Future Prospects for the Treatment of Graves' Hyperthyroidism and Eye Disease.

Although there are adequate therapies for Graves' hyperthyroidism, mild to moderate Graves' orbitopathy (GO) is usually treated symptomatically whereas definitive therapy is reserved for severe, vision-threatening GO. Importantly, none of the treatment regimens for Graves' disease used today are directed at the pathogenesis of the disease. Herein, we review some aspects of what is known about the pathogenesis of these 2 major components of Graves' disease, specifically the apparent important roles of the TSH and IGF-1 receptors, and thereafter describe future therapeutic approaches directed at these receptors. We propose that targeting these receptors will yield effective and better tolerated treatments for Graves' disease, especially for GO.

QTL involved in the modification of cyanidin compounds in black and red raspberry fruit.

Fruit from Rubus species are highly valued for their flavor and nutritive qualities. Anthocyanin content contributes to these qualities, and although many studies have been conducted to identify and quantify the major anthocyanin compounds from various Rubus species, the genetic control of the accumulation of these complex traits in Rubus is not yet well understood. The identification of the regions of the genome involved in the production of anthocyanins is an important first step in identifying the genes underlying their expression. In this study, ultra and high-performance liquid chromatography (UHPLC and HPLC) and two newly developed Rubus linkage maps were used to conduct QTL analyses to explore the presence of associations between concentrations of five anthocyanins in fruit and genotype. In total, 27 QTL were identified on the Rubus linkage maps, four of which are associated with molecular markers designed from transcription factors and three of which are associated with molecular markers designed from anthocyanin biosynthetic pathway candidate genes. The results of this study suggest that, while QTL for anthocyanin accumulation have been identified on six of seven Rubus linkage groups (RLG), the QTL on RLG2 and RLG7 may be very important for genetic control of cyanidin modification in Rubus.

Search for Dark Matter Axions with CAST-CAPP.

The CAST-CAPP axion haloscope, operating at CERN inside the CAST dipole magnet, has searched for axions in the 19.74 µeV to 22.47 µeV mass range. The detection concept follows the Sikivie haloscope principle, where Dark Matter axions convert into photons within a resonator immersed in a magnetic field. The CAST-CAPP resonator is an array of four individual rectangular cavities inserted in a strong dipole magnet, phase-matched to maximize the detection sensitivity. Here we report on the data acquired for 4124 h from 2019 to 2021. Each cavity is equipped with a fast frequency tuning mechanism of 10 MHz/ min between 4.774 GHz and 5.434 GHz. In the present work, we exclude axion-photon couplings for virialized galactic axions down to gaγγ = 8 × 10-14 GeV-1 at the 90% confidence level. The here implemented phase-matching technique also allows for future large-scale upgrades.

The Canadian Neuromuscular Disease Registry 2010-2019: A Decade of Facilitating Clinical Research Througha Nationwide, Pan-NeuromuscularDisease Registry.

We report the recruitment activities and outcomes of a multi-disease neuromuscular patient registry in Canada. The Canadian Neuromuscular Disease Registry (CNDR) registers individuals across Canada with a confirmed diagnosis of a neuromuscular disease. Diagnosis and contact information are collected across all diseases and detailed prospective data is collected for 5 specific diseases: Amyotrophic Lateral Sclerosis (ALS), Duchenne Muscular Dystrophy (DMD), Myotonic Dystrophy (DM), Limb Girdle Muscular Dystrophy (LGMD), and Spinal Muscular Atrophy (SMA). Since 2010, the CNDR has registered 4306 patients (1154 pediatric and 3148 adult) with 91 different neuromuscular diagnoses and has facilitated 125 projects (73 academic, 3 not-for-profit, 3 government, and 46 commercial) using registry data. In conclusion, the CNDR is an effective and productive pan-neuromuscular registry that has successfully facilitated a substantial number of studies over the past 10 years.

