Alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-mediated excitotoxic axonal damage is attenuated in the absence of myelin proteolipid protein

In vivo and in vitro studies have shown that alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-receptor-mediated excitotoxicity causes cytoskeletal damage to axons. AMPA/kainate receptors are present on oligodendrocytes and myelin, but currently there is no evidence to suggest that axon cylinders contain AMPA receptors. Proteolipid protein (PLP) and DM20 are integral membrane proteins expressed by CNS oligodendrocytes and located in compact myelin. Humans and mice lacking normal PLP/DM20 develop axonal swellings and degeneration, suggesting that local interactions between axons and the oligodendrocyte/myelin unit are important for the normal functioning of axons and that PLP/DM20 is involved in this process. To determine whether perturbed glial-axonal interaction affects AMPA-receptor-mediated axonal damage, AMPA (1.5 nmol) was injected into the caudate nucleus of anesthetized Plp knockout and wild-type male mice (n = 13). Twenty-four hours later, axonal damage was detected by using neurofilament 200 (NF 200) immunohistochemistry and neuronal damage detected via histology. AMPA-induced axonal damage, assessed with NF 200 immunohistochemistry, was significantly reduced in Plp knockout mice compared with wild-type mice (P = 0.015). There was no significant difference in the levels of neuronal perikaryal damage between the Plp knockout and wild-type mice. In addition, there was no significant difference in the levels of glutamate receptor subunits GluR1-4 or KA2 in Plp knockout compared with wild-type littermates. The present study suggests that PLP-mediated interactions among oligodendrocytes, myelin, and axons may be involved in AMPA-mediated axonal damage.     

Genetic dissection of oligodendroglial and neuronal Plp1 function in a novel mouse model of spastic paraplegia type 2

Proteolipid protein (PLP) is the most abundant integral membrane protein in compact central nervous system myelin, and null mutations of the PLP1 gene cause spastic paraplegia type 2 (SPG2). SPG2 patients and PLP‐deficient mice exhibit only moderate abnormalities of myelin but progressive degeneration of long axons. Since Plp1 gene products are detected in a subset of neurons it has been suggested that the loss of neuronal Plp1 expression could be the cause of the axonal pathology. To test this hypothesis, we created mice with a floxed Plp1 allele for selective Cre‐mediated recombination in neurons. We find that recombination of Plp1 in excitatory projection neurons does not cause neuropathology, whereas oligodendroglial targeting of Plp1 is sufficient to cause the entire neurodegenerative spectrum of SPG2 including axonopathy and secondary neuroinflammation. We conclude that PLP‐dependent loss of oligodendroglial support is the primary cause of axonal degeneration in SPG2.     

Schwann Cells Provide Iron to Axonal Mitochondria and Its Role in Nerve Regeneration

Iron is an essential cofactor for several metabolic processes, including the generation of ATP in mitochondria, which is required for axonal function and regeneration. However, it is not known how mitochondria in long axons, such as those in sciatic nerves, acquire iron in vivo. Because of their close proximity to axons, Schwann cells are a likely source of iron for axonal mitochondria in the PNS. Here we demonstrate the critical role of iron in promoting neurite growth in vitro using iron chelation. We also show that Schwann cells express the molecular machinery to release iron, namely, the iron exporter, ferroportin (Fpn) and the ferroxidase ceruloplasmin (Cp). In Cp KO mice, Schwann cells accumulate iron because Fpn requires to partner with Cp to export iron. Axons and Schwann cells also express the iron importer transferrin receptor 1 (TfR1), indicating their ability for iron uptake. In teased nerve fibers, Fpn and TfR1 are predominantly localized at the nodes of Ranvier and Schmidt-Lanterman incisures, axonal sites that are in close contact with Schwann cell cytoplasm. We also show that lack of iron export from Schwann cells in Cp KO mice reduces mitochondrial iron in axons as detected by reduction in mitochondrial ferritin, affects localization of axonal mitochondria at the nodes of Ranvier and Schmidt-Lanterman incisures, and impairs axonal regeneration following sciatic nerve injury. These finding suggest that Schwann cells contribute to the delivery of iron to axonal mitochondria, required for proper nerve repair.SIGNIFICANCE STATEMENT This work addresses how and where mitochondria in long axons in peripheral nerves acquire iron. We show that Schwann cells are a likely source as they express the molecular machinery to import iron (transferrin receptor 1), and to export iron (ferroportin and ceruloplasmin [Cp]) to the axonal compartment at the nodes of Ranvier and Schmidt-Lanterman incisures. Cp KO mice, which cannot export iron from Schwann cells, show reduced iron content in axonal mitochondria, along with increased localization of axonal mitochondria at Schmidt-Lanterman incisures and nodes of Ranvier, and impaired sciatic nerve regeneration. Iron chelation in vitro also drastically reduces neurite growth. These data suggest that Schwann cells are likely to contribute iron to axonal mitochondria needed for axon growth and regeneration.     

