Formation and characterisation of neuromuscular junctions between hiPSC derived motoneurons and myotubes.

Striated skeletal muscle cells from humans represent a valuable source for in vitro studies of the motoric system as well as for pathophysiological investigations in the clinical settings. Myoblasts can readily be grown from human muscle tissue. However, if muscle tissue is unavailable, myogenic cells can be generated from human induced pluripotent stem cells (hiPSCs) preferably without genetic engineering. Our study aimed to optimize the generation of hiPSCs derived myogenic cells by employing selection of CD34 positive cells and followed by distinct, stepwise culture conditions. Following the expansion of CD34 positive single cells under myogenic cell culture conditions, serum deprived myoblast-like cells finally fused and formed multinucleated striated myotubes that expressed a set of key markers for muscle differentiation. In addition, these myotubes contracted upon electrical stimulation, responded to acetylcholine (Ach) and were able to generate action potentials. Finally, we co-cultured motoneurons and myotubes generated from identical hiPSCs cell lines. We could observe the early aggregation of acetylcholine receptors in muscle cells of immature co-cultures. At later stages, we identified and characterised mature neuromuscular junctions (NMJs). In summary, we describe here the successful generation of an iPS cell derived functional cellular system consisting of two distinct communicating cells types. This in vitro co-culture system could therefore contribute to research on diseases in which the motoneurons and the NMJ are predominantly affected, such as in amyotrophic lateral sclerosis or spinal muscular atrophy.

CAPE (caffeic acid phenethyl ester)-based propolis extract (Bio 30) suppresses the growth of human neurofibromatosis (NF) tumor xenografts in mice.

Dysfunction of the NF1 gene coding a RAS GAP is the major cause of neurofibromatosis type 1 (NF1), whereas neurofibromatosis type 2 (NF2) is caused primarily by dysfunction of the NF2 gene product called merlin that inhibits directly PAK1, an oncogenic Rac/CDC42-dependent Ser/Thr kinase. It was demonstrated previously that PAK1 is essential for the growth of both NF1 and NF2 tumors. Thus, several anti-PAK1 drugs, including FK228 and CEP-1347, are being developed for the treatment of NF tumors. However, so far no effective NF therapeutic is available on the market. Since propolis, a very safe healthcare product from bee hives, contains anticancer ingredients called CAPE (caffeic acid phenethyl ester) or ARC (artepillin C), depending on the source, both of which block the oncogenic PAK1 signaling pathways, its potential therapeutic effect on NF tumors was explored in vivo. Here it is demonstrated that Bio 30, a CAPE-rich water-miscible extract of New Zealand (NZ) propolis suppressed completely the growth of a human NF1 cancer called MPNST (malignant peripheral nerve sheath tumor) and caused an almost complete regression of human NF2 tumor (Schwannoma), both grafted in nude mice. Although CAPE alone has never been used clinically, due to its poor bioavailability/water-solubility, Bio 30 contains plenty of lipids which solubilize CAPE, and also includes several other anticancer ingredients that seem to act synergistically with CAPE. Thus, it would be worth testing clinically to see if Bio 30 and other CAPE-rich propolis are useful for the treatment of NF patients.

Seasonal closures as a measure of trawling effort control in two Mediterranean trawling grounds: effects on epibenthic communities.

Within the framework of ecosystem-based management, we focused on the use of seasonal closures as effective measures to minimise the degradation of benthic communities by trawling. These closures imply the complete cessation of trawling fleet activity and are commonly used in the Mediterranean to reduce the annual fishing effort, with the ultimate goal of effective resource management. In this study, we aimed to investigate how epibenthic communities respond to seasonal closures. The potential benefits of short-term annual closures in two Mediterranean fishing grounds were evaluated by analysing changes in community structure and composition that were linked to the closure. A decrease of faunal abundance was observed with the resumption of fishing activity after the closure at both fishing grounds. Remarkably, results indicated that some large and mobile fauna were able to respond to these closures. We concluded that the currently planned closures are too short to benefit benthic communities.

