Jessica Ruivo Maximino

Early motor and electrophysiological changes in transgenic mouse model of amyotrophic lateral sclerosis and gender differences on clinical outcome

Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disorder affecting motoneurons and the SOD1(G93A) transgenic mice are widely employed to study disease physiopathology and therapeutic strategies. Despite the cellular and biochemical evidences of an early motor system dysfunction, the conventional behavioral tests do not detect early motor impairments in SOD1 mouse model. We evaluated early changes in motor behavior of ALS mice by doing the analyses of tail elevation, footprint, automatic recording of motor activities by means of an infrared motion sensor activity system and electrophysiological measurements in male and female wild-type (WT) and SOD1(G93A) mice from postnatal day (P) 20 up to endpoint. The classical evaluations of mortality, weight loss, tremor, rotometer, hanging wire and inclined plane were also employed. There was a late onset (after P90) of the impairments of classical parameters and the outcome varied between genders of ALS mice, being tremor, cumulative survival, weight loss and neurological score about 10 days earlier in male than female ALS mice and also about 20 days earlier in ALS males regarding rotarod and hanging wire performances. While diminution of hindpaw base was 10 days earlier in ALS males (P110) compared to females, the steep length decreased 40 days earlier in ALS females (P60) than ALS males. The automatic analysis of motor impairments showed substantial late changes (after P90) of motility and locomotion in the ALS females, but not in the ALS males. It was surprising that the scores of tail elevation were already decreased in ALS males and females by P40, reaching the minimal values at the endpoint. The electrophysiological analyses showed early changes of measures in the ALS mouse sciatic nerve, i.e., decreased values of amplitude (P40) and nerve conduction velocity (P20), and also an increased latency (P20) reaching maximal level of impairments at the late disease phase. The early changes were not accompanied by reductions of neuronal protein markers of neurofilament 200 and ChAT in the ventral part of the lumbar spinal cord of P20 and P60 ALS mice by means of Western blot technique, despite remarkable decreases of those protein levels in P120 ALS mice. In conclusion, early changes of motor behavior and electrophysiological parameters in ALS mouse model must be taken into attention in the analyses of disease mechanisms and therapeutic effects.

Gene expression profiling for human iPS-derived motor neurons from sporadic ALS patients reveals a strong association between mitochondrial functions and neurodegeneration

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that leads to widespread motor neuron death, general palsy and respiratory failure. The most prevalent sporadic ALS form is not genetically inherited. Attempts to translate therapeutic strategies have failed because the described mechanisms of disease are based on animal models carrying specific gene mutations and thus do not address sporadic ALS. In order to achieve a better approach to study the human disease, human induced pluripotent stem cell (hiPSC)-differentiated motor neurons were obtained from motor nerve fibroblasts of sporadic ALS and non-ALS subjects using the STEMCCA Cre-Excisable Constitutive Polycistronic Lentivirus system and submitted to microarray analyses using a whole human genome platform. DAVID analyses of differentially expressed genes identified molecular function and biological process-related genes through Gene Ontology. REVIGO highlighted the related functions mRNA and DNA binding, GTP binding, transcription (co)-repressor activity, lipoprotein receptor binding, synapse organization, intracellular transport, mitotic cell cycle and cell death. KEGG showed pathways associated with Parkinsons disease and oxidative phosphorylation, highlighting iron homeostasis, neurotrophic functions, endosomal trafficking and ERK signaling. The analysis of most dysregulated genes and those representative of the majority of categorized genes indicates a strong association between mitochondrial function and cellular processes possibly related to motor neuron degeneration. In conclusion, iPSC-derived motor neurons from motor nerve fibroblasts of sporadic ALS patients may recapitulate key mechanisms of neurodegeneration and may offer an opportunity for translational investigation of sporadic ALS. Large gene profiling of differentiated motor neurons from sporadic ALS patients highlights mitochondrial participation in the establishment of autonomous mechanisms associated with sporadic ALS.