Lack of association of transforming growth factor (TGF)-beta 1 and beta 2 gene polymorphisms with multiple sclerosis (MS) in Northern Ireland.

OBJECTIVE: To examine the influence of TGF-beta genes on MS susceptibility. BACKGROUND: TGF-beta, of which three homologous isoforms exist (1, 2 and 3), is a strongly immunosuppressive cytokine-inhibiting expression of pro-inflammatory cytokines and blocking cytokine induction of adhesion molecules. TGF-beta delays onset of EAE and TGF-beta 1 gene knockout mice develop fatal multifocal inflammatory disease. High TGF-beta levels exist during MS remission whilst E-selectin, whose expression is inhibited by TGF-beta, is found at higher levels in primary progressive disease (PPMS) and it is postulated that the unremitting course of PPMS may be due to low levels of TGF-beta. METHODS: Gene association studies using separate polymorphic microsatellite markers for TGF-beta 1 and TGF-beta 2 were performed, incorporating 151 relapsing-remitting or secondary progressive MS (RR/SPMS) patients, 104 PPMS patients and 159 normal controls (Nor). Forward primers were 5' end-labelled with 6-Fam, PCR products were analysed on an Applied Biosystems 373A fluorescent fragment analyser and Genescan 672 software was used for allele sizing. RESULTS: No significant differences existed in allele frequencies between either MS group and controls regarding the TGF-beta 1 marker: RR/SPMS vs Nor (P = 0.48, df = 8); PPMS vs Nor (P = 0.34, df = 8). Similarly there were no associations demonstrated with the TGF-beta 2 marker: RR/SPMS vs Nor (P = 0.24, df = 2); PPMS vs Nor (P = 0.53, df = 2). CONCLUSION: These data indicate that TGF-beta 1 and beta 2 genes are not loci influencing MS susceptibility, either RR/SPMS or PPMS, in this population.     

Structural remodeling of nucleus ambiguus projections to cardiac ganglia following chronic intermittent hypoxia in C57BL/6J mice

The baroreflex control of heart rate (HR) is reduced following chronic intermittent hypoxia (CIH). Since the nucleus ambiguus (NA) plays a key role in baroreflex control of HR, we examined whether CIH remodels vagal efferent projections to cardiac ganglia. C57BL/6J mice (3-4 months of age) were exposed to either room air (RA) or CIH for 3 months. Confocal microscopy was used to examine NA axons and terminals in cardiac ganglia following Fluoro-Gold (FG) injections to label cardiac ganglia, and microinjections of tracer DiI into the left NA to anterogradely label vagal efferents. We found that: 1) Cardiac ganglia were widely distributed on the dorsal surface of the atria. Although the total number of cardiac ganglia did not differ between RA and CIH mice, the size of ganglia and the somatic area of cardiac principal neurons (PNs) were significantly decreased (P < 0.01), and the size of the PN nuclei was increased following CIH (P < 0.01). 2) NA axons entered cardiac ganglia and innervated PNs with dense basket endings in both RA and CIH mice, and the percentage of innervated PNs was similar (RA: 50 +/- 1.0%; CIH: 49 +/- 1.0%; P > 0.10). In CIH mice, however, swollen cardiac axons and terminals without close contacts to PNs were found. Furthermore, varicose endings around PNs appeared swollen and the axonal varicose area around PNs was almost doubled in size (CIH: 163.1 +/- 6.4 microm(2); RA: 88 +/- 3.9 microm(2), P < 0.01). Thus, CIH significantly altered the structure of cardiac ganglia and resulted in reorganized vagal efferent projections to cardiac ganglia. Such remodeling of cardiac ganglia and vagal efferent projections provides new insight into the effects of CIH on the brain-heart circuitry of C57BL/6J mice.     

