Fusun Baumann

Lactate promotes glioma migration by TGF-β2–dependent regulation of matrix metalloproteinase-2

Lactate dehydrogenase type A (LDH-A) is a key metabolic enzyme catalyzing pyruvate into lactate and is excessively expressed by tumor cells. Transforming growth factor-beta2 (TGF-beta2) is a key regulator of invasion in high-grade gliomas, partially by inducing a mesenchymal phenotype and by remodeling the extracellular matrix. In this study, we tested the hypothesis that lactate metabolism regulates TGF-beta2-mediated migration of glioma cells. Small interfering RNA directed against LDH-A (siLDH-A) suppresses, and lactate induces, TGF-beta2 expression, suggesting that lactate metabolism is strongly associated with TGF-beta2 in glioma cells. Here we demonstrate that TGF-beta2 enhances expression, secretion, and activation of matrix metalloproteinase-2 (MMP-2) and induces the cell surface expression of integrin alpha(v)beta(3) receptors. In spheroid and Boyden chamber migration assays, inhibition of MMP-2 activity using a specific MMP-2 inhibitor and blocking of integrin alpha(v)beta(3) abrogated glioma cell migration stimulated by TGF-beta2. Furthermore, siLDH-A inhibited MMP2 activity, leading to inhibition of glioma migration. Taken together, we define an LDH-A-induced and TGF-beta2-coordinated regulatory cascade of transcriptional regulation of MMP-2 and integrin alpha(v)beta(3). This novel interaction between lactate metabolism and TGF-beta2 might constitute a crucial mechanism for glioma migration.

Use of respiratory function tests to predict survival in amyotrophic lateral sclerosis

Respiratory function tests (RFTs) are commonly used as a measure of progression in ALS. This study assessed the ability of various RFTs to predict survival in ALS patients. Subjects with ALS had one or more measurements of seated and supine FVC, maximal inspiratory pressure (MIP) and maximal expiratory pressure (MEP). Kaplan-Meier (KM) analysis was used to determine whether patients with abnormal RFTs had shorter survival than those with normal RFTs. The sensitivity and specificity of RFTs as predictors of two-year survival were calculated from receiver operating characteristic (ROC) curves. With KM analysis, subjects with abnormal values of seated FVC, supine FVC, MIP and MEP had significantly reduced survival compared to subjects with normal values. With ROC curves, a normal supine FVC was highly predictive for two-year survival and had superior sensitivity over seated FVC. Slower rates of decline in seated or supine FVC were strong predictors of two-year survival. Our study demonstrates that respiratory function measurements are useful to predict survival in ALS patients. We show that measurements of FVC in the supine position are worth including in the assessment of respiratory function in ALS.

Direct evidence of intra- and interhemispheric corticomotor network degeneration in amyotrophic lateral sclerosis: An automated MRI structural connectivity study

