Impaired proteasome activity and neurodegeneration with brain iron accumulation in FBXO7 defect.

FBXO7 is implicated in the ubiquitin-proteasome system and parkin-mediated mitophagy. FBXO7defects cause a levodopa-responsive parkinsonian-pyramidal syndrome(PPS). METHODS: We investigated the disease molecular bases in a child with PPS and brain iron accumulation. RESULTS: A novel homozygous c.368C>G (p.S123*) FBXO7 mutation was identified in a child with spastic paraplegia, epilepsy, cerebellar degeneration, levodopa nonresponsive parkinsonism, and brain iron deposition. Patient's fibroblasts assays demonstrated an absence of FBXO7 RNA expression leading to impaired proteasome degradation and accumulation of poly-ubiquitinated proteins. CONCLUSION: This novel FBXO7 phenotype associated with impaired proteasome activity overlaps with neurodegeneration with brain iron accumulation disorders.

Biophysical characterization Of Alpers encephalopathy associated mutants of human mitochondrial phenylalanyl-tRNA synthetase.

Mutations in nucleus-encoded mitochondrial aminoacyl-tRNA synthetases (mitaaRSs) lead to defects in mitochondrial translation affecting the expression and function of 13 subunits of the respiratory chain complex leading to diverse pathological conditions. Mutations in the FARS2 gene encoding human mitochondrial phenylalanyl-tRNA synthetase (HsmitPheRS) have been found to be associated with two different clinical representations, infantile Alpers encephalopathy and spastic paraplegia. Here we have studied three pathogenic mutants (Tyr144Cys, Ile329Thr, and Asp391Val) associated with Alpers encephalopathy to understand how these variants affect the biophysical properties of the enzyme. These mutants have already been reported to have reduced aminoacylation activity. Our study established that the mutants are significantly more thermolabile compared to the wild-type enzyme with reduced solubility in vitro. The presence of aggregation-prone insoluble HsmitPheRS variants could have a detrimental impact on organellar translation, and potentially impact normal mitochondrial function. © 2019 IUBMB Life, 71(8): 1141-1149, 2019 © 2019 IUBMB Life, 71(8):1141-1149, 2019.

Elevated Levels of Protein Disulfide Isomerase and Binding Immunoglobulin Protein Implicated in Spinal Cord Injury Paraplegia Patients with Pressure Ulcers.

AIMS: To explore the associations between two endoplasmic reticulum (ER) stress proteins, protein disulfide isomerase (PDI), binding immunoglobulin protein (BIP), and the development and progression of pressure ulcers (PUs) in spinal cord injury (SCI) paraplegia patients. METHODS: ELISA kits were used to measure the levels of serum PDI and BIP in 67 SCI paraplegia patients with PUs and 61 SCI paraplegia patients without PUs. The associations between PDI and BIP, PU formation, PU staging, and pressure ulcer scale for healing (PUSH) score were analyzed. RESULTS: The patients in the PU group had higher levels of PDI and BIP than those in the non-PU group (both p < 0.05). Furthermore, the levels of PDI were positively correlated with those of BIP (r = 0.707, p < 0.0001). There were significant differences in the PDI and BIP levels among the different stages of PU (all p < 0.05). As the PU stages progressed, the levels of PDI and BIP first increased, then decreased, and finally peaked at stage III of the PUs. The PUSH scores significantly declined 7 days after debridement for the PU stage II (p < 0.01) but showed no significant difference between stages III and IV at 7 days after debridement (p > 0.05). The PUSH scores also decreased at 28 days after debridement for stages II, III, and IV (all p < 0.01). Higher PUSH scores indicated a longer time of debridement accompanied by a longer wound surface healing time (p < 0.05). CONCLUSION: ER stress proteins may be involved in the process of PU formation and healing; moreover, the levels of PDI and BIP were also associated with the severity of the PUs. Finally, we found that the PUSH scores can be used as a reference to evaluate PU severity and healing.

Active paraplegics are protected against exercise-induced oxidative damage through the induction of antioxidant enzymes.