Changes in rat cerebral mitochondrial succinate dehydrogenase activity after brain trauma

The objective of this study was to evaluate 2,3,5-triphenyltetrazolium chloride (TTC) staining in the brain tissue of rats submitted to a closed head traumatic injury, in comparison to control rats not submitted to trauma. The closed head, weight drop trauma model described by Marmarou et al. (1994) was used. Animals were all sacrificed 24 h after trauma. Staining of cerebral coronal slices using TTC, coupled to image analysis software, was used to measure the level of staining. An ultrastructural study of the brain region underneath the impact zone, as well as from the correspondent region of control rats, was also done. The TTC image analysis revealed a significant decrease in the percentage of white area, in traumatized rats (mean +/- SEM 23.93% +/- 2.26, n = 4 for control, 12.13% +/- 1.72, n = 9 for traumatized rats, p <.05). The ultrastructural analysis revealed that the number of axons showing at least one mitochondrion was significantly higher in the trauma group (mean +/- SEM 49.3%, n = 4 rats, 75 photographs, 2443 axons) than in control groups (23%, n = 3 rats, 30 photographs, 6220 axons (p <.001). Another difference observed was the larger mitochondrial size in the axons of traumatized rats (mean diameter +/- SEM 0.520 +/- 0.003 microm) compared to the controlled rats (0.368 +/- 0.006 microm; p <.001). The ultrastructural observation of the traumatized brain revealed a significantly higher number of peroxisomes per photograph (mean number +/- SEM 10.58 +/- 1.18, n = 75) compared to the control group (0.19 +/- 0.08, n = 30, p <.001). The results indicate an increase of mitochondrial and peroxysomal relative mass, with a higher succinate dehydrogenase activity, 24 h after the induction of traumatic brain injury.     

Age-dependent acrylamide neurotoxicity in mice: morphology, physiology, and function.

Acrylamide intoxication produces peripheral neuropathy characterized by weakness and ataxia in both humans and experimental animals. Previous studies on animals of different ages and species indicate that the longest and largest nerves are affected earlier with the major pathology in the terminal parts of axons, i.e., distal axonopathy. However, several issues have remained elusive; for example, what are the earliest pathological changes? An equally intriguing question is whether younger animals are more susceptible to acrylamide than older animals. To address these issues, we compared the vulnerability to acrylamide of 3- and 8-week-old mice. These mice were intoxicated with acrylamide in drinking water (400 ppm). The sequence of intoxication could be categorized into three stages. In the initial stage, there was no visible weakness or ataxia. The only noticeable changes were poor performance on the rota-rod test and swelling of motor nerve terminals. Obvious weakness and ataxia of hindlimbs developed gradually (here designated as the early stage). The weakness and ataxia progressed at variable speeds in mice of different ages, and eventually the forelimbs (quadriparesis) were affected in the late stage. Each stage appeared earlier in 3-week-old mice than in 8-week-old mice (7.1 +/- 1.1 vs 15.6 +/- 4.0 days, P < 0.01 for the early stage; and 15.3 +/- 2.1 vs 31.7 +/- 6.0 days, P < 0.01 for the late stage). The progression of neurological deficits was also faster in the younger mice (7.2 +/- 1.8 vs 16.3 +/- 4.2 days, P < 0.01). Pathological changes in the distal parts of motor nerves innervating hindfoot muscles were evaluated by combined cholinesterase histochemistry and immunocytochemistry for neuronal markers to demonstrate motor nerve terminals and neuromuscular junctions simultaneously. In the initial stage, there was axonal swelling in motor nerve terminals. As acrylamide intoxication continued, axonal swelling extended into junctional folds and into the intramuscular nerves, which resulted in Wallerian-like degeneration. Our results indicate that younger mice show a much higher susceptibility to acrylamide intoxication, and pathological changes precede neurological symptoms.     

