Roberto Navarrete

Lack of myostatin results in excessive muscle growth but impaired force generation

The lack of myostatin promotes growth of skeletal muscle, and blockade of its activity has been proposed as a treatment for various muscle-wasting disorders. Here, we have examined two independent mouse lines that harbor mutations in the myostatin gene, constitutive null (Mstn−/−) and compact (Berlin High Line, BEHc/c). We report that, despite a larger muscle mass relative to age-matched wild types, there was no increase in maximum tetanic force generation, but that when expressed as a function of muscle size (specific force), muscles of myostatin-deficient mice were weaker than wild-type muscles. In addition, Mstn−/− muscle contracted and relaxed faster during a single twitch and had a marked increase in the number of type IIb fibers relative to wild-type controls. This change was also accompanied by a significant increase in type IIB fibers containing tubular aggregates. Moreover, the ratio of mitochondrial DNA to nuclear DNA and mitochondria number were decreased in myostatin-deficient muscle, suggesting a mitochondrial depletion. Overall, our results suggest that lack of myostatin compromises force production in association with loss of oxidative characteristics of skeletal muscle.

Umbilical cord blood mesenchymal stromal cells are neuroprotective and promote regeneration in a rat optic tract model

Exploitation of the ability of stem cells to protect damaged neuronal tissue may be a more viable strategy than cell replacement for repair of the central nervous system (CNS). In this study we assessed the capacity of human umbilical cord blood (hUCB)-derived mesenchymal stromal cells (MSCs) to protect and promote regeneration of axotomised neurons within the rat optic system. The optic tract of neonatal rats was transected at the level of the lateral geniculate nucleus, and MSCs were introduced into the lesion site. MSCs survived well up to 2 weeks after grafting, and did not migrate significantly or differentiate. In the presence of MSC grafts, host axonal processes were found to be present in the lesion site, and there was stimulation of an endogenous neural precursor population. Four weeks after grafting, retrograde tracer experiments demonstrated that grafted MSCs, as well as cells of a human fibroblast line, exerted a neuroprotective effect, rescuing a significant percentage of axotomised retinal ganglion cells (RGCs). Further experiments with retrograde and anterograde tracers strongly indicated that MSCs could also promote re-growth of axotomised RGCs to their target, the superior colliculus (SC). Further analysis showed that hUCB-derived MSCs secreted several immunomodulatory and neurotrophic factors in vitro, including TGFbeta1, CNTF, NT-3 and BDNF, which are likely to play a role in neuroprotection. Our data indicate that hUCB-derived MSCs may be an easily accessible, widely available source of cells that can contribute towards neural repair through rescue and regeneration of injured neurons.

Widespread loss of neuronal populations in the spinal ventral horn in sporadic motor neuron disease. A morphometric study

The cytopathology and loss of neurons was studied in 7670 neurons from the ventral horn of the third lumbar segment of the spinal cord of six sporadic motor neuron disease (MND) patients compared with 7568 neurons in seven age matched control subjects. A modified Tomlinson et al. [Tomlinson BE, Irving D, Rebeiz JJ. Total numbers of limb motor neurones in the human lumbosacral cord and an analysis of the accuracy of various sampling procedures. J Neurol Sci 1973;20:313-27] sampling procedure was used for neuronal counts. The ventral horn was divided in quadrants. Neuronal populations were also classified by the maximum cell diameter through the nucleolus. There was widespread loss of neurons in all quadrants of the ventral horn in MND. Size distribution histograms showed similar neuron loss across all populations of neurons. The dorsomedial quadrant contains almost exclusively interneurons and the ventrolateral quadrant mostly motor neurons. The cytopathology of neurons in the dorsomedial quadrant and of large motorneurons in the ventrolateral quadrant MND was similar. In the dorsomedial quadrant, neuron loss (56.7%) was similar to the loss of large motor neurons in the ventrolateral quadrant (64.4%). The loss of presumed motor neurons and interneurons increased with increased disease duration. There was no evidence that loss of presumed interneurons occurred prior, or subsequent, to loss of motor neurons. We conclude that, in sporadic MND, all neuronal populations in the ventral horn are affected and that interneurons are involved to a similar extent and in parallel with motor neurons, as reported in the G86R transgenic mouse model of familial MND. The increasing evidence of loss of neurons other than motor neurons in MND suggests the need for revising the concept of selective motor neuron vulnerability.

Characterization of mesenchymal stromal cells derived from full-term umbilical cord blood

BACKGROUND Multipotent mesenchymal stromal cells (MSC) are of interest for their potential to repair bone and cartilage, and also their immunosuppressive properties. Umbilical cord blood (UCB) is reported to contain MSC, and therefore may be a useful source of these cells for clinical applications. METHODS We evaluated protocols for isolating MSC from UCB and characterized the surface phenotype, differentiation potential and immunoregulatory properties of the cells obtained. RESULTS Ten of 25 UCB units processed yielded MSC-like colonies, with depletion of lineage+ cells providing a higher efficiency. Only two of the cultures could be expanded satisfactorily; the remainder failed to proliferate. One culture generated transformed lines that were grossly aneuploid, had up-regulated TERT transcripts and had lost CD90 expression and the capacity to differentiate. The two propagated UCB-MSC lines were similar to those from bone marrow but were not identical to each other, with differences seen in expression of surface markers and cytoskeletal proteins. Both underwent osteogenesis, but at different rates and to different degrees, while neither generated adipocytes. When added as a third party to a mixed lymphocyte culture, both suppressed proliferation. DISCUSSION MSC-like cells can be isolated from UCB, but at low efficiencies, and they exhibit a variety of morphologies, growth rates and differentiation potentials and can transform in culture.

