Physiology of PNS axons relies on glycolytic metabolism in myelinating Schwann cells.

While lactate shuttle theory states that glial cells metabolize glucose into lactate to shuttle it to neurons, how glial cells support axonal metabolism and function remains unclear. Lactate production is a common occurrence following anaerobic glycolysis in muscles. However, several other cell types, including some stem cells, activated macrophages and tumor cells, can produce lactate in presence of oxygen and cellular respiration, using Pyruvate Kinase 2 (PKM2) to divert pyruvate to lactate dehydrogenase. We show here that PKM2 is also upregulated in myelinating Schwann cells (mSC) of mature mouse sciatic nerve versus postnatal immature nerve. Deletion of this isoform in PLP-expressing cells in mice leads to a deficit of lactate in mSC and in peripheral nerves. While the structure of myelin sheath was preserved, mutant mice developed a peripheral neuropathy. Peripheral nerve axons of mutant mice failed to maintain lactate homeostasis upon activity, resulting in an impaired production of mitochondrial ATP. Action potential propagation was not altered but axonal mitochondria transport was slowed down, muscle axon terminals retracted and motor neurons displayed cellular stress. Additional reduction of lactate availability through dichloroacetate treatment, which diverts pyruvate to mitochondrial oxidative phosphorylation, further aggravated motor dysfunction in mutant mice. Thus, lactate production through PKM2 enzyme and aerobic glycolysis is essential in mSC for the long-term maintenance of peripheral nerve axon physiology and function.

Advances in Organ Preservation for Laryngeal Cancer.

OPINION STATEMENT: At the University of Wisconsin, all treatment of head and neck cancer patients begins with discussion at our multi-disciplinary tumor board. Most patients with T4 disease, with existing laryngeal dysfunction, considered unlikely to complete definitive CRT or who have a high risk of persistent aspiration after non-operative management undergo total laryngectomy. A laryngeal sparing approach is attempted on most other patients. Radiotherapy is delivered over 6.5 weeks, preferably with concurrent weekly cisplatin. If the patient is hesitant of chemotherapy or has contraindications to cisplatin, concurrent cetuximab may be offered. Patients treated with RT alone are often treated to the same dose, but via an accelerated schedule by adding a 6th fraction per week. The 6th fraction is given by delivering two treatments at least 6 h apart on a weekday of the patient's choosing. We consider the following to be major risk factors for clinically significant weight loss during treatment: a 10% or greater loss of weight in the 6 months prior to starting treatment, delivery of concurrent cisplatin, and treatment of the bilateral neck with radiation. Patients who have 2-3 of these characteristics are often given gastrostomy tubes prophylactically. Patients are seen 2 weeks after completion of therapy, and then every 3 months after completion for 2 years. A CT neck and PET-CT are performed at the first 3-month visit. They are seen twice in year three, and then yearly until years 5-7. At each of these visits, we have a low threshold to present the patient at our multidisciplinary tumor board for consideration of salvage laryngectomy if there are signs of progression.

AAV2/9-mediated silencing of PMP22 prevents the development of pathological features in a rat model of Charcot-Marie-Tooth disease 1 A.

Charcot-Marie-Tooth disease 1 A (CMT1A) results from a duplication of the PMP22 gene in Schwann cells and a deficit of myelination in peripheral nerves. Patients with CMT1A have reduced nerve conduction velocity, muscle wasting, hand and foot deformations and foot drop walking. Here, we evaluate the safety and efficacy of recombinant adeno-associated viral vector serotype 9 (AAV2/9) expressing GFP and shRNAs targeting Pmp22 mRNA in animal models of Charcot-Marie-Tooth disease 1 A. Intra-nerve delivery of AAV2/9 in the sciatic nerve allowed widespread transgene expression in resident myelinating Schwann cells in mice, rats and non-human primates. A bilateral treatment restore expression levels of PMP22 comparable to wild-type conditions, resulting in increased myelination and prevention of motor and sensory impairments over a twelve-months period in a rat model of CMT1A. We observed limited off-target transduction and immune response using the intra-nerve delivery route. A combination of previously characterized human skin biomarkers is able to discriminate between treated and untreated animals, indicating their potential use as part of outcome measures.

In vivo real-time dynamics of ATP and ROS production in axonal mitochondria show decoupling in mouse models of peripheral neuropathies.

Mitochondria are critical for the function and maintenance of myelinated axons notably through Adenosine triphosphate (ATP) production. A direct by-product of this ATP production is reactive oxygen species (ROS), which are highly deleterious for neurons. While ATP shortage and ROS levels increase are involved in several neurodegenerative diseases, it is still unclear whether the real-time dynamics of both ATP and ROS production in axonal mitochondria are altered by axonal or demyelinating neuropathies. To answer this question, we imaged and quantified mitochondrial ATP and hydrogen peroxide (H2O2) in resting or stimulated peripheral nerve myelinated axons in vivo, using genetically-encoded fluorescent probes, two-photon time-lapse and CARS imaging. We found that ATP and H2O2 productions are intrinsically higher in nodes of Ranvier even in resting conditions. Axonal firing increased both ATP and H2O2 productions but with different dynamics: ROS production peaked shortly and transiently after the stimulation while ATP production increased gradually for a longer period of time. In neuropathic MFN2R94Q mice, mimicking Charcot-Marie-Tooth 2A disease, defective mitochondria failed to upregulate ATP production following axonal activity. However, elevated H2O2 production was largely sustained. Finally, inducing demyelination with lysophosphatidylcholine resulted in a reduced level of ATP while H2O2 level soared. Taken together, our results suggest that ATP and ROS productions are decoupled under neuropathic conditions, which may compromise axonal function and integrity.