CNTF promotes the regrowth of retinal ganglion cell axons into murine peripheral nerve grafts

Autologous peripheral nerves were transplanted onto transected optic nerves of adult mice. We examined whether intraocular CNTF injections increased retinal ganglion cell (RGC) axon regeneration, and what types of RGCs regrew axons into grafts. After temporal CNTF eye injections there were more fluorogold-labelled regenerating RGCs (mean +/- s.e.m. 342+/-113.1; n=6) than in sham eye-injected mice (133+/-27.6; n=8). Greater numbers of regenerating RGCs (1198+/-367.6; n=6) were seen in mice receiving both nasal and temporal CNTF injections. The range of soma areas in regenerate and normal retinas was similar but the average size of regenerating RGCs was greater (212 microm2 vs 111 microm2). Most regenerating RGCs had large dendritic fields. The data suggest a heterogeneous response to axotomy in adult mice, large RGCs preferentially regrowing axons into PN grafts.     

A study of the HLA-DR region in clinical subgroups of multiple sclerosis and its influence on prognosis.

OBJECTIVE: To investigate the HLA-DR associations in relapsing-remitting/secondary progressive multiple sclerosis (RR/SPMS) and primary progressive MS (PPMS). The HLA-DR2 allele (or its split, HLA-DRB1*15) is felt to be a risk factor for MS, rather than a genetic marker for the population of origin. Some studies have indicated a different HLA-DR antigen profile in PPMS patients compared with those having an initially relapsing-remitting course, only those with relapsing disease showing an increase in HLA-DR2. Association of PPMS with DR4 has been suggested. Several DR alleles have also been felt to influence the prognosis in MS. METHODS: Genomic DNA was prepared from peripheral blood of 202 RR/SPMS patients identified in a population-based prevalence study, 102 PPMS patients identified throughout Northern Ireland and 398 normal controls (Nor) matched for the postcode areas of those identified in the prevalence study. Samples were typed for the HLA-DR antigens using polymerase chain reaction (PCR) technology and sequence specific oligonucleotide probes (SSOP). RESULTS: A high incidence of HLA-DRB1*15 was found in each MS group - PPMS (63.73%), RR/SPMS (66.83%) - compared with normals (32.41%), (PPMS vs. Nor, P<0.0001: RR/SPMS vs. Nor, P<0.0001). HLA-DRB1*04 occurred at a lower incidence in both MS groups compared with controls - RR/SPMS (22%), PPMS (30%), Nor (35%). Overall, highly significant differences existed across the full HLA-DR allele distribution (RR/SPMS vs. Nor, P<0.0001, df=12: PPMS vs. Nor, P=0.0007, df=12). No significant differences existed between PPMS and RR/SPMS (P=0.47, df=12), and the allele distributions in benign and aggressive MS were similar. CONCLUSIONS: These data suggest that in this population, HLA-DRB1*15 is indeed associated with PPMS and that PPMS has a HLA-DR profile distinct from the normal population but not from those with an initially relapsing-remitting course. No single allele is associated with either a good or poor prognosis.     

830 nm laser irradiation induces varicosity formation, reduces mitochondrial membrane potential and blocks fast axonal flow in small and medium diameter rat dorsal root ganglion neurons: implications for the analgesic effects of 830 nm laser

