imaging data, we developed a mathematical model describing cell cycle kinetics to predict the growth curves of cells over time in vitro and the labeling index over time in vivo. Together, these data surprisingly suggest that progenitor cells remaining in the aged SVZ are highly proliferative.

Glial cells in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an adult motor neuron disease characterized by premature death of upper and lower motor neurons. Two percent of ALS cases are caused by the dominant mutations in the gene for superoxide dismutase 1 (SOD1) through a gain of toxic property of mutant protein. Genetic and chimeric mice studies using SOD1 models indicate that non-neuronal cells play important roles in neurodegeneration through non-cell autonomous mechanism. We review the contribution of each glial cell type in ALS pathology from studies of the rodent models and ALS patients. Astrogliosis and microgliosis are not only considerable hallmarks of the disease, but the intensity of microglial activation is correlated with severity of motor neuron damage in human ALS. The impaired astrocytic functions such as clearance of extracellular glutamate and release of neurotrophic factors are implicated in disease. Further, the damage within astrocytes and microglia is involved in accelerated disease progression. Finally, other glial cells such as NG2 cells, oligodendrocytes and Schwann cells are under the investigation to determine their contribution in ALS. Accumulating knowledge of active role of glial cells in the disease should be carefully applied to understanding of the sporadic ALS and development of therapy targeted for glial cells.

Targeted nonviral delivery vehicles to neural progenitor cells in the mouse subventricular zone.

Targeted gene therapy can potentially minimize undesirable off-target toxicity due to specific delivery. Neuron-specific gene delivery in the central nervous system is challenging because neurons are non-dividing and also outnumbered by glial cells. One approach is to transfect dividing neural stem and progenitor cells (NSCs and NPCs, respectively). In this work, we demonstrate cell-specific gene delivery to NPCs in the brains of adult mice using a peptide-modified polymeric vector. Tet1, a 12-amino acid peptide which has been shown to bind specifically to neuronal cells, was utilized as a neuronal targeting ligand. The cationic polymer polyethylenimine (PEI) was covalently modified with polyethylene glycol (PEG) for in vivo salt stability and Tet1 for neuron targeting to yield a Tet1-PEG-PEI conjugate. When plasmid DNA encoding the reporter gene luciferase was complexed with Tet1-PEG-PEI and delivered in vivo via an injection into the lateral ventricle, Tet1-PEG-PEI complexes mediated increased luciferase expression levels in brain tissue when compared to unmodified PEI-PEG complexes. In addition, cells transfected by Tet1-PEG-PEI complexes were found to be exclusively adult NPCs whereas untargeted PEG-PEI complexes were found to transfect a heterogenous population of cells. Thus, we have demonstrated targeted, nonviral delivery of nucleic acids to adult NPCs using the Tet1 targeting ligand. These materials could potentially be used to deliver therapeutic genes for the treatment of neurodegenerative diseases.

Age-related myelin dynamics revealed by increased oligodendrogenesis and short internodes.

Aging is associated with many functional and morphological central nervous system changes. It is important to distinguish between changes created by normal aging and those caused by disease. In the present study we characterized myelin changes within the murine rubrospinal tract and found that internode lengths significantly decrease as a function of age which suggests active remyelination. We also analyzed the proliferation, distribution and phenotypic fate of dividing cells with Bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU). The data reveal a decrease in glial cell proliferation from 1 to 6, 14 and 21 months of age in gray matter 4 weeks post-BrdU injections. However, we found an increase in gliogenesis at 21st month in white matter of the spinal cord. Half of newly generated cells expressed NG2. Most cells were positive for the early oligodendrocyte marker Olig2 and a few also expressed CC1. Very few cells ever became positive for the astrocytic markers S100beta or GFAP. These data demonstrate ongoing oligodendrogenesis and myelinogenesis as a function of age in the spinal cord.

[Associations of emotional state and quality of life with lipid concentration, duration of the disease, and the way of treating the disease in persons with type 2 diabetes mellitus]. / Serganciuju 2 tipo cukriniu diabetu emocines bukles ir gyvenimo kokybes sasajos su lipidu koncentracija, ligos trukme bei vartojamais vaistais.

The aim of the study was to evaluate associations of emotional state and quality of life with lipid concentration, duration of the disease, and the way of treating the disease in males and females with type 2 diabetes mellitus. A total of 53 persons with type 2 diabetes mellitus (27 males and 26 females; mean age, 58.7+/-8.9 years) and 56 healthy persons (26 males and 30 females; mean age, 54.7+/-8.3 years) participated in the study. Emotional state was evaluated by means of Profile of Mood State and quality of life by means of WHO Brief Quality of Life Questionnaire. Emotional state and quality of life were significantly worse, tension-anxiety and fatigue-inertia were significantly higher, vigor-activity was significantly lower in male patients with type 2 diabetes mellitus than in healthy males. In females, no significant differences in emotional state and quality of life comparing type 2 diabetes mellitus group and controls were detected. In females with type 2 diabetes mellitus, emotional state and quality of life were significantly better, scores of tension-anxiety, depression dejection, anger-hostility, and fatigue-inertia were significantly lower, and score of vigor-activity was significantly higher than in males with type 2 diabetes mellitus. Some significant correlations were found. In males, vigor-activity correlated with total cholesterol level and negatively correlated with triglyceride level. In females, significant correlations were found between scores of emotional state (tension-anxiety, depression-dejection, confusion-bewilderment, and total score of emotional state) and lipid levels (total cholesterol, triglyceride, and low-density lipoprotein cholesterol levels). There were no significant associations of emotional state and quality of life with duration of the disease in males and females with type 2 diabetes mellitus. No significant differences in emotional state and quality of life were found between males and females with type 2 diabetes mellitus, who were treated with oral antidiabetic preparations and insulin preparations.