Serine proteases purified from sera of patients with amyotrophic lateral sclerosis (ALS) induce contrasting cytopathology in murine motoneurones to IgG.

Affinity purified IgG from sera of patients with amyotrophic lateral sclerosis (ALS) is claimed to enhance transmitter release, induce apoptotic death of cultured motoneurones, and elicit a distinctive cytopathology with raised Ca(2+) in mouse motoneurones. An alternative hypothesis attributes these events to serine proteases in ALS sera. To test this, motoneurones in BALB/c mice injected intraperitoneally with plasminogen affinity purified from sera of ALS patients and healthy controls were analysed using immunochemical and ultrastructural morphometric methods. The responses were validated in motoneurones of mice injected with commercially purified plasminogen, tissue plasminogen activator (tPA), or plasmin. Motoneurones in non-injected mice had normal morphology and ultrastructure without evidence of electron-dense degeneration. Purified plasminogen from both ALS patients and healthy controls, evoked electron-dense motoneurone degeneration, as did commercially purified plasminogen and tPA. The common cytopathology comprised disruption and distension of Nissl body rough endoplasmic reticulum, cytoplasmic polyribosomal proliferation, and significant Ca(2+) enhancement in mitochondria. By contrast, using affinity purified serum immunoglobulins, ALS-IgG but not IgG from healthy or disease controls, elicited necrosis, with 30% of ALS-IgGs tested evoking electron-dense degeneration in 40% of motoneurones. The primary cytopathology was extensive swelling of Golgi endoplasmic reticulum and mitochondria, with enhancement of Ca(2+) in Golgi endoplasmic reticulum and presynaptic boutons. We conclude that serine proteases purified from sera of ALS patients elicits a distinctive cytopathology and pattern of Ca(2+) enhancement in motoneurones different from that found on passive transfer of affinity purified ALS-IgG.

Characterization of matrix metalloproteinases in denervated muscle.

In a nerve crush model of denervation, we examined muscle matrix metalloproteinase (MMP) expression, localization and activity. In normal muscle, MMP mRNA levels were low, and immunohistochemically MMPs were distributed around the muscle fibre with MMPs-3, -7 and -9 also staining at the neuromuscular junction. Seven days after nerve crush, muscle MMP immunoreactivity, especially MMP-12 and MMP-14, became irregularly distributed. At 20 days reinnervation of the muscle was observed, and some restitution of the normal pattern of immunoreactivity was noted concomitant with a higher level of MMP mRNA expression. In situ zymography showed that MMP activity was very weak in normal muscle whereas it was increased up to 40 days following denervation. Our results suggest that MMPs in muscle are involved in the tissue changes following denervation. Further experiments are required to test the hypothesis that MMP inhibition may be beneficial in protecting muscle from excessive remodelling following denervation and therefore improve reinnervation.

Time-course expression of CNS inflammatory, neurodegenerative tissue repair markers and metallothioneins during experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model for multiple sclerosis (MS). EAE and MS are characterized by CNS inflammation, demyelination and neurodegeneration. The inflammatory response occurring within the CNS leads to glial activation, dysfunction and death, as well as axonal damage and neurological deficit. Although the pathogenic mechanisms involved in EAE/MS are not well understood, accumulating data suggest that oxidative stress plays a major role in lesion development, and contributes to axonal dysfunction and degeneration. Metallothionein-I and -II are anti-inflammatory, neuroprotective, antioxidant proteins expressed during EAE and MS, in which they might play a protective role. The present study aimed to describe the expression profile of a group of inflammatory, neurodegenerative and tissue repair markers as well as metallothioneins during proteolipid protein-induced EAE, and to establish the time-relationships these molecules had during EAE. Interestingly, we found two marker expression profiles. In the first, marker expression increased as clinical signs worsened and reverted to baseline expression during recovery; in the second, marker expression increased at a later point during relapse, peaked at highest clinical score, and remained elevated throughout recovery. Of note, metallothionein expression was found to be related to the second profile, which would suggest that metallothionein proteins are implicated in the clinical recovery of EAE and perhaps these antioxidant proteins may provide therapeutic benefits in MS.