Increased incidence of gap junctional coupling between spinal motoneurones following transient blockade of NMDA receptors in neonatal rats.

Neonatal rat motoneurones are electrically coupled via gap junctions and the incidence of this coupling declines during postnatal development. The mechanisms involved in this developmental regulation of gap junctional communication are largely unknown. Here we have studied the role of NMDA receptor‐mediated glutamatergic synaptic activity in the regulation of motoneurone coupling. Gap junctional coupling was demonstrated by the presence of graded, short latency depolarising potentials following ventral root stimulation, and by the transfer of the low molecular weight tracer Neurobiotin to neighbouring motoneurones. Sites of close apposition between the somata and/or dendrites of the dye‐coupled motoneurones were identified as potential sites of gap junctional coupling. Early postnatal blockade of the NMDA subtype of glutamate receptors using the non‐competitive antagonist dizocilpine maleate (MK801) arrested the developmental decrease in electrotonic and dye coupling during the first postnatal week. These results suggest that the postnatal increase in glutamatergic synaptic activity associated with the onset of locomotion promote the loss of gap junctional connections between developing motoneurones.

Heat shock protein 27 rescues motor neurons following nerve injury and preserves muscle function

Heat shock proteins (HSPs) are a family of ubiquitously expressed proteins that are up-regulated in response to a range of stresses and play an important role in cellular defence mechanisms. In previous studies, we demonstrated that overexpression of heat shock protein 27 (HSP27) in transgenic mice has significant cytoprotective properties in vivo, reducing caspase-3-mediated cell death in the hippocampus associated with limbic seizures and reducing infarct size in cardiac ischaemia. In motor neurons, HSP27 is also implicated as a survival promoting factor; however, it remains to be established whether HSP27 is able to exert long-term neuroprotective effects following neonatal nerve injury. We now show that, following neonatal nerve crush, HSP27 overexpression in vivo provides a substantial rescue of motor neurons 5-6 months following nerve injury. Furthermore, in vivo isometric tension recordings demonstrate that surviving motor neurons were able to regenerate, resulting in a 90% improvement (P < 0.0005) in motor unit number in HSP27 mice. Moreover, this increase in motor unit number was associated with improved muscle weight, muscle force, contractile speeds, and histochemical markers of muscle activity. These properties of HSP27 therefore have considerable potential for improving long-term muscle function in motor neuron disorders.

Early expression of glycine and GABAA receptors in developing spinal cord neurons. Effects on neurite outgrowth

Using fluorometric and immunocytochemical techniques, we found that high glycine concentrations or blockade of glycine receptors increases neurite outgrowth in developing mouse spinal cord neurons. Glycine- and GABA(A)-activated currents were demonstrated during applications of glycine and GABA (50-100 microM) in 5 days in vitro (DIV) neurons. Long application (> or =10 min) of 100 microM glycine desensitized the membrane response by more than 95%. Application of glutamate in the absence of external Mg(2+), at several membrane potentials, did not produce any detectable membrane response in these cells. Immunocytochemical studies with NR1 and GluR1 antibodies showed a delayed appearance of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors respectively. Spontaneous synaptic activity was readily observed in 5 DIV neurons. The use of various receptor antagonists (strychnine, bicuculline, DL-2-amino-5-phosphonovalerate [APV], 6-cyano-7-nitroquinoxaline-2,3-dione [CNQX]) revealed that this activity was predominantly glycinergic, and to a smaller extent, GABAergic. In the presence of bicuculline, APV and CNQX, we detected abundant spontaneous depolarizing potentials which often reached the action potential threshold. Further evidence for functional synaptic activity was provided by the detection of co-localization of gephyrin and synaptophysin at 5 DIV using confocal microscopy. Fluorometric studies with Fluo-3, a Ca(2+) indicator, in 5 DIV cultures showed the presence of spontaneous fluctuations associated with tetrodotoxin-sensitive synaptic events. The number of neurons displaying these fluctuations was significantly increased (>100%) when the cells were bathed in a strychnine-containing solution. On the other hand, these synaptically mediated Ca(2+) events were blocked by the co-application of strychnine and bicuculline. This suggests that glycine and GABA(A) receptors provide a fundamental regulation of both neuronal excitability and intracellular Ca(2+) at this early time of development.The neurotrophic effects of agonists and antagonists for glycine, GABA(A) and glutamate receptors were examined in neurons cultured for 2 or 5 DIV. From all the agonists used, only high concentrations of glycine increased neurite outgrowth in 5 DIV neurons. We found that strychnine also increased neurite outgrowth, whereas tetrodotoxin (1 microM), nimodipine (4 microM) and bicuculline (20 microM) completely blocked it. On the other hand, APV (50 microM) and CNQX (20 microM) were unable to affect neurite outgrowth. These data suggest that spinal glycine receptors depress neurite outgrowth by shunting neuronal excitability. Outgrowth induction possibly results from the enhanced activity found after the inhibition of glycinergic activity. We postulate that this resets the intracellular calcium at a concentration that favors neurite outgrowth.