Magnetic resonance microscopy and immunohistochemistry of the CNS of the mutant SOD murine model of ALS reveals widespread neural deficits.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that primarily affects motor neurons and descending motor tracts of the CNS. We have evaluated the CNS of a murine model of familial ALS based on the over-expression of mutant human superoxide dismutase (mSOD; G93A) using magnetic resonance microscopy (MRM) and immunohistochemistry (IHC). Three-dimensional volumetric analysis was performed from 3D T2*-weighted images acquired at 17.6 T at isotropic resolutions of 40 mum. Compared to controls, mSOD mice had significant reductions in the volumes of total brain, substantia nigra, striatum, hippocampus, and internal capsule, with decreased cortical thickness in primary motor and somatosensory cortices. In the spinal cord, mSOD mice had significantly decreased volume of both the total grey and white matter; in the latter case, the volume change was confined to the dorsal white matter. Increased apoptosis, GFAP positive astrocytes, and/or activated microglia were observed in all those CNS regions that showed volume loss except for the hippocampus. The MRM findings in mSOD over-expressing mice are similar to data previously obtained from a model of ALS-parkinsonism dementia complex (ALS-PDC), in which neural damage occurred following a diet of washed cycad flour containing various neurotoxins. The primary difference between the two models involves a significantly greater decrease in spinal cord white matter volume in mSOD mice, perhaps reflecting variations in degeneration of the descending motor tracts. The extent to which several CNS structures are impacted in both murine models of ALS argues for a reevaluation of the nature of the pathogenesis of ALS since CNS structures involved in Parkinson's and Alzheimer's diseases appear to be affected as well.

Identification, distribution, and quantification of biominerals in a deciduous forest.

Biomineralization is a common process in most vascular plants, but poorly investigated for trees. Although the presence of calcium oxalate and silica accumulation has been reported for some tree species, the chemical composition, abundance, and quantification of biominerals remain poorly documented. However, biominerals may play important physiological and structural roles in trees, especially in forest ecosystems, which are characterized by nutrient-poor soils. In this context, our study aimed at investigating the morphology, distribution, and relative abundance of biominerals in the different vegetative compartments (foliage, branch, trunk, and root) of Fagus sylvatica L. and Acer pseudoplatanus L. using a combination of scanning electron microscopy and tomography analyses. Biomineral crystallochemistry was assessed by X-ray diffraction and energy-dispersive X-ray analyses, while calcium, silicon, and oxalic acid were quantified in the compartments and at the forest scale. Our analyses revealed that biominerals occurred as crystals or coating layers mostly in bark and leaves and were identified as opal, whewellite, and complex biominerals. In both tree species, opal was mostly found in the external tissues of trunk, branch, and leaves, but also in the roots of beech. In the stand, opal represents around 170 kg/ha. Whewellite was found to suit to conductive tissues (i.e., axial phloem parenchyma, vascular bundles, vessel element) in all investigated compartments of the two tree species. The shape of whewellite was prismatic and druses in beech, and almost all described shapes were seen in sycamore maple. Notably, the amount of whewellite was strongly correlated with the total calcium in all investigated compartments whatever the tree species is, suggesting a biologic control of whewellite precipitation. The amount of whewellite in the aboveground biomass of Montiers forest was more important than that of opal and was around 1170 kg/ha. Therefore, biominerals contribute in a substantial way to the biogeochemical cycles of silicon and calcium.

Intracellular Mg2+ concentrations following metabolic inhibition in opossum kidney cells.

Intracellular magnesium is associated with intracellular ATP concentrations as Mg-ATP2- and is involved with many enzymes in energy utilization. Intracellular Mg2+ has also been postulated to be involved with various Ca2+ actions. We determined adenine nucleotide concentrations (ATP, ADP and AMP) by HPLC and the associated changes in intracellular free Mg2+ ([Mg2+]i) by fluorescent methods in an epithelial cell line (opossum kidney cells). CCCP (a mitochondrial uncoupler), iodoacetate and amobarbital resulted in marked and rapid falls in [ATP]i with disproportionate increases in [Mg2+]i. These studies indicate that we are able to distinguish Mg2+ movements from Ca2+ by fluorescent techniques and suggests that intracellular regulation of [Mg2+]i is distinctive from those of [Ca2+]i. As CCCP plus amobarbital are reversible, we removed these inhibitors and tested the effect of Mg(2+)-availability on ATP depletion and recovery. The response of magnesium-depleted cells (basal [Mg2+]i 231 +/- 10 microM) following inhibitor-induced energy depletion and ATP recovery were similar to control cells. Accordingly, intracellular [Mg2+]i does not appear to be a limiting factor in ATP regeneration following removal of the chemical hypoxic insult. Finally, exogenous application of Na2ATP2- altered intracellular energy levels in normal and energy depleted cells but was without effect on [Mg2+]i. These studies suggest that intracellular ATP levels do not directly alter intracellular [Mg2+]i control and, in turn, intracellular free Mg2+ is not a limiting factor in ATP regeneration following energy depletion with chemical hypoxia.