ALS-IgG-induced selective motor neurone apoptosis in rat mixed primary spinal cord cultures.

There is evidence that in sporadic amyotrophic lateral sclerosis (ALS) immunological mechanisms may be involved in the pathophysiology of the disease. We tested whether purified IgG from ALS patients induce cell death in rat mixed primary spinal cord cultures and compared this with the effect of IgG purified from patients with Guillain-Barré syndrome (GBS) or from healthy donors. Treatment with ALS-IgG increases caspase-3 apoptosis when compared with control IgG or with GBS-IgG, but does not induce death by necrosis. Because ALS is characterized by the selective loss of motor neurones, we next assessed the differential effect of ALS-IgG on motor neurones or astrocytes. We showed, semiquantitatively, that motor neurones are more susceptible to apoptosis when cultures were treated with ALS-IgG compared with control-IgG. In conclusion, we have demonstrated in primary spinal cord cultures that IgG from patients with ALS induces apoptosis selectively in motor neurones, and that the caspase-3 pathway is involved. This suggests that immunological mechanisms may contribute to the selective loss of motor neurones in ALS.

The pro and the active form of matrix metalloproteinase-9 is increased in serum of patients with amyotrophic lateral sclerosis.

Pro and active-matrix metalloproteinase-9 (MMP-9) was measured in sera from patients with amyotrophic lateral sclerosis (ALS), Guillain-Barre syndome (GBS), and healthy subjects. Both forms of MMP-9 were elevated in sera of ALS and GBS patients, compared with healthy controls. It has been postulated that elevated MMP-9 reflects damage to peripheral nerve and muscle. This possibility was investigated in sera, and tissue extracts of sciatic nerves and muscle from mice 5 and 12 days after axotomy of the sciatic nerve. Pro-MMP-9 was elevated in sera and extracts of damaged nerve and muscle, suggesting such damage may be followed by elevated pro-MM9-9 in sera. Active MMP-9 was only elevated in the sera. However, in situ activation of MMP-9 is tightly regulated and localised, and probably difficult to demonstrate by ELISA, resulting in a short half-life active MMP-9, implying any active MMP-9 in the serum may have a more immediate origin than injured muscle or nerve, for example circulating blood cells.

Characterisation of matrix metalloproteinases and the effects of a broad-spectrum inhibitor (BB-1101) in peripheral nerve regeneration.

The effect of treatment with a broad-spectrum inhibitor (BB1101) of the matrix metalloproteinases (MMPs) on nerve regeneration and functional recovery after nerve crush was examined. Drug treatment had no effect on latency but from 63 days the compound muscle action potential was significantly increased and was no different to that in the sham-operated controls at 72 days. Levels of MMP mRNA expression, and the localisation and activity of MMP proteins, were examined in rats for a 2 month period following a nerve crush injury, and compared with sham-operated controls. The mRNA of all the MMPs studied was up-regulated by 5-10 days after nerve crush, and they remained up-regulated for 40-63 days, except for MMP-9 which was down-regulated by 10 days. MMP immunoreactivity was localised to Schwann cells, macrophages and endothelial cells, and with the exception of membrane type 1-MMP (MT1-MMP), it was more intense after nerve crush compared with sham-operated controls. Regenerating axons showed immunoreactivity for MMP-2 and MMP-3. In situ zymography confirmed that the activity of MMPs in the nerve was increased following crush but that the activity was greatly reduced in rats treated with BB-1101. Thus despite the inhibition of MMPs by BB-1101, the drug did not appear to essentially affect nerve degeneration or regeneration following nerve crush but that it could be beneficial in promoting the more effective reinnervation of muscles possibly by actions at the level of the muscles.

Passive transfer of purified IgG from patients with amyotrophic lateral sclerosis to mice results in degeneration of motor neurons accompanied by Ca2+ enhancement.