Analysis of neural potential of human umbilical cord blood–derived multipotent mesenchymal stem cells in response to a range of neurogenic stimuli

We investigated the neurogenic potential of full‐term human umbilical cord blood (hUCB)–derived multipotent mesenchymal stem cells (MSCs) in response to neural induction media or coculture with rat neural cells. Phenotypic and functional changes were assessed by immunocytochemistry, RT‐PCR, and whole‐cell patch‐clamp recordings. Naive MSCs expressed both mesodermal and ectodermal markers prior to neural induction. Exposure to retinoic acid, basic fibroblast growth factor, or cyclic adenosine monophosphate (cAMP) did not stimulate neural morphology, whereas exposure to dibutyryl cAMP and 3‐isobutyl‐1‐methylxanthine stimulated a neuron‐like morphology but also appeared to be cytotoxic. All protocols stimulated increases in expression of the neural precursor marker nestin, but expression of mature neuronal or glial markers MAP2 and GFAP was not observed. Nestin expression increases were serum level dependent. Electrophysiological properties of MSCs were studied with whole‐cell patch‐clamp recordings. The MSCs possessed no ionic currents typical of neurons before or after neural induction protocols. Coculture of hUCB‐derived MSCs and rat neural cells induced some MSCs to adopt an astrocyte‐like morphology and express GFAP protein and mRNA. Our data suggest hUCB‐derived MSCs do not transdifferentiate into mature functioning neurons in response to the above neurogenic protocols; however, coculture with rat neural cells led to a minority adopting an astrocyte‐like phenotype.

Changes in expression of NMDA receptor subunits in the rat lumbar spinal cord following neonatal nerve injury

The vulnerability of motoneurones to glutamate has been implicated in neurological disorders such as amyotrophic lateral sclerosis but it is not known whether specific receptor subtypes mediate this effect. In order to investigate this further, the expression of N‐methyl‐ d‐aspartate (NMDA) receptor subunits was studied during the first three post‐natal weeks when motoneurones are differentially vulnerable to injury following neonatal nerve crush compared to the adult. Unilateral nerve crush was carried out at day 2 after birth (P2) which causes a decrease of 66% in motoneurone number by 14 days (P14). To study receptor expression in identified motoneurones, serial section analysis was carried out on retrogradely labelled common peroneal (CP) motoneurones by combined immunocytochemistry and in situ hybridization (ISH). mRNA levels were also quantified in homogenates from lumbar spinal cords in which the side ipsilateral to the crush was separated from the contralateral side. The NR1 subunit of the NMDA receptor was widely distributed in the spinal cord being expressed most strongly in motoneurone somata particularly during the neonatal period (P3–P7). The NR2 subunits were also expressed at higher levels in the somata and dendrites of neonatal motoneurones compared to older animals. NR2B mRNA was expressed at low to moderate levels throughout the studied period whereas NR2A mRNA levels were low until P21. Following unilateral nerve crush, an initial decrease in NR1 mRNA occurred at one day after nerve crush (P3) in labelled CP motoneurones ipsilateral to the crush which was followed by a significant increase in NR1 subunit expression at 5 days post‐injury. This increase was bilateral although reaching greater significance ipsilateral to the crush compared with sham‐operated animals. A significant increase in NR1 and NR2B mRNA post injury was also detected in spinal cord homogenates. In addition, the changes in levels of NR1 and NR2B mRNA were reflected by comparable bilateral changes at P7 in receptor protein determined by quantitative immunocytochemical analysis of NR1 and NR2 subunit expression in identified CP motoneurones indicating a co‐ordinated regulation of receptor subunits in response to injury.

Neurite outgrowth in developing mouse spinal cord neurons is modulated by glycine receptors.

The effect of glycine receptor activation on neurite outgrowth and survival was studied in 5 DIV (days in vitro) spinal neurons. These neurons were depolarized by spontaneous synaptic activity and by glycine, but not by glutamate. These responses were accompanied by increases in intracellular calcium concentration measured with Indo-1 and Fluo-3. Glycine (100 μM, 48h) increased (46 ± 6%) the number of primary neurites and total neuritic length. This effect was mediated by synaptic activity and calcium influx because TTX (1 μM) and nimodipine (4 μM) blocked the stimulatory effect of glycine. Neuronal survival, on the other hand, was not affected. This study shows for the first time the modulatory effect of glycine receptors on spinal neuron development.