Occurrence of two molecular forms of human acid sphingomyelinase

Human acid sphingomyelinase (ASM) hydrolyses sphingomyelin to ceramide and phosphocholine. Metabolic studies on COS-1 cells transfected with ASM cDNA revealed the occurrence of an enzymically inactive precursor which is differentially processed to two predominant native glycoprotein forms: a 70 kDa polypeptide corresponding to human urinary protein and a 57 kDa form. Formation of these potentially active forms was shown to be restricted to distinct compartments. Maturation of the ASM precursor to a predominant 70 kDa form occurs exclusively inside acidic organelles, whereas variable amounts of 57 kDa ASM are detectable immediately after biosynthesis. Metabolic labelling of transfected COS-1 cells with [32P]Pi further suggests that this form obviously does not carry oligomannosylphosphate residues, in contrast with the mature lysosomal ASM. In order to verify that this early form of active ASM results from co-post-translational proteolysis of the ASM precursor and not from the use of different translation-initiation sites on the ASM mRNA, appropriate 5'-mutagenized cDNA constructs were transiently expressed. These results clearly indicate that the first potential in-frame AUG is exclusively used for translation initiation in vivo and that deletion of the proposed signal sequence for endoplasmic reticulum import completely eliminates the ability of the translation product to enter the vacuolar apparatus. As there are two different subcellular sites of maturation of the ASM precursor, and intracellular targeting of the two processed forms appears to be different, the two ASM proteins may contribute to distinct physiological functions

Peripheral Nerve Ultrasound for the Differentiation between ALS, Inflammatory, and Hereditary Polyneuropathies

Background and Objectives: Ultrasound (US) is a non-invasive tool for the in vivo detection of peripheral nerve alterations. Materials and Methods: In this study, we applied nerve US to assist the discrimination between the spectrum of amyotrophic lateral sclerosis (ALS, n = 11), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP, n = 5), and genetically confirmed Charcot–Marie–Tooth disease (CMT, n = 5). All participants and n = 15 controls without neurological diseases underwent high-resolution US of the bilateral tibial nerve. The nerve cross-sectional area (CSA) and nerve microvascular blood flow were compared between the groups and related to cerebrospinal fluid (CSF) measures, clinical symptoms, and nerve conduction studies. The analyses are part of a larger multimodal study on the comparison between US and 7 Tesla (7T) magnetic resonance neurography (MRN). Results: The patients and controls were matched with respect to their demographical data. CMT had the longest disease duration, followed by CIDP and ALS. CSA was related to age, weight, and disease duration. CSA was larger in CMT and CIDP compared to ALS and controls. The blood flow was greatest in CIDP, and higher than in CMT, ALS, and controls. In ALS, greater CSA was correlated with greater CSF total protein and higher albumin quotient. The US measures did not correlate with clinical scores or nerve conduction studies in any of the subgroups. Conclusion: Our results point towards the feasibility of CSA and blood flow to discriminate between ALS, CIDP, and CMT, even in groups of small sample size. In ALS, larger CSA could indicate an inflammatory disease subtype characterized by reduced blood–nerve barrier integrity. Our upcoming analysis will focus on the additive value of 7T MRN in combination with US to disentangle the spectrum between more inflammatory or more degenerative disease variants among the disease groups

Cognitive Profiles of Amyotrophic Lateral Sclerosis Differ in Resting-State Functional Connectivity: An fMRI Study

BackgroundHalf of all amyotrophic lateral sclerosis-frontotemporal spectrum disorder (ALS-FTSD) patients are classified as cognitively impaired, of which 10% have frontotemporal dementia (FTD), and an additional 40% suffer from a frontotemporal syndrome not severe enough to be described as dementia (cognitively impaired/ALSci). As changes in cerebral function measured by resting-state magnet resonance imaging (rs-fMRI) are known in ALS, we investigated whether group differences in resting-state functional connectivity (RSFC) networks could be observed between ALS patients with different cognitive profiles against healthy controls (HC). Furthermore, we correlated cognition and motor functioning with network connectivity.MethodsHealthy controls, 69, and 97 ALS patients underwent functional MRI scanning and cognitive assessment. The ALS patients were categorized as non-impaired (ALSni; n = 68), cognitively impaired (ALSci; n = 21), and ALS-FTD (n = 8). Group differences in connectivity of the default mode network (DMN), motor network (MN), and ventral attention network (VAN) were investigated using a full-factorial model; correlations between global cognitive performance, shifting, and motor symptom severity were established using Pearson’s correlation.ResultsAt a liberal alpha level of uncorrected p < 0.005 and a cluster size exceeding 20 voxels, we found widespread decreases in functional connectivity in all three networks when comparing ALS patients to HC. Similar patterns of hypoconnectivity in the bilateral motor cortices and frontotemporal emerged when comparing the ALSci and ALS-FTD patients to those not cognitively impaired. Hyperconnectivity in the DMN temporal gyrus correlated with worse global cognition; moreover, hyperconnectivity in the VAN thalamus, insula, and putamen correlated with worse shifting ability. Better-preserved motor function correlated with higher MN connectivity. Only the motor-related effects prevailed at a more conservative significance level of pFDR< 0.001.ConclusionResting-state functional connectivity differs between cognitive profiles of ALS and is directly associated with clinical presentation, specifically with motor function, and cognitive shifting