Topography and axon arbor architecture in the visual callosal pathway: effects of deafferentation and blockade of TV-methyl-D-aspartate receptors

Visual callosal fibers link cortical loci in opposite hemispheres that represent the same visual field but whose locations are not mirror-symmetric with respect to the brain midline. Presence of the eyes from postnatal day 4 (P4) to P6 is required for this map to be specified. We tested the hypothesis that specification of the callosal map requires the activation of A'-methyl-D-aspartate receptors (NMDARs). Our results show that blockade of NMDARs with MK-801 during this critical period did not induce obvious abnormalities in callosal connectivity patterns, suggesting that retinal influences do not operate through NMDAR-mediated processes to specify normal callosal topography. In contrast, we found that interfering with NMDAR function either through MK801-induced blockade of NMDARs starting at P6 or neonatal enucleation significantly increases the length of axon branches and total length of arbors, without major effects on the number of branch tips. Our results further suggest that NMDARs act by altering the initial elaboration of arbors rather than by inhibiting a later-occurring remodeling process. Since the callosal map is present by P6, just as axonal branches of simple architecture grow into gray matter, we suggest that regulation of arbor development by NMDAR-mediated processes is important for maintaining the precision of this map.

No evidence for chronic demyelination in spared axons after spinal cord injury in a mouse.

The pattern of remyelination after traumatic spinal cord injury remains elusive, with animal and human studies reporting partial to complete demyelination followed by incomplete remyelination. In the present study, we found that spared rubrospinal tract (RST) axons of passage traced with actively transported dextrans and examined caudally to the lesion 12 weeks after mouse spinal cord contusion injury were fully remyelinated. Spared axons exhibited a marginally reduced myelin thickness and significantly shorter internodes. CASPR (contactin-associated protein) and K(v)1.2 channels were used to identify internodes and paranodal protein distribution properties were used as an index of myelin integrity. This is the first time the CNS myelin internode length was measured in a mouse. To better understand the significance of shortened internodes and thinner myelin in spared axons, we modeled conduction properties using McIntyre's et al. model of myelinated axons. Mathematical modeling predicted a 21% decrease in the conduction velocity of remyelinated RST axons attributable to shortened internodes. To determine whether demyelination could be present on axons exhibiting a pathological transport system, we used the retroviral reporter system. Virally delivered green fluorescent protein unveiled a small population of dystrophic RST axons that persist chronically with evident demyelination or abnormal remyelination. Collectively, these data show that lasting demyelination in spared axons is rare and that remyelination of axons of passage occurs in the chronically injured mouse spinal cord.

Topography and axon arbor architecture in the visual callosal pathway: effects of deafferentation and blockade of N-methyl-D-aspartate receptors.

Visual callosal fibers link cortical loci in opposite hemispheres that represent the same visual field but whose locations are not mirror-symmetric with respect to the brain midline. Presence of the eyes from postnatal day 4 (P4) to P6 is required for this map to be specified. We tested the hypothesis that specification of the callosal map requires the activation of N-methyl-D-aspartate receptors (NMDARs). Our results show that blockade of NMDARs with MK-801 during this critical period did not induce obvious abnormalities in callosal connectivity patterns, suggesting that retinal influences do not operate through NMDAR-mediated processes to specify normal callosal topography. In contrast, we found that interfering with NMDAR function either through MK801-induced blockade of NMDARs starting at P6 or neonatal enucleation significantly increases the length of axon branches and total length of arbors, without major effects on the number of branch tips. Our results further suggest that NMDARs act by altering the initial elaboration of arbors rather than by inhibiting a later-occurring remodeling process. Since the callosal map is present by P6, just as axonal branches of simple architecture grow into gray matter, we suggest that regulation of arbor development by NMDAR-mediated processes is important for maintaining the precision of this map.

Neuron-specific delivery of nucleic acids mediated by Tet1-modified poly(ethylenimine).

BACKGROUND: The development of minimally invasive, non-viral gene delivery vehicles for the central nervous system (CNS) is an important technology goal in the advancement of molecular therapies for neurological diseases. One approach is to deliver materials peripherally that are recognized and retrogradely transported by motor neurons toward the CNS. Tet1 is a peptide identified by Boulis and coworkers to possess the binding characteristics of tetanus toxin, which interacts specifically with motor neurons and undergoes fast, retrograde delivery to cell soma. In this work, Tet1-poly(ethylenimine) (Tet1-PEI) was synthesized and evaluated as a neurontargeted delivery vehicle. METHODS: Tet1-PEI and NT-PEI (neurotensin-PEI) were synthesized and complexed with plasmid DNA to form polyplexes. Polyplexes were assessed for binding and uptake in differentiated neuron-like PC-12 cells by flow cytometry and confocal microscopy. In order to determine gene delivery efficiency, polyplexes were exposed to PC-12 cells at various stages of differentiation. Targeted binding of polyplexes with primary neurons was studied using dorsal root ganglion cells. RESULTS: Tet1-PEI and NT-PEI polyplexes bound specifically to differentiated PC-12 cells. The specificity of the interaction was confirmed by delivery to non-neuronal cells and by competition studies with free ligands. Tet1-PEI polyplexes preferentially transfected PC-12 cells undergoing NGF-induced differentiation. Finally, neuron-specific binding of Tet1-PEI polyplexes was confirmed in primary neurons. CONCLUSIONS: These studies demonstrate the potential of Tet1-PEI as a neuron-targeted material for non-invasive CNS delivery. Tet1-PEI binds specifically and is internalized by neuron-like PC-12 cells and primary dorsal root ganglion. Future work will include evaluation of siRNA delivery with these vectors.