Activation of protein kinase C by intracellular free calcium in the motoneuron cell line NSC-19.

The relationship between intracellular free calcium ([Ca2+]i) and the activation of protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) was investigated in the NSC-19 motoneuron cell line. Increased extracellular calcium ([Ca2+]o) up to 10 mM resulted in sustained elevations of [Ca2+]i. Control cell cultures (1.3 mM [Ca2+]o, [Ca2+]i = 83 +/- 17 nM) contained Ca2+- and PS/DO lipid-dependent PKC activity predominantly in the cytosol. However, elevation of [Ca2+]o up to 5 mM ([Ca2+]i = 232 +/- 24 nM) resulted in almost complete loss of cytosolic PKC activity. Cells incubated in 10 mM [Ca2+]o ([Ca2+]i = 365 +/- 13 nM) showed increased levels of both cytosolic and membrane PKC activity compared to control. These alterations in PKC activity appeared to be translocation-independent, since PKC protein levels were unchanged as demonstrated by Western blotting analysis. When cells were exposed to 25 or 50 mM [Ca2+]o, [Ca2+]i rose transiently to over 600 and 900 nM, respectively, and then returned to near basal values. Under these conditions, total PKC activity decreased, and increased amounts of the catalytic fragment of PKC, protein kinase M, were generated. Extracts from cells exposed to [Ca2+]o between 1.3 and 25 mM did not differ significantly in the levels of measurable CaMKII activity 10 min following the change in [Ca2+]o.

Amyotrophic lateral sclerosis: the involvement of intracellular Ca2+ and protein kinase C.

The neurodegenerative disease, amyotrophic lateral sclerosis (ALS), is characterized by the selective death of motoneurones and corticospinal tract neurones. Abnormalities in excitatory amino acids and their receptors, as well as disordered function of voltage-dependent Ca2+ channels and superoxide dismutase have been reported in ALS patients. Furthermore, the activity of protein kinase C (PKC), a Ca2+, phospholipid-dependent enzyme, is also substantially increased in tissue from ALS patients, suggesting that alterations in intracellular free Ca2+ may be central to many of the diverse pathogenic mechanisms potentially responsible for ALS as discussed here by Charles Krieger and colleagues. Increased PKC activity, in turn, may have direct or indirect effects on neuronal viability and influence the pathogenic process in ALS by modifying the phosphorylation of voltage-dependent Ca2+ channels, neurotransmitter receptors and structural proteins.

Increased phospho-adducin immunoreactivity in a murine model of amyotrophic lateral sclerosis.

Adducins alpha, beta and gamma are proteins that link spectrin and actin in the regulation of cytoskeletal architecture and are substrates for protein kinase C and other signaling molecules. Previous studies have shown that expressions of phosphorylated adducin (phospho-adducin) and protein kinase C are increased in spinal cord tissue from patients who died with amyotrophic lateral sclerosis, a neurodegenerative disorder of motoneurons and other cells. However, the distribution of phospho-adducin immunoreactivity has not been described in the mammalian spinal cord. We have evaluated the distribution of immunoreactivity to serine/threonine-dependent phospho-adducin at a region corresponding to the myristoylated alanine-rich C kinase substrate-related domain of adducin in spinal cords of mice over-expressing mutant human superoxide dismutase, an animal model of amyotrophic lateral sclerosis, and in control littermates. We find phospho-adducin immunoreactivity in control spinal cord in ependymal cells surrounding the central canal, neurons and astrocytes. Phospho-adducin immunoreactivity is localized to the cell bodies, dendrites and axons of some motoneurons, as well as to astrocytes in the gray and white matter. Spinal cords of mutant human superoxide dismutase mice having motoneuron loss exhibit significantly increased phospho-adducin immunoreactivity in ventral and dorsal horn spinal cord regions, but not in ependyma surrounding the central canal, compared with control animals. Increased phospho-adducin immunoreactivity localizes predominantly to astrocytes and likely increases as a consequence of the astrogliosis that occurs in the mutant human superoxide dismutase mouse with disease progression. These findings demonstrate increased immunoreactivity against phosphorylated adducin at the myristoylated alanine-rich C kinase substrate domain in a murine model of amyotrophic lateral sclerosis. As adducin is a substrate for protein kinase C at the myristoylated alanine-rich C kinase substrate domain, the increased phospho-adducin immunoreactivity is likely a consequence of protein kinase C activation in neurons and astrocytes of the spinal cord and evidence for aberrant phosphorylation events in mutant human superoxide dismutase mice that may affect neuron survival.