STUDY DESIGN: Experimental study. OBJECTIVES: Exercise improves functional capacity in spinal cord injury (SCI). However, exhaustive exercise, especially when sporadic, is linked to the production of reactive oxygen species that may have a detrimental effect on SCI. We aimed to study the effect of a single bout of exhaustive exercise on systemic oxidative stress parameters and on the expression of antioxidant enzymes in individuals with paraplegia. SETTING: The study was conducted in the Physical Therapy department and the Physical Education and Sports department of the University of Valencia. METHODS: Sixteen paraplegic subjects were submitted to a graded exercise test (GET) until volitional exhaustion. They were divided into active or non-active groups. Blood samples were drawn immediately, 1 and 2 h after the GET. We determined plasma malondialdehyde (MDA) and protein carbonylation as markers of oxidative damage. Antioxidant gene expression (catalase and glutathione peroxidase-GPx) was determined in peripheral blood mononuclear cells. RESULTS: We found a significant increase in plasma MDA and protein carbonyls immediately after the GET (P<0.05). This increment correlated significantly with the lactate levels. Active paraplegics showed lower levels of exercise-induced oxidative damage (P<0.05) and higher exercise-induced catalase (P<0.01) and GPx (P<0.05) gene expression after the GET. CONCLUSIONS: These results suggest that exercise training may be useful in SCI patients to develop systemic antioxidant defenses that may protect them against exercise-induced oxidative damage.

Erythropoietin activates the phosporylated cAMP [adenosine 3'5' cyclic monophosphate] response element-binding protein pathway and attenuates delayed paraplegia after ischemia-reperfusion injury.

OBJECTIVE: Paraplegia remains a devastating complication of complex aortic surgery. Erythropoietin (EPO) has been shown to prevent paraplegia after ischemia reperfusion, but the protective mechanism remains poorly described in the spinal cord. We hypothesized that EPO induces the CREB (cAMP [adenosine 3'5' cyclic monophosphate] response element-binding protein) pathway and neurotrophin production in the murine spinal cord, attenuating functional and cellular injury. METHODS: Adult male mice were subjected to 4 minutes of spinal cord ischemia via an aortic and left subclavian cross-clamp. Experimental groups included EPO treatment 4 hours before incision (n = 7), ischemic control (n = 7), and shams (n = 4). Hind-limb function was assessed using the Basso motor score for 48 hours after reperfusion. Spinal cords were harvested and analyzed for neuronal viability using histology and staining with a fluorescein derivative. Expression of phosphorylated (p)AKT (a serine/threonine-specific kinase), pCREB, B-cell lymphoma 2, and brain-derived neurotrophic factor were determined using immunoblotting. RESULTS: By 36 hours of reperfusion, EPO significantly preserved hind-limb function after ischemia-reperfusion injury (P < .01). Histology demonstrated preserved cytoarchitecture in the EPO treatment group. Cords treated with EPO expressed significant increases in pAKT (P = .021) and pCREB (P = .038). Treatment with EPO induced expression of both of the neurotrophins, B-cell lymphoma 2, and brain-derived neurotrophic factor, beginning at 12 hours. CONCLUSIONS: Erythropoietin-mediated induction of the CREB pathway and production of neurotrophins is associated with improved neurologic function and increased neuronal viability following spinal cord ischemia reperfusion. Further elucidation of EPO-derived neuroprotection will allow for expansion of adjunct mechanisms for spinal cord protection in high-risk thoracoabdominal aortic intervention.

Spastic paraplegia gene 7 in patients with spasticity and/or optic neuropathy.

Mutations in the spastic paraplegia 7 (SPG7) gene encoding paraplegin are responsible for autosomal recessive hereditary spasticity. We screened 135 unrelated index cases, selected in five different settings: SPG7-positive patients detected during SPG31 analysis using SPG31/SPG7 multiplex ligation-dependent probe amplification (n = 7); previously reported ambiguous SPG7 cases (n = 5); patients carefully selected on the basis of their phenotype (spasticity of the lower limbs with cerebellar signs and/or cerebellar atrophy on magnetic resonance imaging/computer tomography scan and/or optic neuropathy and without other signs) (n = 24); patients with hereditary spastic paraparesis referred consecutively from attending neurologists and the national reference centre in a diagnostic setting (n = 98); and the index case of a four-generation family with autosomal dominant optic neuropathy but no spasticity linked to the SPG7 locus. We identified two SPG7 mutations in 23/134 spastic patients, 21% of the patients selected according to phenotype but only 8% of those referred directly. Our results confirm the pathogenicity of Ala510Val, which was the most frequent mutation in our series (65%) and segregated at the homozygous state with spastic paraparesis in a large family with autosomal recessive inheritance. All SPG7-positive patients tested had optic neuropathy or abnormalities revealed by optical coherence tomography, indicating that abnormalities in optical coherence tomography could be a clinical biomarker for SPG7 testing. In addition, the presence of late-onset very slowly progressive spastic gait (median age 39 years, range 18-52 years) associated with cerebellar ataxia (39%) or cerebellar atrophy (47%) constitute, with abnormal optical coherence tomography, key features pointing towards SPG7-testing. Interestingly, three relatives of patients with heterozygote SPG7 mutations had cerebellar signs and atrophy, or peripheral neuropathy, but no spasticity of the lower limbs, suggesting that SPG7 mutations at the heterozygous state might predispose to late-onset neurodegenerative disorders, mimicking autosomal dominant inheritance. Finally, a novel missense SPG7 mutation at the heterozygous state (Asp411Ala) was identified as the cause of autosomal dominant optic neuropathy in a large family, indicating that some SPG7 mutations can occasionally be dominantly inherited and be an uncommon cause of isolated optic neuropathy. Altogether, these results emphasize the clinical variability associated with SPG7 mutations, ranging from optic neuropathy to spastic paraplegia, and support the view that SPG7 screening should be carried out in both conditions.