It has been reported that immunoglobulins (IgG) in sera of patients with amyotrophic lateral sclerosis (ALS) kill cultured motoneurones (MN), but whether they also cause MN degeneration in vivo is unclear. To test this, protein-A affinity purified and dialysed IgGs were prepared from sera of 44 ALS patients without paraproteinemias, 20 healthy controls and 15 disease controls. Control and ALS-IgGs were injected intraperitoneally into groups of mice for 5 consecutive days and examined at day 8. IgG was localised immunocytochemically and spinal MN were characterised histologically and ultrastructurally and by comparative counts of Ca(2+) containing organelles revealed with oxylate-pyroantimonate histochemistry. ELISA revealed no differences in IgG concentration between ALS patients and control subjects. Immunocytochemistry showed IgG was present in MN of mice injected with control or ALS-IgG, but densitometry showed immunostaining in MN was stronger in mice injected with ALS-IgG. Compared to MN of non-injected mice, control-IgG-treated mice showed near normal MN morphology and numbers of Ca(2+)-containing organelles. Disease control IgGs evoked negligible or minor morphological changes according to disease, but normal numbers of Ca(2+) containing organelles. Ultrastructurally, about 70% of ALS-derived IgGs induced a population of MN with electron lucent cytoplasm, distended Golgi, disrupted Nissl and mitochondria (i.e., necrosis). However 30% of ALS-IgGs additionally induced electron-dense degeneration in 40% of the MN. These MN exhibited shrinkage, condensed nuclear chromatin and ill-defined nuclear membranes and resembled preliminary stages of apoptosis. We conclude that passive transfer of ALS-derived, but not control IgGs, does result in MN degeneration in the recipient mice. This appears to be associated with abnormal calcium homeostasis, but the exact target of ALS-IgG remains conjectural, and the possibilities are discussed.

Relation between cerebral blood flow and extracellular glucose in rat striatum during mild hypoxia and hyperoxia.

Rats were exposed to mild hyperoxia and hypoxia by the administration of oxygen/air and nitrogen/air mixtures through plastic tubing held close to their snouts for periods of 3 min. Changes in tissue oxygen were monitored at an implanted carbon paste electrode; local cerebral blood flow (rCBF) at an implanted platinum electrode using the hydrogen clearance technique; extracellular brain glucose at an implanted glucose oxidase-based biosensor and changes in lactate were measured using microdialysis. The nitrogen/air mixture led to a decrease in tissue oxygen, an increase in rCBF, a decrease in extracellular glucose, and an increase in lactate. The oxygen/air mixture led to an increase in tissue oxygen and extracellular glucose but no change in lactate or rCBF. The effects in unanaesthetised rats were compared with those in rats given 350 mg/kg chloral hydrate. The increase in lactate was greater in unanaesthetised than anaesthetised rats. The results show a dissociation between changes in rCBF and extracellular glucose and suggest that changes in oxygen affect utilisation rather than supply of glucose to the extracellular compartment.

Stimulated release of lactate in freely moving rats is dependent on the uptake of glutamate.

1. Physiological stimulation of neuronal activity induces an increase in extracellular lactate. Experiments were designed to determine the role of the reuptake of neuronally released glutamate in lactate delivery to the extracellular compartment. 2. In vivo microdialysis was used in freely moving rats. The lactate concentration in striatal dialysate was assayed using an enzyme-based on-line assay at 1 min intervals. Drugs were given locally through the dialysis probe. 3. The extracellular concentration of lactate, determined using the zero net flux method, was 346 +/- 21 microM. 4. Induced grooming caused a maximal increase in lactate concentration in striatal dialysate of 58 +/- 10%. 5. Administration of 100 microM glutamate caused a transient increase in dialysate lactate concentration of 72 +/- 17%. 6. A 20 min infusion of the glutamate uptake blockers beta-D,L-threohydroxyaspartate (THA) or pirrolidine-2-4-dicarboxylate (PDC) produced an increase in basal lactate, which was sustained in response to THA and transient in response to PDC. 7. Grooming induced during the infusion of PDC produced no significant increase in lactate. 8. Grooming induced after the infusion of the glutamate uptake blockers gave rise to a reduced increase in lactate. 9. These results support the hypothesis that stimulated release of lactate is dependent on the uptake of glutamate.