[Prospective study of the new oral anticoagulants in private practice: cautious optimism]. / Enquête prospective sur les nouveaux anticoagulants oraux en médecine libérale : un enthousiasme prudent.

BACKGROUND: The new direct oral anticoagulants (DOA) such as dabigatran, rivaroxaban or apixaban are an evolution in the management of patients requiring curative anticoagulation. However, behind the simplicity of prescribing and monitoring, several questions remain about their daily use. The aim of this prospective study was to measure the feelings of general practitioners (GP), angiologists (AP) and cardiologists (CP), potential prescribers of this new anticoagulant family. METHOD: Between December 2012 and May 2013, a questionnaire including five open questions and 11 questions using a positioning on an analogic visual scale (AVS 0 to 10) was subjected to GP, AP and CP in Alsace. RESULTS: Responses from 224 physicians (150 GP, 35 AP and 39 CP) were collected. Thus, 83% of GP, 83% of AP and 100% of CP were prescribers of DOA. However, among these prescribing doctors, the feeling was not the same and the trend of prescription was lower in GP (2.0 [1.1-3.2] AVS units) than in AP (3.1 [2.0-5.6]) and in CP (5.0 [1.2-8.7]) (P<0.0001 in multivariate analysis). The female doctors tended to prescribe DOA in younger patients than male doctors (respectively 66.1 [52.5-76.7] vs. 75.0 [65.7-81.0] years; P=0.004). The DOA were more considered as progress by AP and CP (respectively 7.8 [5.3-9.0] and 7.9 [7.0-8.7] AVSu) than by GP (6.1 [4.8-8.2] AVSu; P=0.02 in multivariate analysis). The answer about the eventual replacement of vitamin K antagonists by the DOA was very mixed whatever the practitioner group (5.1 [3.0-7.8] AVSu; P=0.139). The ease to use and the lack of biological monitoring were the main arguments leading to the prescription but the attitude of practitioners was very balanced by the lack of experience on the bleeding risk and the lack of available antidote. CONCLUSIONS: If the DOA are considered as an improvement for the physicians, the enthusiasm remains cautious whatever the type of practiced medicine. The results of clinical trials and the clinical experience should better appreciate the ongoing change in the field of anticoagulation.

Haematopoietic toxicity of radium-223 in patients with high skeletal tumour burden.

UNLABELLED: In patients with metastasized, castration resistant prostate cancer (mCRPC) treatment with radium-223 (Xofigo) is an attractive therapeutic option. In particular, patients with high tumour load seem to profit from this treatment in regard of survival and quality of live. Aim of this study was to stratify mCRPC patients according to a quantitative imaging marker derived from routine bone scans (EXINI bone) and analyze haematopoietic toxicity of Xofigo in these patients. PATIENTS, METHODS: Toxicity and oncologic outcome were investigated in a cohort of 14 patients with high tumour load. Additionally, based on a web survey, experience of toxicity in 41 high tumour load patients in Germany in 2014 was collected. RESULTS: In patients with a bone scan index (BSI) greater than 5, significant toxicity occurred in more patients than expected from the ALSYMPCA trial. This was associated with application of fewer cycles. Similar experiences have been made in other centers in Germany. Approximately 7% of these patients will need very long time or will not recover from grade ≥ 3 toxicity. CONCLUSION: Close follow-up of haematopoietic indices and, in case of toxicity, early termination of therapy is in particular necessary in late stage disease where limited bone marrow reserve is likely.

PTH and Vitamin D Repress DMP1 in Cementoblasts.