CSF Neurofilament Light Chain Levels in Primary Progressive MS: Signs of Axonal Neurodegeneration

Objectives: Elevated neurofilament light chain (NFL) levels within the cerebrospinal fluid (CSF) are a biomarker representing axonal neurodegeneration in rapid progressive neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). It is unclear to what extent the levels of NFL increase in the CSF (CSF-NFL) in a chronic neuroinflammatory process with axonal neurodegeneration, as found in primary progressive multiple sclerosis (PPMS).Methods: We used a multicenter approach to statistically compare CSF-NFL levels between PPMS patients (n = 50), ALS patients (n = 50), and healthy controls (n = 50). Clinical findings, including disease duration, expanded disability status scale (EDSS), electrophysiological recordings such as visual evoked potentials or spinal and cerebral MRI, and previously administered treatment were selected as experimental parameters retrospectively.Results: Median [range] CSF-NFL concentrations in PPMS patients were significantly higher than in the controls [1724 (799–4275) pg/ml vs. 1202 (612–2934) pg/ml, p = 0.015], and significantly lower compared to ALS patients [1724 (799–4275) pg/ml vs. 10238 (2610–35138) pg/ml, p < 0.001]. There was no correlation between CSF-NFL and disease duration (p = 0.5), EDSS (p = 0.2) or treatment (p = 0.3).Conclusion: We conclude that CSF-NFL may mirror the proposed slow axonal degeneration in PPMS, but does not reflect the disease severity

Casein kinase I δ/ɛ phosphorylates topoisomerase IIα at serine-1106 and modulates DNA cleavage activity

We previously reported that phosphorylation of topoisomerase (topo) IIα at serine-1106 (Ser-1106) regulates enzyme activity and sensitivity to topo II-targeted drugs. In this study we demonstrate that phosphorylation of Ser-1106, which is flanked by acidic amino acids, is regulated in vivo by casein kinase (CK) Iδ and/or CKIɛ, but not by CKII. The CKI inhibitors, CKI-7 and IC261, reduced Ser-1106 phosphorylation and decreased formation of etoposide-stabilized topo II–DNA cleavable complex. In contrast, the CKII inhibitor, 5,6-dichlorobenzimidazole riboside, did not affect etoposide-stabilized topo II–DNA cleavable complex formation. Since, IC261 specifically targets the Ca2+-regulated isozymes, CKIδ and CKIɛ, we examined the effect of down-regulating these enzymes on Ser-1106 phosphorylation. Down-regulation of these isozymes with targeted si-RNAs led to hypophosphorylation of the Ser-1106 containing peptide. However, si-RNA-mediated down-regulation of CKIIα and α′ did not alter Ser-1106 phosphorylation. Furthermore, reduced phosphorylation of Ser-1106, observed in HRR25 (CKIδ/ɛ homologous gene)-deleted Saccharomyces cerevisiae cells transformed with human topo IIα, was enhanced following expression of human CKIɛ. Down-regulation of CKIδ and CKIɛ also led to reduced formation of etoposide stabilized topo II–DNA cleavable complex. These results provide strong support for an essential role of CKIδ/ɛ in phosphorylating Ser-1106 in human topo IIα and in regulating enzyme function

Mitochondrial abnormalities and low grade inflammation are present in the skeletal muscle of a minority of patients with amyotrophic lateral sclerosis; an observational myopathology study

