Molecular classification of amyotrophic lateral sclerosis by unsupervised clustering of gene expression in motor cortex

AbstractAmyotrophic lateral sclerosis (ALS) is a rapidly progressive and ultimately fatal neurodegenerative disease, caused by the loss of motor neurons in the brain and spinal cord. Although 10% of ALS cases are familial (FALS), the majority are sporadic (SALS) and probably associated to a multifactorial etiology. Currently there is no cure or prevention for ALS. A prerequisite to formulating therapeutic strategies is gaining understanding of its etio-pathogenic mechanisms. In this study we analyzed whole-genome expression profiles of 41 motor cortex samples of control (10) and sporadic ALS (31) patients. Unsupervised hierarchical clustering was able to separate control from SALS patients. In addition, SALS patients were subdivided in two different groups that were associated to different deregulated pathways and genes, some of which were previously associated to familiar ALS. These experiments are the first to highlight the genomic heterogeneity of sporadic ALS and reveal new clues to its pathogenesis and potential therapeutic targets

The BH4 domain of Bcl-XL rescues astrocyte degeneration in amyotrophic lateral sclerosis by modulating intracellular calcium signals

Collective evidence indicates that motor neuron degeneration in amyotrophic lateral sclerosis (ALS) is non-cell-autonomous and requires the interaction with the neighboring astrocytes. Recently, we reported that a subpopulation of spinal cord astrocytes degenerates in the microenvironment of motor neurons in the hSOD1G93A mouse model of ALS. Mechanistic studies in vitro identified a role for the excitatory amino acid glutamate in the gliodegenerative process via the activation of its inositol 1,4,5-triphosphate (IP3)-generating metabotropic receptor 5 (mGluR5). Since non-physiological formation of IP3 can prompt IP3 receptor (IP3R)-mediated Ca2+ release from the intracellular stores and trigger various forms of cell death, here we investigated the intracellular Ca2+ signaling that occurs downstream of mGluR5 in hSOD1G93A-expressing astrocytes. Contrary to wild-type cells, stimulation of mGluR5 causes aberrant and persistent elevations of intracellular Ca2+ concentrations ([Ca2+]i) in the absence of spontaneous oscillations. The interaction of IP3Rs with the anti-apoptotic protein Bcl-XL was previously described to prevent cell death by modulating intracellular Ca2+ signals. In mutant SOD1-expressing astrocytes, we found that the sole BH4 domain of Bcl-XL, fused to the protein transduction domain of the HIV-1 TAT protein (TAT-BH4), is sufficient to restore sustained Ca2+ oscillations and cell death resistance. Furthermore, chronic treatment of hSOD1G93A mice with the TAT-BH4 peptide reduces focal degeneration of astrocytes, slightly delays the onset of the disease and improves both motor performance and animal lifespan. Our results point at TAT-BH4 as a novel glioprotective agent with a therapeutic potential for AL

Neuroinflammatory targets and treatments for epilepsy validated in experimental models

A large body of evidence that has accumulated over the past decade strongly supports the role of inflammation in the pathophysiology of human epilepsy. Specific inflammatory molecules and pathways have been identified that influence various pathologic outcomes in different experimental models of epilepsy. Most importantly, the same inflammatory pathways have also been found in surgically resected brain tissue from patients with treatment-resistant epilepsy. New antiseizure therapies may be derived from these novel potential targets. An essential and crucial question is whether targeting these molecules and pathways may result in anti-ictogenesis, antiepileptogenesis, and/or disease-modification effects. Therefore, preclinical testing in models mimicking relevant aspects of epileptogenesis is needed to guide integrated experimental and clinical trial designs. We discuss the most recent preclinical proof-of-concept studies validating a number of therapeutic approaches against inflammatory mechanisms in animal models that could represent novel avenues for drug development in epilepsy. Finally, we suggest future directions to accelerate preclinical to clinical translation of these recent discoveries

Atherosclerosis in the circle of Willis: Spatial differences in composition and in distribution of plaques