A complex feedback mechanism between parathyroid hormone (PTH), 1,25(OH)2D3 (1,25D), and fibroblast growth factor 23 (FGF-23) maintains mineral homeostasis, in part by regulating calcium and phosphate absorption/reabsorption. Previously, we showed that 1,25D regulates mineral homeostasis by repressing dentin matrix protein 1 (DMP1) via the vitamin D receptor pathway. Similar to 1,25D, PTH may modulate DMP1, but the underlying mechanism remains unknown. Immortalized murine cementoblasts (OCCM.30), similar to osteoblasts and known to express DMP1, were treated with PTH (1-34). Real-time quantitative polymerase chain reaction (PCR) and Western blot revealed that PTH decreased DMP1 gene transcription (85%) and protein expression (30%), respectively. PTH mediated the downregulation of DMP1 via the cAMP/protein kinase A (PKA) pathway. Immunohistochemistry confirmed the decreased localization of DMP1 in vivo in cellular cementum and alveolar bone of mice treated with a single dose (50 µg/kg) of PTH (1-34). RNA-seq was employed to further identify patterns of gene expression shared by PTH and 1,25D in regulating DMP1, as well as other factors involved in mineral homeostasis. PTH and 1,25D mutually upregulated 36 genes and mutually downregulated 27 genes by ≥2-fold expression (P ≤ 0.05). Many identified genes were linked with the regulation of bone/tooth homeostasis, cell growth and differentiation, calcium signaling, and DMP1 transcription. Validation of RNA-seq results via PCR array confirmed a similar gene expression pattern in response to PTH and 1,25D treatment. Collectively, these results suggest that PTH and 1,25D share complementary effects in maintaining mineral homeostasis by mutual regulation of genes/proteins associated with calcium and phosphate metabolism while also exerting distinct roles on factors modulating mineral metabolism. Furthermore, PTH may modulate phosphate homeostasis by downregulating DMP1 expression via the cAMP/PKA pathway. Targeting genes/proteins mutually governed by PTH and 1,25D may be a viable approach for designing new therapies for preserving mineralized tissue health.

Amyotrophic lateral sclerosis: fasting plasma levels of cysteine and inorganic sulfate are normal, as are brain contents of cysteine.

Recent reports suggest that amyotrophic lateral sclerosis (ALS) is caused by one or more unidentified neurotoxins that are poorly metabolized in patients to less toxic and more readily excreted compounds, and that a genetically determined defect in cysteine degradation and in inorganic sulfate production is the mechanism underlying a failure to metabolize xenobiotics normally in ALS. We measured concentrations of total cysteine and of inorganic sulfate in the plasma of age-matched groups of ALS patients and healthy control subjects and found no differences. L-Cysteine, a putative endogenous neurotoxin in ALS, was present in equal concentrations in autopsied brain from ALS patients and controls.

Phosphatidylinositol 3-kinase activity in murine motoneuron disease: the progressive motor neuropathy mouse.

A murine model of motoneuron disease, the pmn/pmn mouse, shows a reduction in the retrograde transport of fluorescent probes applied directly onto the cut end of sciatic nerve. Brain-derived neurotrophic factor (BDNF), when co-applied with fluorescent tracers, increases the number of retrograde labelled motoneurons. We demonstrate here that spinal cord tissue from pmn/pmn mice had significantly reduced phosphatidylinositol 3-kinase activity and expression in the particulate fraction compared to controls, without changes in the activities or expression of the downstream kinases, protein kinase B/Akt or Erk1. Systemic administration of BDNF augmented phosphatidylinositol 3-kinase specific activity in spinal cord tissue from pmn/pmn and control mice, with a greater elevation in the particulate fractions of pmn/pmn mice than in controls. We examined the effect of inhibitors of phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase on the retrograde labelling of motoneurons, 24h following the direct application of inhibitors and Fluorogold to the cut end of sciatic nerve in control and pmn/pmn mice (labelling index). The mitogen-activated protein kinase kinase inhibitor PD 98059 had no effect on the labelling index in control or pmn/pmn mice. In the absence of exogenous BDNF, phosphatidylinositol 3-kinase inhibitors reduced the number of labelled motoneurons in control mice, without changing the labelling index in pmn/pmn. Co-application of phosphatidylinositol 3-kinase inhibitors with BDNF to the cut end of sciatic nerve blocked the action of BDNF on retrograde labelling in pmn/pmn mice. These results indicate that the retrograde labelling of motoneurons is mediated by phosphatidylinositol 3-kinase-dependent and -independent pathways. In pmn/pmn mice, phosphatidylinositol 3-kinase activity in spinal neurons is below the level required for optimal retrograde labelling of motoneurons and labelling can be augmented by the administration of growth factors stimulating phosphatidylinositol 3-kinase activity. The data indicate that phosphatidylinositol 3-kinase activity is important in the uptake and/or retrograde transport of substances by motoneurons and is altered in this model of motoneuron diseases.