Transcriptional and post-transcriptional regulation of SPAST, the gene most frequently mutated in hereditary spastic paraplegia.

Hereditary spastic paraplegias (HSPs) comprise a group of neurodegenerative disorders that are characterized by progressive spasticity of the lower extremities, due to axonal degeneration in the corticospinal motor tracts. HSPs are genetically heterogeneous and show autosomal dominant inheritance in ∼70-80% of cases, with additional cases being recessive or X-linked. The most common type of HSP is SPG4 with mutations in the SPAST gene, encoding spastin, which occurs in 40% of dominantly inherited cases and in ∼10% of sporadic cases. Both loss-of-function and dominant-negative mutation mechanisms have been described for SPG4, suggesting that precise or stoichiometric levels of spastin are necessary for biological function. Therefore, we hypothesized that regulatory mechanisms controlling expression of SPAST are important determinants of spastin biology, and if altered, could contribute to the development and progression of the disease. To examine the transcriptional and post-transcriptional regulation of SPAST, we used molecular phylogenetic methods to identify conserved sequences for putative transcription factor binding sites and miRNA targeting motifs in the SPAST promoter and 3'-UTR, respectively. By a variety of molecular methods, we demonstrate that SPAST transcription is positively regulated by NRF1 and SOX11. Furthermore, we show that miR-96 and miR-182 negatively regulate SPAST by effects on mRNA stability and protein level. These transcriptional and miRNA regulatory mechanisms provide new functional targets for mutation screening and therapeutic targeting in HSP.

The vulnerability of nitrergic neurons to transient spinal cord ischemia: a quantitative immunohistochemical and histochemical study.

Spinal cord ischemia belongs to serious and relatively frequent diseases of CNS. The aim of the present study was to find out the vulnerability of nitrergic neurons to 15 min transient spinal cord ischemia followed by 1 and 2 weeks of reperfusion. We studied neuronal nitric oxide synthase (nNOS) and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) in structural elements of lumbosacral spinal cord along its rostrocaudal axis. In addition, a neurological deficit of experimental animals was evaluated. Spinal cord ischemia, performed on the rabbit, was induced by abdominal aorta occlusion using Fogarty catheter introduced into the right femoral artery for a period of 15 min. After surgical intervention the animals survived for 7 and 14 days. nNOS-immunoreactivity (nNOS-IR) was measured by immunohistochemical and NADPHd-positivity by histochemical method, and both immunohistochemical and histochemical stainings were quantified by densitometric analyses. Neurological deficit was evaluated according Zivin's criteria. The number of nNOS-IR and/or NADPH-d positive neurons and the density of neuropil were markedly increased in superficial dorsal horn (laminae I-III) after 15 min ischemia and 7 days of reperfusion. However, ischemia followed by longer time of survival (14 days) returned the number of nNOS-IR and NADPH-d positive neurons to control. In the pericentral region (lamina X) containing interneurons and crossing fibers of spinal tracts, than in lamina VII and in dorsomedial part of the ventral horn (lamina VIII) we recorded a decreased number of nNOS-IR and NADPH-d positive neurons after both ischemia/reperfusion periods. In the medial portion of lamina VII and dorsomedial part of the ventral horn (lamina VIII) we observed many necrotic loci. This area was the most sensitive to ischemia/reperfusion injury. Fifteen minute ischemia caused a marked deterioration of neurological function of hind limbs, often developing into paraplegia. A quantitative immunohistochemical and histochemical study have shown a strong vulnerability of nitrergic neurons in intermediate zone to transient spinal cord ischemia.