We report the formation of 830 nm (cw) laser-induced, reversible axonal varicosities, using immunostaining with beta-tubulin, in small and medium diameter, TRPV-1 positive, cultured rat DRG neurons. Laser also induced a progressive and statistically significant decrease (p<0.005) in MMP in mitochondria in and between static axonal varicosities. In cell bodies of the neuron, the decrease in MMP was also statistically significant (p<0.05), but the decrease occurred more slowly. Importantly we also report for the first time that 830 nm (cw) laser blocked fast axonal flow, imaged in real time using confocal laser microscopy and JC-1 as mitotracker. Control neurons in parallel cultures remained unaffected with no varicosity formation and no change in MMP. Mitochondrial movement was continuous and measured along the axons at a rate of 0.8 microm/s (range 0.5-2 microm/s), consistent with fast axonal flow. Photoacceptors in the mitochondrial membrane absorb laser and mediate the transduction of laser energy into electrochemical changes, initiating a secondary cascade of intracellular events. In neurons, this results in a decrease in MMP with a concurrent decrease in available ATP required for nerve function, including maintenance of microtubules and molecular motors, dyneins and kinesins, responsible for fast axonal flow. Laser-induced neural blockade is a consequence of such changes and provide a mechanism for a neural basis of laser-induced pain relief. The repeated application of laser in a clinical setting modulates nociception and reduces pain. The application of laser therapy for chronic pain may provide a non-drug alternative for the management of chronic pain.     

Increased axonal mitochondrial activity as an adaptation to myelin deficiency in the Shiverer mouse

Axonal pathology in multiple sclerosis (MS) has been described for over a century, but new insights into axonal loss and disability have refocused interest in this area. There is evidence of oxidative damage to mitochondrial DNA in chronic MS plaques, suggesting that mitochondrial failure may play a role in MS pathology. We propose that in the chronic absence of myelin the maintenance of conduction relies partially on an increase in mitochondria to provide energy. This increased energy requirement also promotes reactive oxygen species (ROS), because most intraaxonal ROS are generated by mitochondria. If antioxidant defenses are overwhelmed by an excess of ROS, this may result in damage to the axon. Our aim was to investigate whether a chronic lack of myelin results in adaptive changes involving mitochondria within the axon. We investigated this in the shiverer mouse. This myelin basic protein gene mutant provides a model of how adult central nervous system (CNS) axons cope with the chronic absence of a compact myelin sheath. Cytochrome c histochemistry demonstrated a twofold increase in mitochondrial activity in white matter tracts of shiverer, and electron microscopy confirmed a significantly higher number of mitochondria within the dysmyelinated axons. Our data demonstrate that there are adaptive changes involving mitochondria occurring within CNS axons in shiverer mice in response to a lack of myelin. This work contributes to our understanding of the adaptive changes occurring in response to a lack of myelin in a noninflammatory environment similar to the situation seen in chronically demyelinated MS plaques.     

Quantitative pathological evidence for axonal loss in normal appearing white matter in multiple sclerosis

We assessed axonal loss in the normal appearing white matter of the corpus callosum in postmortem brains of patients with multiple sclerosis, using quantitative measures of both axonal density and white matter atrophy. The calculated total number of axons was reduced significantly (mean +/- SD, 5.4 x 10(7) +/- 3.1 x 10(7)) compared with normal controls (11.6 x 10(7) +/- 2.2 x 10(7), p = 0.001) with a reduction both in axonal density (median, 34%; range, 16-56%; p = 0.004) and area (mean +/- SD: multiple sclerosis, 584 +/- 170 mm2; controls, 871 +/- 163 mm2; p = 0.004). These results confirm substantial axonal loss in the normal appearing white matter and demonstrate that measures of both axonal density and white matter volume are necessary to appreciate the full extent of axonal loss.     

Evidence for a mitochondrial oxidative phosphorylation defect in brains from patients with schizophrenia