Effects of ATP and elevated K+ on K+ currents and intracellular Ca2+ in human microglia.

We have used whole-cell patch-clamp recordings and calcium microfluorescence measurements to study the effects of ATP and elevated external K+ on properties of human microglia. The application of ATP (at 0.1 mM) led to the activation of a transient inward non-selective cationic current at a cell holding potential of -60 mV and a delayed, transient expression of an outward K+ current activated with depolarizing steps applied from holding level. The ATP response included an increase in inward K+ conductance and a depolarizing shift in reversal potential as determined using a voltage ramp waveform applied from -120 to -50 mV. Fura-2 microspectrofluorescence measurements showed intracellular calcium to be increased following the application of ATP. This response was characterized by an initial transient phase, which persisted in Ca2+-free media and was due to release of Ca2+ from intracellular storage sites. The response had a later plateau phase, consistent with Ca2+ influx. In addition, ATP-induced changes in intracellular Ca2+ exhibited prominent desensitization. Elevated external K+ (at 40 mM) increased inward K+ conductance and shifted the reversal potential in the depolarizing direction, with no effect on outward K+ current or the level of internal Ca2+. The results of these experiments show the differential responses of human microglia to ATP and elevated K+, two putative factors associated with neuronal damage in the central nervous system.

Analysis of GABA(A)- and GABA(B)-receptor mediated effects on intracellular Ca(2+) in DRG hybrid neurones.

1. Using pharmacological analysis and fura-2 spectrofluorimetry, we examined the effects of gamma-aminobutyric acid (GABA) and related substances on intracellular Ca(2+) concentration ([Ca(2+)]i) of hybrid neurones, called MD3 cells. The cell line was produced by fusion between a mouse neuroblastoma cell and a mouse dorsal root ganglion (DRG) neurone. 2. MD3 cells exhibited DRG neurone-like properties, such as immunoreactivity to microtubule-associated protein-2 and neurofilament proteins. Bath applications of capsaicin and alpha, beta-methylene adenosine triphosphate reversibly increased [Ca(2+)]i. However, repeated applications of capsaicin were much less effective. 3. Pressure applications of GABA (100 microM), (Z)-3-[(aminoiminomethyl) thio] prop-2-enoic acid sulphate (ZAPA; 100 microM), an agonist at low affinity GABA(A)-receptors, or KCl (25 mM), transiently increased [Ca(2+)]i. 4. Bath application of bicuculline (100 nM - 100 microM), but not picrotoxinin (10 - 25 microM), antagonized GABA-induced increases in [Ca(2+)]i in a concentration-dependent manner (IC(50)=9.3 microM). 5. Ca(2+)-free perfusion reversibly abolished GABA-evoked increases in [Ca(2+)]i. Nifedipine and nimodipine eliminated GABA-evoked increases in [Ca(2+)]i. These results imply GABA response dependence on extracellular Ca(2+). 6. Baclofen (500 nM - 100 microM) activation of GABA(B)-receptors reversibly attenuated KCl-induced increases in [Ca(2+)]i in a concentration-dependent manner (EC(50)=1.8 microM). 2-hydroxy-saclofen (1 - 20 microM) antagonized the baclofen-depression of the KCl-induced increase in [Ca(2+)]i. 7. In conclusion, GABA(A)-receptor activation had effects similar to depolarization by high external K(+), initiating Ca(2+) influx through high voltage-activated channels, thereby transiently elevating [Ca(2+)]i. GABA(B)-receptor activation reduced Ca(2+) influx evoked by depolarization, possibly at Ca(2+)-channel sites in MD3 cells.