Although the pathogenesis of amyotrophic lateral sclerosis (ALS) is uncertain, there is mounting neuroimaging evidence to suggest a mechanism involving the degeneration of multiple white matter (WM) motor and extramotor neural networks. This insight has been achieved, in part, by using MRI Diffusion Tensor Imaging (DTI) and the voxelwise analysis of anisotropy indices, along with DTI tractography to determine which specific motor pathways are involved with ALS pathology. Automated MRI structural connectivity analyses, which probe WM connections linking various functionally discrete cortical regions, have the potential to provide novel information about degenerative processes within multiple white matter (WM) pathways. Our hypothesis is that measures of altered intra- and interhemispheric structural connectivity of the primary motor and somatosensory cortex will provide an improved assessment of corticomotor involvement in ALS. To test this hypothesis, we acquired High Angular Resolution Diffusion Imaging (HARDI) scans along with high resolution structural images (sMRI) on 15 patients with clinical evidence of upper and lower motor neuron involvement, and 20 matched control participants. Whole brain probabilistic tractography was applied to define specific WM pathways connecting discrete corticomotor targets generated from anatomical parcellation of sMRI of the brain. The integrity of these connections was interrogated by comparing the mean fractional anisotropy (FA) derived for each WM pathway. To assist in the interpretation of results, we measured the reproducibility of the FA summary measures over time (6months) in control participants. We also incorporated into our analysis pipeline the evaluation and replacement of outlier voxels due to head motion and physiological noise. When assessing corticomotor connectivity, we found a significant reduction in mean FA within a number of intra- and interhemispheric motor pathways in ALS patients. The abnormal intrahemispheric pathways include the corticospinal tracts involving the left and right precentral gyri (lh.preCG, rh.preCG) and brainstem (bs); right postcentral gyrus (rh.postCG) and bs; lh.preCG and left posterior cingulate gyrus (lh.PCG); rh.preCG and right posterior cingulate gyrus (rh.PCG); and the rh.preCG and right paracentral gyrus (rh.paraCG). The abnormal interhemispheric pathways included the lh.preCG and rh.preCG; lh.preCG and rh.paraCG; lh.preCG and right superior frontal gyrus (rh.supFG); lh.preCG and rh.postCG; rh.preCG and left paracentral gyrus (lh.paraCG); rh.preCG and left superior frontal gyrus (lh.supFG); and the rh.preCG and left caudal middle frontal gyrus (lh.caudMF). The reproducibility of the measurement of these pathways was high (variation less than 5%). Maps of the outlier rejection voxels, revealed clusters within the corpus callosum and corticospinal projections. This finding highlights the importance of correcting for motion artefacts and physiological noise when studying clinical populations. Our novel findings, many of which are consistent with known pathology, show extensive involvement and degeneration of multiple corticomotor pathways in patients with upper and lower motor neuron signs and provide support for the use of automated structural connectivity techniques for studying neurodegenerative disease processes.

The relationship between Bayesian motor unit number estimation and histological measurements of motor neurons in wild-type and SOD1G93A mice

OBJECTIVE To assess the relationship between Bayesian MUNE and histological motor neuron counts in wild-type mice and in an animal model of ALS. METHODS We performed Bayesian MUNE paired with histological counts of motor neurons in the lumbar spinal cord of wild-type mice and transgenic SOD1(G93A) mice that show progressive weakness over time. We evaluated the number of acetylcholine endplates that were innervated by a presynaptic nerve. RESULTS In wild-type mice, the motor unit number in the gastrocnemius muscle estimated by Bayesian MUNE was approximately half the number of motor neurons in the region of the spinal cord that contains the cell bodies of the motor neurons supplying the hindlimb crural flexor muscles. In SOD1(G93A) mice, motor neuron numbers declined over time. This was associated with motor endplate denervation at the end-stage of disease. CONCLUSION The number of motor neurons in the spinal cord of wild-type mice is proportional to the number of motor units estimated by Bayesian MUNE. In SOD1(G93A) mice, there is a lower number of estimated motor units compared to the number of spinal cord motor neurons at the end-stage of disease, and this is associated with disruption of the neuromuscular junction. SIGNIFICANCE Our finding that the Bayesian MUNE method gives estimates of motor unit numbers that are proportional to the numbers of motor neurons in the spinal cord supports the clinical use of Bayesian MUNE in monitoring motor unit loss in ALS patients.

Structural hemispheric asymmetries in the human precentral gyrus hand representation.