Adrenalectomy counteracts the local modulation of astroglial fibroblast growth factor system without interfering with the pattern of 6-OHDA-induced dopamine degeneration in regions of the ventral midbrain

The present study investigated the effects of bilateral adrenalectomy (ADX) on the synthesis of basic fibroblast growth factor (bFGF, FGF-2) mRNA and on the expression of its FGF receptor subtype-2 (FGFR2) mRNA after a 6-hydroxydopamine (6-OHDA)-induced lesion of nigrostriatal dopamine system. In previous papers we have demonstrated that corticosterone increases FGF-2 immunoreactivity mainly in the astrocytes of the substantia nigra [Chadi, G., Rosen, L., Cintra, A., Tinner, B., Zoli, M., Pettersson, R.F., Fuxe, K., 1993b. Corticosterone increases FGF-2 (bFGF) immunoreactivity in the substantia nigra of the rat. Neuroreport 4, 783-786.] and that 6-OHDA injected in the ventral midbrain upregulates FGF-2 synthesis in reactive astrocytes in the ascending dopamine pathways [Chadi, G., Cao, Y., Pettersson, R.F., Fuxe, K., 1994. Temporal and spatial increase of astroglial basic fibroblast growth factor synthesis after 6-hydroxydopamine-induced degeneration of the nigrostriatal dopamine neurons. Neuroscience 61, 891-910.]. Rats were adrenalectomized and received a 6-OHDA stereotaxical injection in the ventral midbrain 2 days later. Seven days after the dopamine lesion, Western blot analysis showed a decreased level of tyrosine hydroxylase in the lesioned side of the midbrain, an event that was not altered by ADX or corticosterone replacement. Moreover, the degeneration of nigral dopamine neurons, which was confirmed by the disappearance of acidic FGF (FGF-1) mRNA and the decrement of tyrosine hydroxylase mRNA labeled nigral neurons, was not altered by ADX. The FGF-2 protein (23 kDa isoform but not 21 kDa fraction) levels increased in the lesioned side of the ventral midbrain. This elevation was counteracted by ADX, an effect that was fully reversed by corticosterone replacement. In situ hybridization revealed that ADX counteracted the elevated FGF-2 mRNA levels in putative glial cells of the ipsilateral pars compacta of the substantia nigra and in the ventral tegmental area. The ADX also counteracted the increased density and intensity of the astroglial FGF-2 immunoreactive profiles within the lesioned pars compacta of the substantia nigra and the ventral tegmental area as determined by stereology. The stereotaxical mechanical needle insertion triggered the expression of FGFR2 mRNA in putative glial cells, spreading to the entire ipsilateral ventral midbrain from the region of needle track, an occurrence that was partially reversed by ADX. In conclusion, bilateral ADX counteracted the increased astroglial FGF-2 synthesis in the dopamine regions of the ventral midbrain following a 6-OHDA-induced local lesion and interfered with FGF receptor regulation around injury. These findings give further evidence that adrenocortical hormones may regulate the astroglial FGF-2-mediated trophic mechanisms and wound repair events in the lesioned central nervous system.

Deregulated expression of cytoskeleton related genes in the spinal cord and sciatic nerve of presymptomatic SOD1G93A Amyotrophic Lateral Sclerosis mouse model

Early molecular events related to cytoskeleton are poorly described in Amyotrophic Lateral Sclerosis (ALS), especially in the Schwann cell (SC), which offers strong trophic support to motor neurons. Database for Annotation, Visualization and Integrated Discovery (DAVID) tool identified cytoskeleton-related genes by employing the Cellular Component Ontology (CCO) in a large gene profiling of lumbar spinal cord and sciatic nerve of presymptomatic SOD1G93A mice. One and five CCO terms related to cytoskeleton were described from the spinal cord deregulated genes of 40 days (actin cytoskeleton) and 80 days (microtubule cytoskeleton, cytoskeleton part, actin cytoskeleton, neurofilament cytoskeleton, and cytoskeleton) old transgene mice, respectively. Also, four terms were depicted from the deregulated genes of sciatic nerve of 60 days old transgenes (actin cytoskeleton, cytoskeleton part, microtubule cytoskeleton and cytoskeleton). Kif1b was the unique deregulated gene in more than one studied region or presymptomatic age. The expression of Kif1b [quantitative polymerase chain reaction (qPCR)] elevated in the lumbar spinal cord (40 days old) and decreased in the sciatic nerve (60 days old) of presymptomatic ALS mice, results that were in line to microarray findings. Upregulation (24.8 fold) of Kif1b was seen in laser microdissected enriched immunolabeled motor neurons from the spinal cord of 40 days old presymptomatic SOD1G93A mice. Furthermore, Kif1b was dowregulated in the sciatic nerve Schwann cells of presymptomatic ALS mice (60 days old) that were enriched by means of cell microdissection (6.35 fold), cell sorting (3.53 fold), and primary culture (2.70 fold) technologies. The gene regulation of cytoskeleton molecules is an important occurrence in motor neurons and Schwann cells in presymptomatic stages of ALS and may be relevant in the dying back mechanisms of neuronal death. Furthermore, a differential regulation of Kif1b in the spinal cord and sciatic nerve cells emerged as key event in ALS.