AbstractBackground and aimsIntracranial atherosclerosis is one of the main causes of ischemic stroke. However, the characteristics of intracranial arteries and atherosclerosis have rarely been studied. Therefore, we systematically investigated atherosclerotic changes in all arteries of the Circle of Willis (CoW).MethodsSixty-seven CoWs obtained at autopsy from randomly chosen hospital patients (mean age, 67.3 ± 12.5 years), of which a total of 1220 segments were collected from 22 sites. Atherosclerotic plaques were classified according to the revised American Heart Association classification and were related to local vessel characteristics, such as the presence of an external and internal elastic lamina and the elastic fibre density of the media.Results181 out of the 1220 segments had advanced plaques (15%), which were mainly observed in large arteries such as the internal carotid, middle cerebral, basilar and vertebral artery. Only 11 out of 1220 segments (1%) showed complicated plaques (p < 0.001). Six of these were intraplaque hemorrhages (IPH) and observed only in patients who had cardiovascular-related events (p = 0.015). The frequency of characteristics such as the external elastic lamina and a high elastin fibre density in the media was most often associated with the vertebral artery. Only 3% (n = 33) of the CoW arteries contained calcification (p < 0.001), which were mostly observed in the vertebral artery (n = 13, 12%).ConclusionsAdvanced atherosclerotic plaques in the CoW are relatively scarce and mainly located in the 4 large arteries, and mostly characterized by an early and stable phenotype, a low calcific burden, and a low frequency of IPH

Comparative interactomics analysis of different ALS-associated proteins identifies converging molecular pathways

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease with no effective treatment available. An increasing number of genetic causes of ALS are being identified, but how these genetic defects lead to motor neuron degeneration and to which extent they affect common cellular pathways remains incompletely understood. To address these questions, we performed an interactomic analysis to identify binding partners of wild-type (WT) and ALS-associated mutant versions of ATXN2, C9orf72, FUS, OPTN, TDP-43 and UBQLN2 in neuronal cells. This analysis identified several known but also many novel binding partners of these proteins

Dissecting the molecular determinants of GABAA receptors current rundown, a hallmark of refractory human epilepsy

GABAA receptors-(Rs) are fundamental for the maintenance of an efficient inhibitory function in the central nervous system (CNS). Their dysfunction is associated with a wide range of CNS disorders, many of which characterized by seizures and epilepsy. Recently, an increased use-dependent desensitization due to a repetitive GABA stimulation (GABAA current rundown) of GABAARs has been associated with drug-resistant temporal lobe epilepsy (TLE). Here, we aimed to investigate the molecular determinants of GABAA current rundown with two different heterologous expression systems (Xenopus oocytes and human embryonic kidney cells; HEK) which allowed us to manipulate receptor stoichiometry and to study the GABAA current rundown on different GABAAR configurations. To this purpose, we performed electrophysiology experiments using two-electrode voltage clamp in oocytes and confirming part of our results in HEK. We found that different degrees of GABAA current rundown can be associated with the expression of different GABAAR beta-subunits reaching the maximum current decrease when functional alpha1beta2 receptors are expressed. Furthermore, the blockade of phosphatases can prevent the current rundown observed in alpha1beta2 GABAARs. Since GABAAR represents one important therapeutic target in the treatment of human epilepsy, our results could open new perspectives on the therapeutic management of drug-resistant patients showing a GABAergic impairment

Regulation of Kir4.1 expression in astrocytes and astrocytic tumors: a role for interleukin-1 beta