The superior region of the precentral gyrus (preCG) is known to be actively involved with hand function and has been proposed as a possible neural correlate of handedness. To test this hypothesis, we used a combined voxel-based morphometric (VBM) asymmetry analysis of structural MRI, along with diffusion MRI (dMRI) tractography to investigate laterality indices of corticomotor white matter (WM) pathways, based on measures of fractional anisotropy (FA). The relationship between measures of motor performance and FA laterality indices was also investigated. In a cohort of 14 right-handed healthy participants, the VBM asymmetry analysis revealed an area within the preCG associated with hand representation. The tractography analysis revealed that this region possessed a number of major WM intrahemispheric connections to the brain stem, thalamus, cerebellum, postcentral, caudal middle and superior frontal, and superior and inferior parietal corticomotor regions. Within the corticospinal tracts, we found FA was significantly higher in the left hemisphere compared with the right. Furthermore, significant correlations were found between FA asymmetry measures projecting from this region, namely corticospinal tracts and those connecting the postcentral gyri, with grip strength and finger-tapping performance, respectively. A number of the motor pathways projecting from this region also exhibited leftward asymmetry of FA distributions. The findings from this study highlight the role of the left motor cortex in skilled motor performance and provide a framework for the study of the relationship between handedness and preCG hand representation in larger normative populations.

Quantitative studies of lower motor neuron degeneration in amyotrophic lateral sclerosis: evidence for exponential decay of motor unit numbers and greatest rate of loss at the site of onset

OBJECTIVE To use our Bayesian method of motor unit number estimation (MUNE) to evaluate lower motor neuron degeneration in ALS. METHODS In subjects with ALS we performed serial MUNE studies. We examined the repeatability of the test and then determined whether the loss of MUs was fitted by an exponential or Weibull distribution. RESULTS The decline in motor unit (MU) numbers was well-fitted by an exponential decay curve. We calculated the half life of MUs in the abductor digiti minimi (ADM), abductor pollicis brevis (APB) and/or extensor digitorum brevis (EDB) muscles. The mean half life of the MUs of ADM muscle was greater than those of the APB or EDB muscles. The half-life of MUs was less in the ADM muscle of subjects with upper limb than in those with lower limb onset. CONCLUSIONS The rate of loss of lower motor neurons in ALS is exponential, the motor units of the APB decay more quickly than those of the ADM muscle and the rate of loss of motor units is greater at the site of onset of disease. SIGNIFICANCE This shows that the Bayesian MUNE method is useful in following the course and exploring the clinical features of ALS.

CMAP decrement in ALS.

CMAP DECREMENT IN ALS In previous studies the compound muscle action potential (CMAP) decrement in amyotrophic lateral sclerosis (ALS) subjects was thought to arise from degenerating neurons, from failure of conduction down axon branches, or at the neuromuscular junctions of regenerating neurons undergoing collateral sprouting.2,3,5 To explore the significance of a decrement in ALS subjects, we gave a short train of 2 Hz stimulation in 27 subjects (mean age 54 years, 15 male) with definite ALS.1 The CMAP amplitude was measured in response to five stimuli at supramaximal stimulus intensity from either the median, ulnar, or peroneal nerves on one to four occasions using techniques previously described.3 CMAP decrement was defined as a greater than 10% difference between the first and fifth CMAP negative peak amplitude. A total of 106 studies were obtained from 60 ulnar, 35 median, and 11 peroneal nerves with mean CMAP amplitudes of 4.2 mV, 3.7 mV, and 2.4 mV, respectively. A decrement was observed in studies of 12/35 median (34%), 6/60 ulnar (10%), and 1/11 peroneal (9%) nerves. Using a lower limit for normal CMAP amplitude (median nerve, 4 mV; ulnar nerve, 6 mV; and peroneal nerve, 2 mV), a reduced CMAP amplitude was variably associated with a decrement (Fig. 1A,B). There was a negative correlation between the size of the CMAP decrement and the amplitude of the CMAP (r: 0.59, P-value 0.0001). Except for one subject, a normal amplitude ulnar, median or peroneal CMAP was not associated with a decrement, i.e., the decrement was only present when there was significant loss of motor units. In a subgroup of 20 subjects, serial studies were obtained from 30 nerves. Three ALS subjects had a decrement on the initial study, and the CMAP decrement in these subjects continued to be greater than 10% on subsequent studies over 2–13 months (Fig. 1C). Of the 17 ALS subjects who lacked a decrement on the first study, six subjects showed a decrement on a subsequent study. The CMAP decrement detected in nine subjects had increased by an average of 10.8% despite a fall in CMAP amplitude by an average of 24.3% on subsequent studies. Three ALS subjects with a CMAP decrement in one nerve had studies from other nerves that did not show a decrement. No apparent clinical difference in survival was noted between the ALS subjects with and without a decrement. This is consistent with a previous report.6 Our serial studies were performed to find evidence to distinguish between the mechanisms that may cause decrement in ALS subjects. The ALS subjects were studied over a reasonable period in which dying motor neurons would likely have disappeared, and this is supported by the observation of reduced CMAP amplitudes over time. If the decrement arose from individual dying motor neurons, then we would have similarly expected the decrement to disappear once that neuron was no longer present. Our finding that the decrement persists despite falling CMAP amplitude favors the theory that a CMAP decrement arises from regenerating neurons. It is doubtfulwhether the decrement is associated with reduced survival.4,6