BACKGROUND Amyotrophic lateral sclerosis (ALS) is a primary progressive neurodegenerative disease characterised by neuronal loss of lower motor neurons (in the spinal cord and brainstem) and/or upper motor neurons (in the motor cortex) and subsequent denervation atrophy of skeletal muscle. AIM A comprehensive examination of muscle pathology from a cohort of clinically confirmed ALS patients, including an investigation of inflammation, complement activation, and deposition of abnormal proteins in order to compare them with findings from an age-matched, control group. MATERIAL AND METHODS 31 muscle biopsies from clinically confirmed ALS patients and 20 normal controls underwent a comprehensive protocol of histochemical and immunohistochemical stains, including HLA-ABC, C5b-9, p62, and TDP-43. RESULTS Neurogenic changes were confirmed in 30/31 ALS cases. In one case, no neurogenic changes could be detected. Muscle fibre necrosis was seen in 5/31 cases and chronic mononuclear inflammatory cell infiltration in 5/31 (2 of them overlapped with those showing muscle necrosis). In four biopsies there was an increase in the proportion of cytochrome oxidase (COX) negative fibres (2-3%). p62 faintly stained cytoplasmic bodies in eight cases and none were immunoreactive to TDP-43. CONCLUSION This large series of muscle biopsies from patients with ALS demonstrates neurogenic atrophy is a nearly uniform finding and that mild mitochondrial abnormalities and low-grade inflammation can be seen and do not rule out the diagnosis of ALS. These findings could lend support to the notion that ALS is a complex and heterogeneous disorder

Interactions between Casein Kinase Iε (CKIε) and Two Substrates from Disparate Signaling Pathways Reveal Mechanisms for Substrate-Kinase Specificity

Members of the Casein Kinase I (CKI) family of serine/threonine kinases regulate diverse biological pathways. The seven mammalian CKI isoforms contain a highly conserved kinase domain and divergent amino- and carboxy-termini. Although they share a preferred target recognition sequence and have overlapping expression patterns, individual isoforms often have specific substrates. In an effort to determine how substrates recognize differences between CKI isoforms, we have examined the interaction between CKIepsilon and two substrates from different signaling pathways.CKIepsilon, but not CKIalpha, binds to and phosphorylates two proteins: Period, a transcriptional regulator of the circadian rhythms pathway, and Disheveled, an activator of the planar cell polarity pathway. We use GST-pull-down assays data to show that two key residues in CKIalpha's kinase domain prevent Disheveled and Period from binding. We also show that the unique C-terminus of CKIepsilon does not determine Dishevelled's and Period's preference for CKIepsilon nor is it essential for binding, but instead plays an auxillary role in stabilizing the interactions of CKIepsilon with its substrates. We demonstrate that autophosphorylation of CKIepsilon's C-terminal tail prevents substrate binding, and use mass spectrometry and chemical crosslinking to reveal how a phosphorylation-dependent interaction between the C-terminal tail and the kinase domain prevents substrate phosphorylation and binding.The biochemical interactions between CKIepsilon and Disheveled, Period, and its own C-terminus lead to models that explain CKIepsilon's specificity and regulation

Activation of BKCa Channels in Zoledronic Acid-Induced Apoptosis of MDA-MB-231 Breast Cancer Cells

BACKGROUND: Zoledronic acid, one of the most potent nitrogen-containing biphosphonates, has been demonstrated to have direct anti-tumor and anti-metastatic properties in breast cancer in vitro and in vivo. In particular, tumor-cell apoptosis has been recognized to play an important role in the treatment of metastatic breast cancer with zoledronic acid. However, the precise mechanisms remain less clear. In the present study, we investigated the specific role of large conductance Ca(2+)-activated potassium (BK(Ca)) channel in zoledronic acid-induced apoptosis of estrogen receptor (ER)-negative MDA-MB-231 breast cancer cells. METHODOLOGY/PRINCIPAL FINDINGS: The action of zoledronic acid on BK(Ca) channel was investigated by whole-cell and cell-attached patch clamp techniques. Cell apoptosis was assessed with immunocytochemistry, analysis of fragmented DNA by agarose gel electrophoresis, and flow cytometry assays. Cell proliferation was investigated by MTT test and immunocytochemistry. In addition, such findings were further confirmed with human embryonic kidney 293 (HEK293) cells which were transfected with functional BK(Ca) α-subunit (hSloα). Our results clearly indicated that zoledronic acid directly increased the activities of BK(Ca) channels, and then activation of BK(Ca) channel by zoledronic acid contributed to induce apoptosis in MDA-MB-231 cells. The possible mechanisms were associated with the elevated level of intracellular Ca(2+) and a concomitant depolarization of mitochondrial membrane potential (Δψm) in MDA-MB-231 cells. CONCLUSIONS: Activation of BK(Ca) channel was here shown to be a novel molecular pathway involved in zoledronic acid-induced apoptosis of MDA-MB-231 cells in vitro