Protective effects of cold spinoplegia with fasudil against ischemic spinal cord injury in rabbits.

OBJECTIVE: Paraplegia remains a serious complication after surgical repair of thoracoabdominal aortic aneurysms. The aim of this study was to evaluate the neuroprotective efficacy of fasudil, a Rho kinase (ROCK) inhibitor, by reducing the number of infiltrating cells in the ventral horn and increasing the induction of eNOS against ischemic spinal cord injury in rabbits. METHODS: Eighteen Japanese white rabbits were divided into three groups: saline (group 1, n = 7, 4 degrees C) and fasudil (group 2, n = 6, 4 degrees C) were immediately infused into the isolated segmental lumbar arteries over 30 seconds after aortic clamping. Group 3 (n = 5) was the sham-operated group. Hind limb function was evaluated 4 and 8 hours, and 1 and 2 days after 15 minutes of transient ischemia. Cell damage was analyzed by hematoxylin and eosin staining and temporal profiles of endothelial nitric oxide synthase immunoreactivity were performed. The number of intact motor neuron cells and infiltrating cells in the ventral horn were compared. RESULTS: Two days after reperfusion, group 2 and group 3 showed better neurologic function, a greater number of intact motor neuron cells, and a smaller number of infiltrating cells in the ventral horn than group 1. The induction of endothelial nitric oxide synthase (eNOS) was prolonged up to 2 days after reperfusion in group 2. CONCLUSION: These results indicate that fasudil has neuroprotective effects against ischemic spinal cord injury in rabbits by reducing the number of infiltrating cells in the ventral horn and prolonging the expression of eNOS.

Chronic paraplegia-induced muscle atrophy downregulates the mTOR/S6K1 signaling pathway.

Ribosomal S6 kinase 1 (S6K1) is a downstream component of the mammalian target of rapamycin (mTOR) signaling pathway and plays a regulatory role in translation initiation, protein synthesis, and muscle hypertrophy. AMP-activated protein kinase (AMPK) is a cellular energy sensor, a negative regulator of mTOR, and an inhibitor of protein synthesis. The purpose of this study was to determine whether the hypertrophy/cell growth-associated mTOR pathway was downregulated during muscle atrophy associated with chronic paraplegia. Soleus muscle was collected from male Sprague-Dawley rats 10 wk following complete T(4)-T(5) spinal cord transection (paraplegic) and from sham-operated (control) rats. We utilized immunoprecipitation and Western blotting techniques to measure upstream [AMPK, Akt/protein kinase B (PKB)] and downstream components of the mTOR signaling pathway [mTOR, S6K1, SKAR, 4E-binding protein 1 (4E-BP1), and eukaryotic initiation factor (eIF) 4G and 2alpha]. Paraplegia was associated with significant soleus muscle atrophy (174 +/- 8 vs. 240 +/- 13 mg; P < 0.05). There was a reduction in phosphorylation of mTOR, S6K1, and eIF4G (P < 0.05) with no change in Akt/PKB or 4E-BP1 (P > 0.05). Total protein abundance of mTOR, S6K1, eIF2alpha, and Akt/PKB was decreased, and increased for SKAR (P < 0.05), whereas 4E-BP1 and eIF4G did not change (P > 0.05). S6K1 activity was significantly reduced in the paraplegic group (P < 0.05); however, AMPKalpha2 activity was not altered (3.5 +/- 0.4 vs. 3.7 +/- 0.5 pmol x mg(-1) x min(-1), control vs. paraplegic rats). We conclude that paraplegia-induced muscle atrophy in rats is associated with a general downregulation of the mTOR signaling pathway. Therefore, in addition to upregulation of atrophy signaling during muscle wasting, downregulation of muscle cell growth/hypertrophy-associated signaling appears to be an important component of long-term muscle loss.

Na+,K+-ATPase concentration and fiber type distribution after spinal cord injury.