In-vivo imaging studies and post-mortem studies have demonstrated an impairment of energy metabolism in brains of patients with schizophrenia. Decreased oxidative metabolism has been consistently documented in the frontal lobes. However, the biochemical basis of these changes is unclear. The changes could be caused by reduced requirement of the cells for metabolic energy or an abnormality in energy generation. Neurons generate energy through the respiratory chain in the mitochondria. The respiratory chain consists of five enzyme complexes (I-V). The purpose of the present study was to assess mitochondrial function and test the hypothesis of an underlying oxidative phosphorylation defect in schizophrenia. We analysed spectrophotometrically post-mortem brain specimens of frontal cortex, temporal cortex, basal ganglia, and cerebellum of 12 patients who met the DSM-IV criteria for schizophrenia and of 13 healthy controls for the specific activities of respiratory chain enzymes in the mitochondria. The major finding was that the activity of complex IV was significantly reduced in the frontal cortex (40.9+/-6.7 vs. 87.3+/-12, P=0.003) and in the temporal cortex (39.5+/-6.8 vs. 78+/-10.8, P=0.006) of schizophrenics. In addition, the activity of complexes I+III was significantly reduced in the temporal cortex (2.2+/-0.6 vs. 4.4+/-0.5, P=0.01) and basal ganglia (1.6+/-0.5 vs. 3.4+/-0.3, P=0.015) in schizophrenia. All other enzyme activities showed no differences to healthy controls. The results confirm a defect of oxidative phosphorylation in brains from patients with schizophrenia, which may contribute to impaired energy generation.     

Dysregulation of axonal sodium channel isoforms after adult-onset chronic demyelination

Demyelination results in conduction block through changes in passive cable properties of an axon and in the expression and localization of axonal ion channels. We show here that adult-onset chronic demyelination, such as occurs in demyelinating disorders and after nerve injury, alters the complement of axonal voltage-dependent Na+ (Nav) channel isoforms and their localization. As a model, we used heterozygous transgenic mice with two extra copies of the proteolipid protein gene (Plp/-). Retinal ganglion cell axons in these mice myelinate normally, with young Plp/- and wild-type mice expressing Nav1.2 at low levels, whereas Nav1.6 is clustered in high densities at nodes of Ranvier. At 7 months of age, however, Plp/- mice exhibit severe demyelination and oligodendrocyte cell death, leading to a profound reduction in Nav1.6 clusters, loss of the paranodal axoglial apparatus, and a marked increase in Nav1.2. We conclude that myelin is crucial not only for node of Ranvier formation, but also to actively maintain the proper localization and complement of distinct axonal Nav channel isoforms throughout life. The altered Nav channel isoform localization and complement induced by demyelination may contribute to the pathophysiology of demyelinating disorders and nerve injury.     

Clinically benign multiple sclerosis despite large T2 lesion load: can we explain this paradox?

Magnetic resonance imaging (MRI) techniques such as magnetization transfer imaging and magnetic resonance spectroscopy (MRS) may reveal otherwise undetectable tissue damage in multiple sclerosis (MS) and can serve to explain more severe disability than expected from conventional MRI. That an inverse situation may exist where non-conventional quantitative MRI and MRS metrics would indicate less abnormality than expected from T2 lesion load to explain preserved clinical functioning was hypothesized. Quantitative MRI and MRS were obtained in 13 consecutive patients with clinically benign MS (BMS; mean age 44 +/- 9 years) despite large T 2 lesion load and in 15 patients with secondary progressive MS (SPMS; mean age 47 +/- 6 years) matched for disease duration. The magnetization transfer ratio (MTR), magnetization transfer rate (kfor), brain parenchymal fraction (BPF) and brain metabolite concentrations from proton MRS were determined. BMS patients were significantly less disabled than their SPMS counterparts (mean expanded disability status score: 2.1 +/- 1.1 versus 6.2 +/- 1.1; P < 0.001) and had an even somewhat higher mean T2 lesion load (41.2 +/- 27.1 versus 27.9 +/- 24.8 cm3; P = 0.19). Normal appearing brain tissue histogram metrics for MTR and kfor, mean MTR and kfor of MS lesions and mean BPF were similar in BMS and SPMS patients. Levels of N-acetyl-aspartate, choline and myoinositol were comparable between groups. This study thus failed to explain the preservation of function in our BMS patients with large T2 lesion load by a higher morphologic or metabolic integrity of the brain parenchyma. Functional compensation must come from other mechanisms such as brain plasticity.     