A Targeted Mutation Disrupting Mitochondrial Complex IV Function in Primary Afferent Neurons Leads to Pain Hypersensitivity Through P2Y1 Receptor Activation.

As mitochondrial dysfunction is evident in neurodegenerative disorders that are accompanied by pain, we generated inducible mutant mice with disruption of mitochondrial respiratory chain complex IV, by COX10 deletion limited to sensory afferent neurons through the use of an Advillin Cre-reporter. COX10 deletion results in a selective energy-deficiency phenotype with minimal production of reactive oxygen species. Mutant mice showed reduced activity of mitochondrial respiratory chain complex IV in many sensory neurons, increased ADP/ATP ratios in dorsal root ganglia and dorsal spinal cord synaptoneurosomes, as well as impaired mitochondrial membrane potential, in these synaptoneurosome preparations. These changes were accompanied by marked pain hypersensitivity in mechanical and thermal (hot and cold) tests without altered motor function. To address the underlying basis, we measured Ca2+ fluorescence responses of dorsal spinal cord synaptoneurosomes to activation of the GluK1 (kainate) receptor, which we showed to be widely expressed in small but not large nociceptive afferents, and is minimally expressed elsewhere in the spinal cord. Synaptoneurosomes from mutant mice showed greatly increased responses to GluK1 agonist. To explore whether altered nucleotide levels may play a part in this hypersensitivity, we pharmacologically interrogated potential roles of AMP-kinase and ADP-sensitive purinergic receptors. The ADP-sensitive P2Y1 receptor was clearly implicated. Its expression in small nociceptive afferents was increased in mutants, whose in vivo pain hypersensitivity, in mechanical, thermal and cold tests, was reversed by a selective P2Y1 antagonist. Energy depletion and ADP elevation in sensory afferents, due to mitochondrial respiratory chain complex IV deficiency, appear sufficient to induce pain hypersensitivity, by ADP activation of P2Y1 receptors.

Mitochondrial dysfunction and axon degeneration in progressive multiple sclerosis.

The neuron is the target of inflammatory demyelinating processes in multiple sclerosis (MS). In progressive MS, however, there is a gathering body of evidence indicating that molecular changes converge on mitochondria within neuronal cell bodies. The most reproducible change relates to mitochondrial respiratory chain complex deficiency, which compromises the capacity of neurons to generate ATP. The resulting energy failure state is coupled with an increase in demand for energy by the demyelinated axon, being particularly relevant to the long tracts such as corticospinal tracts with long projection axons. Recent work in our laboratory and that of our collaborators indicates the limited reflection of the mitochondria changes within neurons in experimental disease models. The mitochondrial changes within neuronal compartments are likely to offer novel targets for the improvement in neuronal function in patients with progressive MS.

Oxidative Injury and Iron Redistribution Are Pathological Hallmarks of Marmoset Experimental Autoimmune Encephalomyelitis.

Oxidative damage and iron redistribution are associated with the pathogenesis and progression of multiple sclerosis (MS), but these aspects are not entirely replicated in rodent experimental autoimmune encephalomyelitis (EAE) models. Here, we report that oxidative burst and injury as well as redistribution of iron are hallmarks of the MS-like pathology in the EAE model in the common marmoset. Active lesions in the marmoset EAE brain display increased expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (p22phox, p47phox, and gp91phox) and inducible nitric oxide synthase immunoreactivity within lesions with active inflammation and demyelination, coinciding with enhanced expression of mitochondrial heat-shock protein 70 and superoxide dismutase 1 and 2. The EAE lesion-associated liberation of iron (due to loss of iron-containing myelin) was associated with altered expression of the iron metabolic markers FtH1, lactoferrin, hephaestin, and ceruloplasmin. The enhanced expression of oxidative damage markers in inflammatory lesions indicates that the enhanced antioxidant enzyme expression could not counteract reactive oxygen and nitrogen species-induced cellular damage, as is also observed in MS brains. This study demonstrates that oxidative injury and aberrant iron distribution are prominent pathological hallmarks of marmoset EAE thus making this model suitable for therapeutic intervention studies aimed at reducing oxidative stress and associated iron dysmetabolism.

Incidence of plantar lateral foot pain before and after the use of trial metal wedges in lateral column lengthening.

BACKGROUND: One of the major concerns with lateral column lengthening (LCL) in symptomatic flatfoot deformity treatment is the risk of postoperative plantar lateral foot discomfort. We evaluated whether this risk can be minimized by using trial metal wedges. Using our study's evaluation tools, the incidence of postoperative plantar lateral foot discomfort before and after using trial metal wedges was determined. MATERIALS AND METHODS: The incidence of planter lateral foot pain after LCL was retrospectively assessed in 122 consecutive patients (132 feet) after they had undergone flatfoot reconstruction with LCL between 2001 and 2007. To determine if the incidence could be reduced, levels of pain or revision were compared before and after the use of trial metal wedges. The ratio of wedge size to preoperative radiographic calcaneal length was also determined. RESULTS: The overall incidence of plantar lateral discomfort was 11.2%. The incidence of pain or revision was lower after the introduction of trial metal wedges (6.3% compared to 14.7%), but did not reach significance (p = 0.084). There was no significant difference found in the ratio of the size of bone graft wedge to calcaneal length between the two groups (p = 0.805). CONCLUSION: The incidence of plantar lateral foot discomfort overall was 11.2% after LCL. We believe this risk may be reduced using trial metal wedges, properly judging eversion stiffness and carefully assessing the position of the foot intraoperatively.