EFFECTS OF PROLONGED REPETITIVE STIMULATION OF MEDIAN, ULNAR AND PERONEAL NERVES

It is important to know the effects of prolonged repetitive nerve stimulation (RNS) when it is used in neurophysiological studies. RNS with up to 100 supramaximal stimuli was given to the median, ulnar, and peroneal nerves of normal subjects and the ulnar nerves of subjects with early amyotrophic lateral sclerosis (ALS), recording evoked compound muscle action potentials (CMAPs). In all nerves, there was a decline in the CMAP area and a decrease in CMAP duration. For the peroneal nerve there was a decline in the CMAP amplitude, but a similar decline was not seen in the median or ulnar nerves. Cooling of the muscles resulted in decrement of both the amplitude and area with RNS. In ALS subjects, CMAP amplitude and area both declined after RNS of the ulnar nerve. In this study we describe the changes in CMAP with prolonged RNS among commonly tested normal nerves. Our findings have important implications with regard to RNS. Muscle Nerve, 2010

Versican isoform V1 regulates proliferation and migration in high-grade gliomas

Versican is a large chondroitin sulphate proteoglycan produced by several tumor cell types, including high-grade gliomas. Increased expression of distinct versican isoforms in the extracellular matrix plays a role in tumor cell growth, adhesion and migration. We have recently shown that transforming growth factor (TGF-beta)2, an important modulator of glioma invasion, interacts with versican isoforms V0/V1 during malignant progression of glioma in vitro. However, the distinct subtype of versican that modulates these effects could not be specified. Here, we show that transient down-regulation of V1 by siRNA leads to a significant reduction of proliferation and migration in glioblastoma cell lines and glioblastoma progenitor cells, whereas tumor cell attachment stays unaffected. We conclude that V1 plays a predominant role in modulating central pathophysiological mechanisms as proliferation and migration in glioblastoma. Considering that TGF-beta is a master regulator of glioma pathophysiology, and that V0/1 is induced by TGF-beta2, therapeutic regulation of V1 may induce meaningful effects on glioma cell migration not only in vitro, but also in vivo.

Biomarkers of disease in a case of familial lower motor neuron ALS

Abstract ALS is a fatal disease with variable clinical course. There is no single reliable marker of disease progression. Sufficient records were available to study the case history of four family members with the uncommon G93V SOD1 mutation. Distal lower motor neuron (LMN) involvement occurred in all family members with onset from 30 to 51 years of age, with progression over more than six years. Between 2002 and 2009, we used electrophysiology as a biomarker to study disease progression in one patient, assessing the number of motor units in three nerves from different limbs. The loss of motor units showed an exponential decline with different half-lives in different nerves. Diffusion tractography was compared with a control to assess upper motor neuron (UMN) involvement and showed asymmetric evidence of abnormalities of the corticospinal tracts, providing evidence of central involvement despite the absence of UMN signs.