Distribution and levels of insulin-like growth factor (IGF-I and IGF-II) and insulin receptor binding sites in the spinal cords of amyotrophic lateral sclerosis (ALS) patients.

The structurally related peptides, insulin and insulin-like growth factors (IGF-I and IGF-II), have neurotrophic properties and potentially could be of therapeutic value in human neurodegenerative disorders. In this study, we compared the anatomical distribution of [125I]IGF-I, [125I]IGF-II and [125I]insulin binding sites in thoracic spinal cords from patients who died of amyotrophic lateral sclerosis (ALS) and normal controls. For these three ligands, the greatest amounts of specific binding were located in the deeper layers of the dorsal horn > intermediate zone > lamina X > ventral horn > superficial layers of the dorsal horn > white matter of the spinal cord. Highly significant (P < 0.001) increases in the density of [125I]IGF-I and [125I]IGF-II binding were apparent in various laminae of the cord of ALS patients with increased binding being particularly evident in the ventral horn and the intermediate zone. Significant (P < 0.05) increases were also seen in lamina X and in the dorsal horn. In contrast, no significant differences in [125I]insulin binding were observed between ALS and control spinal cords. Taken together, these data reveal significant increases in both [125I]IGF-I and [125I]IGF-II binding levels in the spinal cords of ALS patients albeit to different extents. These findings may be of relevance for future therapeutic strategies aimed at slowing the progression of this chronic neurodegenerative disease, as recently suggested by the beneficial therapeutic effects of an IGF-I treatment in ALS patients.

Differential changes of bax, caspase-3 and p21 mRNA expression after transient focal brain ischemia in the rat.

Recent studies of transient focal ischemia have focused interest on apoptotic mechanisms of neuronal cell death involving constitutive pro-apoptotic proteins. The finding of specific patterns of novel gene expression might indicate the activation of pro-apoptotic genes in previously ischemic areas. Thus, we investigated gene expression for the pro-apoptotic regulators, Bax and caspase-3, after transient focal brain ischemia, together with the p53-regulated cell cycle inhibitor, p21/WAF1/CIP1. Reversible occlusion of the middle cerebral artery for 2 h was carried out in halothane-anesthetized rats using the poly-L-lysine coated filament method. In situ hybridization was performed at 0, 1, 3, 6 h and 1, 3 and 7 d of recirculation and in sham controls. Radioactive antisense probes served for detection of bax, p21 and caspase-3 mRNAs on brain sections, and quantitative film autoradiography was combined with image-averaging techniques. Bax mRNA tended to decline after focal brain ischemia within 1 d. p21 mRNA was upregulated with a perifocal pattern at 3 h and 1 d after ischemia whereas the ischemic regions themselves failed to show significant upregulation. Caspase-3 mRNA was elevated in the resistant dorsomedial cortex at 1 d. A pro-apoptotic pattern of novel gene expression, involving Bax and caspase-3, was not observed after transient focal brain ischemia. Rather, the perifocal expression of p21 and caspase-3 mRNAs observed at 1 d after ischemia points to reactive changes in resistant brain areas.

[3H]phorbol 12,13-dibutyrate/PKC binding in thoracic spinal cord: no change in amyotrophic lateral sclerosis.

[3H]phorbol 12,13-dibutyrate ([3H]PDBu), a selective ligand for various protein kinase C isozymes (PKC), was used to investigate the distribution of [3H]PDBu/PKC binding sites in thoracic spinal cords of patients who died with amyotrophic lateral sclerosis (ALS) and subjects free of neurological disease. In controls, binding of [3H]PDBu was mostly concentrated in the substantia gelatinosa and to a lesser extent in other dorsal horn regions (laminae III and IV). [3H]PDBu binding sites were also present in the ventral horn and laminae X but in somewhat lower quantities. The distribution of [3H]PDBu binding in thoracic spinal cords of patients who died with ALS was unchanged compared to controls and no significant differences were observed in the amount of specific binding in ALS. The present results on the distribution of [3H]PDBu binding sites in human spinal cord are consistent with previous studies of [3H]PDBu binding in rodents indicating that high levels of PKC are present in the dorsal horn. The results also suggest that levels of PKC isozymes are not altered in ALS spinal cord.