Altered ionic conductances in axons of transgenic mouse expressing the human neurofilament heavy gene: A mouse model of amyotrophic lateral sclerosis

Neurofilaments (NFs; made by copolymerization of three intermediate filament proteins NF-L, NF-M, and NF-H, for light, medium, and heavy) constitute the most abundant cytoskeletal structure in large myelinated axons. The presence of aberrant NF accumulation has been associated with neurodegenerative diseases (such as ALS). The possible causal role of NF in neurodegeneration has been supported by studies on recently available transgenic mice in which expression of human NF-H (hNF-H +/+) leads to overt neuropathy. We have examined electrophysiological properties of myelinated axons in hNF-H +/+ mice using intraaxonal microelectrode recording from isolated sciatic and tibial nerves. Transgenic mice showed several deficits in physiological properties of low threshold myelinated fibers: conduction velocity and resting membrane potential were significantly decreased (20 +/- 1.6 vs 40 +/- 2 m/s; -71.3 +/- 0.9 vs -75.5 +/- 0.5 m/s; mean +/- SE; n = 25; 22 degrees C). While the amplitude of action potentials was of comparable size (82 +/- 5 vs 86 +/- 3 mV) duration of action potential (at half-amplitude, AP/2) in hNF-H +/+ was significantly prolonged (0.82 +/- 0.02 vs 0.65 +/- 0.02 ms). Voltage-current properties of axonal membrane indicate a significant decrease in inward and outward rectification. Occasionally, impaled axons of hNF-H +/+ showed membrane oscillations and repetitive activity (reminiscent of fasciculations) never observed in normal animals. These results are compatible with an imbalance between ion conductances in axons from transgenic animals (an increase in Na(+) and a decrease in K(+) conductances), in agreement with recent suggestion based on clinical studies on ALS patients (H. Bostock et al., 1995, Brain 118, 217-225). One may hypothesize that these changes could contribute to neurodegenerative processes (i.e., via an increase in [Na(+)](i)), as well as clinical symptoms (fasciculations) observed in patients with degenerative motor neuron diseases.     

High Dietary Fat Consumption Impairs Axonal Mitochondrial Function In Vivo

Peripheral neuropathy (PN) is the most common complication of prediabetes and diabetes. PN causes severe morbidity for Type 2 diabetes (T2D) and prediabetes patients, including limb pain followed by numbness resulting from peripheral nerve damage. PN in T2D and prediabetes is associated with dyslipidemia and elevated circulating lipids; however, the molecular mechanisms underlying PN development in prediabetes and T2D are unknown. Peripheral nerve sensory neurons rely on axonal mitochondria to provide energy for nerve impulse conduction under homeostatic conditions. Models of dyslipidemia in vitro demonstrate mitochondrial dysfunction in sensory neurons exposed to elevated levels of exogenous fatty acids. Herein, we evaluated the effect of dyslipidemia on mitochondrial function and dynamics in sensory axons of the saphenous nerve of a male high-fat diet (HFD)-fed murine model of prediabetes to identify mitochondrial alterations that correlate with PN pathogenesis in vivo. We found that the HFD decreased mitochondrial membrane potential (MMP) in axonal mitochondria and reduced the ability of sensory neurons to conduct at physiological frequencies. Unlike mitochondria in control axons, which dissipated their MMP in response to increased impulse frequency (from 1 to 50 Hz), HFD mitochondria dissipated less MMP in response to axonal energy demand, suggesting a lack of reserve capacity. The HFD also decreased sensory axonal Ca²⁺ levels and increased mitochondrial lengthening and expression of PGC1α, a master regulator of mitochondrial biogenesis. Together, these results suggest that mitochondrial dysfunction underlies an imbalance of axonal energy and Ca²⁺ levels and impairs impulse conduction within the saphenous nerve in prediabetic PN.SIGNIFICANCE STATEMENT Diabetes and prediabetes are leading causes of peripheral neuropathy (PN) worldwide. PN has no cure, but development in diabetes and prediabetes is associated with dyslipidemia, including elevated levels of saturated fatty acids. Saturated fatty acids impair mitochondrial dynamics and function in cultured neurons, indicating a role for mitochondrial dysfunction in PN progression; however, the effect of elevated circulating fatty acids on the peripheral nervous system in vivo is unknown. In this study, we identify early pathogenic events in sensory nerve axons of mice with high-fat diet-induced PN, including alterations in mitochondrial function, axonal conduction, and intra-axonal calcium, that provide important insight into potential PN mechanisms associated with prediabetes and dyslipidemia in vivo.     