The importance of molecular histology to study glial influence on neurodegenerative disorders. Focus on recent developed single cell laser microdissection

Neuron-glia interaction is involved in physiological function of neurons, however recent evidences have suggested glial cells as participants in neurotoxic and neurotrophic mechanisms of neurodegenerative/neuroregenerative processes. Histological techniques employing immunolabeling, historadiography and in situ hybridization have been useful to localize at cell levels molecules in normal and pathological situations. The intercellular accomplishment leading to neuronal injury in central nervous system disorders implies the performance of quantitative assays to better interpret the role of related molecules or signal pathways, however one limitation employing the whole tissue is the loss of cellular resolution. The laser capture microdissection was developed recently and allows the selection of specific cell types from their original environment after freezing and sectioning the tissue sampling, leading to the quantification of gene expression in individual cells, thus providing a unique opportunity to get new informations on cell signaling related to neurodegeneration. Here we reviewed the role of glial cell signaling on neurodegenerative disorders like ischemia, Parkinson and Alzheimer diseases, and also amyotrophic lateral sclerosis and what has been published with regards to single cell laser capture microdissection technique in the molecular biology investigation on these issues.

The review of the methods to obtain non-neuronal cells to study glial influence on Amyotrophic Lateral Sclerosis pathophysiology at molecular level in vitro

PURPOSE Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that displays a rapid evolution. Current treatments have failed to revert clinical symptoms because the mechanisms involved in the death of motoneuron are still unknown. Recent publications have put non-neuronal cells, particularly, astrocyte and microglia, in the scenario of pathophisiology of the disease. Animal models for ALS, particularly transgenic mice expressing the human SOD1 gene with a G93A mutation (hSOD1), are available and display the phenotype of the disease at cellular and clinical levels. However, it is a lack of detailed information regarding the methods to study the disease in vitro to better understand the contribution of non-neuronal cells in the onset and progression of the pathology. METHODS Colonies of Swiss mice and transgenic mice expressing hSOD1 mutation as well as non-transgenic controls (wild-type) were amplified after a genotyping evaluation. Disease progression was followed behaviorally and mortality was registered. Highly purified primary cultures of astrocytes and microglia from mouse spinal cord were obtained. Cells were identified by means of GFAP and CD11B immunocytochemistry. The purity of astroglial and microglial cell cultures was also accompanied by means of Western blot and RT-PCR analyses employing a number of markers. RESULTS The disease onset was about 105 days and the majority of transgenic mice displayed the disease symptoms by 125 days of age and reached the endpoint 20 days later. A substantial motor weakens was registered in the transgenic mice compared to wild-type at the end point. Immunocytochemical, biochemical and RT-PCR analyses demonstrated a highly purified primary cultures of spinal cord astrocytes and microglia. CONCLUSION It is possible to achieve highly purified primary cultures of spinal cord astrocytes and microglia to be employed in cellular and molecular analyses of the influence of such non-neuronal cells in the pathophysiology of ALS.

Decreases in the expression of CGRP and galanin mRNA in central and peripheral neurons related to the control of blood pressure following experimental hypertension in rats

Calcitonin gene-related peptide (alpha CGRP) and galanin (GAL) are peptides known to participate in central mechanisms of blood pressure control. Nonetheless, variations in the synthesis of the peptides in response to a hypertensive challenge are not well described, specially using a model, which allows acute and chronic analyses. In this study, we have employed in situ hybridization to analyse changes in mRNA expression of alpha CGRP and GAL in the nucleus tractus solitarii (NTS), hypothalamic paraventricular nucleus (PVN) as well as petrosal and nodose ganglia after aortic coarctation-induced hypertension in rats. Acute (2h) and chronic (3 and 7 days) analyses were performed in order to evaluate the involvement of both peptides in different periods of hypertension. The analysis of relative mRNA levels showed significant differences between sham-operated and aortic coarcted hypertensive rats. alpha CGRP mRNA expression was decreased 2h (40%) and 3 days (42%) in nodose and petrosal ganglia, respectively, after coarctation. No changes in CGRP mRNA signal were seen in the NTS and PVN in the analysed periods. GAL mRNA expression was decreased in the NTS (19%) and PVN (55%), 3 and 7 days, respectively, after coarctation-induced hypertension. No changes in GAL mRNA expression were observed in petrosal and nodose ganglia following aortic coarctation. Data suggest that alpha CGRP and GAL may participate in the mechanisms involved in the establishment/maintenance of hypertension induced by aortic coarctation. Acute changes might be involved with the adaptation to the hypertensive state, while changes at the chronic phase might be related to counteraction of hypertension.