<p>Abstract</p> <p>Objective</p> <p>Decreased expression of inwardly rectifying potassium (Kir) channels in astrocytes and glioma cells may contribute to impaired K⁺ buffering and increased propensity for seizures. Here, we evaluated the potential effect of inflammatory molecules, such as interleukin-1β (IL-1β) on Kir4.1 mRNA and protein expression.</p> <p>Methods</p> <p>We investigated Kir4.1 (Kcnj10) and IL-1β mRNA expression in the temporal cortex in a rat model of temporal lobe epilepsy 24 h and 1 week after induction of status epilepticus (SE), using real-time PCR and western blot analysis. The U373 glioblastoma cell line and human fetal astrocytes were used to study the regulation of Kir4.1 expression in response to pro-inflammatory cytokines. Expression of Kir4.1 protein was also evaluated by means of immunohistochemistry in surgical specimens of patients with astrocytic tumors (<it>n</it> = 64), comparing the expression in tumor patients with (<it>n</it> = 38) and without epilepsy (<it>n</it> = 26).</p> <p>Results</p> <p>Twenty-four hours after onset of SE, Kir4.1 mRNA and protein were significantly down-regulated in temporal cortex of epileptic rats. This decrease in expression was followed by a return to control level at 1 week after SE. The transient downregulation of Kir4.1 corresponded to the time of prominent upregulation of IL-1β mRNA. Expression of Kir4.1 mRNA and protein in glial cells in culture was downregulated after exposure to IL-1β. Evaluation of Kir4.1 in tumor specimens showed a significantly lower Kir4.1 expression in the specimens of patients with epilepsy compared to patients without epilepsy. This paralleled the increased presence of activated microglial cells, as well as the increased expression of IL-1β and the cytoplasmic translocation of high mobility group box 1 (HMGB1).</p> <p>Conclusions</p> <p>Taken together, these findings indicate that alterations in expression of Kir4.1 occurring in epilepsy-associated lesions are possibly influenced by the local inflammatory environment and in particular by the inflammatory cytokine IL-1β.</p

miR-193b-3p/ PGC-1α pathway regulates an insulin dependent anti-inflammatory response in Parkinson's disease

It has been shown that many miRNAs, including miR-193b-3p, are differentially expressed in Parkinson's disease (PD). Dysregulation of miR-193b-3p/PGC-1α axis may alter homeostasis in cells and can induce an inflammatory response commonly accompanied by metabolic disturbances. The aim of the present study is to investigate if dysregulation of the miR-193-3p/PGC-1α axis may contribute to the pathological changes observed in the PD brain. Brain tissue were obtained from middle frontal gyrus of non-demented controls and individuals with a PD diagnosis. RT-qPCR was used to determine the expression of miR-193b-3p and in situ hybridization (ISH) and immunological analysis were employed to establish the cellular distribution of miR-193b-3p. Functional assays were performed using SH-SY5Y cells, including transfection and knock-down of miR-193b-3p. We found significantly lower expression of miR-193b-3p in the early stages of PD (PD4) which increased throughout disease progression. Furthermore, altered expression of PGC-1α suggested a direct inhibitory effect of miR-193b-3p in the brain of individuals with PD. Moreover, we observed changes in expression of insulin after transfection of SH-SY5Y cells with miR-193b-3p, which led to dysregulation in the expression of several pro- or anti - inflammatory genes. Our findings indicate that the miR-193b-3p/PGC-1α axis is involved in the regulation of insulin signaling. This regulation is crucial, since insulin induced inflammatory response may serve as a protective mechanism during acute situations but potentially evolve into a pathological process in chronic conditions. This novel regulatory mechanism may represent an interesting therapeutic target with potential benefits for various neurodegenerative diseases.</p

PAK2 is an effector of TSC1/2 signaling independent of mTOR and a potential therapeutic target for Tuberous Sclerosis Complex

Tuberous sclerosis complex (TSC) is caused by inactivating mutations in either TSC1 or TSC2 and is characterized by uncontrolled mTORC1 activation. Drugs that reduce mTOR activity are only partially successful in the treatment of TSC, suggesting that mTOR-independent pathways play a role in disease development. Here, kinome profiles of wild-type and Tsc2-/- mouse embryonic fibroblasts (MEFs) were generated, revealing a prominent role for PAK2 in signal transduction downstream of TSC1/2. Further investigation showed that the effect of the TSC1/2 complex on PAK2 is mediated through RHEB, but is independent of mTOR and p21RAC. We also demonstrated that PAK2 over-activation is likely responsible for the migratory and cell cycle abnormalities observed in Tsc2-/- MEFs. Finally, we detected high levels of PAK2 activation in giant cells in the brains of TSC patients. These results show that PAK2 is a direct effector of TSC1-TSC2-RHEB signaling and a new target for rational drug therapy in TSC