Nerve Growth Factor- and Neurotrophin-3-Releasing Guidance Channels Promote Regeneration of the Transected Rat Dorsal Root

Dorsal roots have a limited regeneration capacity after transection. To improve nerve regeneration, the growth-promoting effects of the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) were evaluated. The proteins were continuously released by synthetic nerve guidance channels bridging a 4-mm gap in the transected dorsal root. Four weeks after lesion, the regenerated nerve cables were analyzed for the presence of myelinated and unmyelinated axons. While BDNF showed a limited effect on axonal regeneration (863 +/- 39 axons/regenerated nerve, n = 6), NGF (1843 +/- 482) and NT-3 (1495 +/- 449) powerfully promoted regeneration of myelinated axons compared to channels releasing the control protein bovine serum albumin (293 +/- 39). In addition, NGF, but not BDNF nor NT-3, had a potent effect on the regeneration of unmyelinated axons (NGF, 55 +/- 1.4; BDNF, 4 +/- 0.3; NT-3, 4.7 +/- 0.3 axons/100 microm(2); n = 6). The present study suggests that synthetic nerve guidance channels slowly and continuously releasing the neurotrophins NGF and NT-3 can overcome the limited regeneration of transected dorsal root.     

GDNF and NGF released by synthetic guidance channels support sciatic nerve regeneration across a long gap

The present work was performed to determine the ability of neurotrophic factors to allow axonal regeneration across a 15-mm-long gap in the rat sciatic nerve. Synthetic nerve guidance channels slowly releasing NGF and GDNF were fabricated and sutured to the cut ends of the nerve to bridge the gap. After 7 weeks, nerve cables had formed in nine out of ten channels in both the NGF and GDNF groups, while no neuronal cables were present in the control group. The average number of myelinated axons at the midpoint of the regenerated nerves was significantly greater in the presence of GDNF than NGF (4942 +/-1627 vs. 1199 +/-431, P < or = 0.04). A significantly greater number of neuronal cells in the GDNF group, when compared to the NGF group, retrogradely transported FluoroGold injected distal to the injury site before explantation. The total number of labelled motoneurons observed in the ventral horn of the spinal cord was 98.1 +/-23.4 vs. 20.0 +/-8.5 (P < or = 0.001) in the presence of GDNF and NGF, respectively. In the dorsal root ganglia, 22.7% +/- 4.9% vs. 3.2% +/-1.9% (P +/-0.005) of sensory neurons were labelled retrogradely in the GDNF and NGF treatment groups, respectively. The present study demonstrates that, sustained delivery of GDNF and NGF to the injury site, by synthetic nerve guidance channels, allows regeneration of both sensory and motor axons over long gaps; GDNF leads to better overall regeneration in the sciatic nerve.     

Mitochondrial accumulation in the distal part of the initial segment of chicken spinal motoneurons

The axonal initial segment is the initiation site of action potentials and is characterized morphologically by a dense undercoating and fascicles of microtubules connected by cross-bridges. In order to analyze subcellular structures in the initial segment, we made serial transverse sections of initial segments of identified chicken motoneurons by retrograde transport of horseradish peroxidase (HRP) injected into the muscle. The mean (+/-SD) length of the initial segment was 28.1+/-2.3 microm (n=6). Mitochondria accumulated in the distal part of the initial segment, which was 1.4-6.9 microm in length (5-23% of the total length of the initial segment). In the transverse section of the distal part, mitochondrial density was 15.8+/-6.2% (n=5), while in the middle and proximal parts it was 6.1+/-1.6% and 5.6+/-1.4%, respectively. Mitochondrial accumulation was observed in common in phasic and tonic motoneurons in the chicken, and also observed in the distal part of the initial segment of the large ventral horn neurons of the chicken without HRP injection. These findings suggest that accumulated mitochondria play an important role in maintaining the physiological function of the distal part of the motoneuron initial segment.