The Effects of Omega-3 Fatty Acid Supplementation on Dexamethasone-Induced Muscle Atrophy

Corticosteroids cause muscle atrophy by acting on proteasomal and lysosomal systems and by affecting pathways related to muscular trophysm, such as the IGF-1/PI-3k/Akt/mTOR. Omega-3 fatty acid (n-3) has been used beneficially to attenuate muscle atrophy linked to sepsis and cachexia; however, its effect on dexamethasone-induced muscle atrophy has not been evaluated. Objectives. We evaluated whether n-3 supplementation could mitigate the development of dexamethasone-induced muscle atrophy. Methods. Two groups of Wistar rats were orally supplemented with n-3 or vehicle solution for 40 days. In the last 10 days, dexamethasone, or saline solution, was administrated establishing four groups: control, dexamethasone, n-3, and dexamethasone + n-3. The cross-sectional areas of muscle fibers, gene expression (MyoD, Myogenin, MuRF-1, and Atrogin-1), and protein expression (Akt, GSK3β, FOXO3a, and mTOR) were assessed. Results. Dexamethasone induced a significant loss in body and muscle weight, atrophy in type 2B fibers, and decreased expression of P-Akt, P-GSK3β, and P-FOXO3a. N-3 supplementation did not attenuate the negative effects of dexamethasone on skeletal muscle; instead, it caused atrophy in type 1, 2A, reduced the expression of Myogenin, and increased the expression of Atrogin-1. Conclusion. Food supplements containing n-3 are usually healthful, but they may potentiate some of the side effects of glucocorticoids.

Genetic analysis of patients with familial and sporadic amyotrophic lateral sclerosis in a Brazilian Research Center

Abstract Objective: To investigate gene mutations in familial form (FALS) and sporadic form (SALS) of amyotrophic lateral sclerosis (ALS) in a highly miscegenated population. Methods: Frequencies of mutations in the C9orfF72, TARDBP, SOD1, FUS and VAPB genes were investigated in a cohort of FALS (n = 39) and SALS (n = 189) subjects from the Research Centre of the University of São Paulo School of Medicine. All patients were subjected to C9orf72 and TARDBP analyses. SOD1, FUS and VAPB were also evaluated in FALS subjects. Results: Mutations were identified in FALS (61.3%) and SALS (5.3%) patients. Mutations in C9orf72 (12.8%, >45 GGGGCC hexanucleotide repeats), VAPB (43.6%, P56S) and SOD1 (7.7%, L145S) were identified in FALS subjects. Pathogenic C9orf72 expansions (2.64%) were identified in some SALS patients. Similar changes of TARDBP were found in SALS (2.64%) but not in FALS subjects. No FUS mutations were seen in any FALS subjects. Conclusions: TARDBP and C9orf72 mutations in this cohort were similar to those found in other centres worldwide. VAPB mutation (P56S) was highly prevalent in Brazilian FALS patients.

Motor recovery and cortical plasticity after functional electrical stimulation in a rat model of focal stroke.

ObjectiveThe aim of this study was to investigate the functional responses and plastic cortical changes in a sample of animals with sequelae of cerebral ischemia that were subjected to a model of functional electrical stimulation (FES). DesignRats received an ischemic cortical lesion (Rose Bengal method) and were randomized and submitted to an FES stimulation (1–2 mA, 30 Hz, 20–40 mins for 14 days) or sham stimulation. The Foot Fault Test was performed before inducing the cortical lesion and also before and after FES. Brain immunochemistry labeling with microtubule-associated protein–2 and neurofilament-200 markers was performed after FES. ResultsThe authors found a decreased percentage of errors in the Foot Fault Test (P < 0.001) in the stimulated group compared with the sham group after FES. FES has not altered the lesion size. Spontaneous motor parameters returned to basal values in both groups. The qualitative analysis showed an increased amount of radial microtubule-associated protein–2 immunoreactive fibers in the preserved cortex adjacent to stroke site in the stimulated animals. Regarding the measurements of neurofilament-200 immunostaining, there were no differences between the hemispheres or groups in area or intensity. ConclusionsAcute and short period of FES led to motor recovery of ankle joint neurodisability. The extent to which compensatory plasticity occurs after stroke or after FES and the extent to which it contributes to functional recovery are yet unclear. The changes induced by the stimulation may improve the ability of the nervous system to undergo spontaneous recovery, which is of substantial interest for neurorehabilitation strategies.