Complete spinal cord injury (SCI) is characterized, in part, by reduced fatigue-resistance of the paralyzed skeletal muscle during stimulated contractions, but the underlying mechanisms are not fully understood. The effects of complete SCI on skeletal muscle Na(+),K(+)-adenosine triphosphatase (ATPase) concentration, and fiber type distribution were therefore investigated. Six individuals (aged 32.0 +/- 5.3 years) with complete paraplegia (T4-T10; 1-19 years since injury) participated. There was a significantly lower Na(+),K(+)-ATPase concentration in the paralyzed vastus lateralis (VL) when compared to either the subjects' own unaffected deltoid or literature values (from our laboratory, utilizing the same methodology) of VL Na(+),K(+)-ATPase concentration for the healthy able-bodied (141.6 +/- 50.0, 213.4 +/- 23.9, 339 +/- 16 pmol/g wet wt., respectively; P < 0.05). There was also a significant negative correlation between the Na(+),K(+)-ATPase concentration in the paralyzed VL and years since injury (r = -0.75, P < 0.05). These findings are clinically relevant as they suggest that reductions in Na(+),K(+)-ATPase contribute to the fatigability of paralyzed muscle after SCI. Unexpectedly, the VL muscles of our subjects had a higher proportion of their area represented by type I fibers compared to literature values for the VL of the healthy able-bodied (52.6 +/- 25.3% vs. 36 +/- 11.3%, respectively; P < 0.05). As all our subjects had upper motor neuron injuries and, therefore, experienced muscle spasticity, our findings warrant further investigation into the relationship between muscle spasticity and fiber type expression after SCI.

[Electro-acupuncture effects on experimental spinal cord injury of the cat as evaluated by acid phosphatase detection].

The spinal cords of adult cats were injured at about L1 level by using Allen's method and the animals were divided randomly into 2 groups: a) electro-acupuncture treatment group and b) control group. The acid phosphatase (ACP) was detected at 3 and 7 days after spinal cord injury. The more ACP positive labelings were found in the treatment group than in the control group at both 3 (P < 0.05) and 7 days (P < 0.01) after injury. The results suggested that electro-acupuncture could increase the concentration of ACP of the injured spinal cord and the increased ACP concentration could make worse to the injured spinal cord at early time and improve regeneration during recovery period.

Increased endothelin and creatine kinase after electrical stimulation of paraplegic muscle.

The purpose of this study was to assess changes in creatine kinase (CK) and endothelin (ET) in individuals with spinal cord injury (SCI) after computerized functional electrical stimulation leg ergometry (CFES LE). Eight subjects (7 male and 1 female) with complete spinal cord lesions (C7 to L1) completed zero-loaded CFES LE tests at baseline, after 3, 6, and 12 wk of CFES LE training (30 min, 3 times/wk), and also after detraining (DT) (n = 5). Venous blood samples were drawn 24, 48, and 72 h after CFES LE for measurement of ET and CK. The CK response was largest (peak CK) 72 h after baseline tests (28.2 +/- 6.0 to 895.7 +/- 345.9 ktals/l) and was no different from baseline by weeks 3, 6, and 12. After DT, CK was similar before and after CFES LE (153.8 +/- 19.0 and 189.7 +/- 34.5 ktals/l, respectively). CFES LE also significantly increased peak ET after baseline (from 11.7 +/- 1.5 to 18.0 +/- 2.5 pg/ml). During the subsequent training, peak ET remained significantly higher than the baseline value at weeks 3, 6, and 12 (20.2 +/- 1.8, 18.0 +/- 1.1, and 16.9 +/- 2.2 pg/ml, respectively). After DT, peak ET increased significant relationship (r = 0.44) existed between ln peak CK activity and peak ET. In summary, the increase in circulating ET in spinal cord-injured individuals may have implications for baroreceptor function and therefore blood pressure control in SCI. Further research into CFES LE, ET, and baroreceptor function in SCI is warranted.

Impaired blood superoxide dismutase in the traumatic paraplegic patients.

Blood superoxide dismutase (SOD) activity and blood copper, zinc-superoxide dismutase (Cu.Zn-SOD) content were measured by luminol chemiluminescence assay and by single radial immunodiffusion assay, respectively, in 50 patients with paraplegia due to traumatic injury to the spinal cord by the Tangshan Earthquake on July 28, 1976, compared with 20 age-matched healthy subjects. We found that blood SOD activity and blood Cu.Zn-SOD content in the paraplegic patients were significantly lower than those in healthy subjects (p < 0.01). In healthy subjects, blood Cu.Zn-SOD fully expressed the enzymatic activity, whereas only 77% of blood Cu.Zn-SOD in the paraplegic patients expressed the enzymatic activity, indicating that in the patients, part of blood Cu.Zn-SOD protein is in a state without function. Also the serum lipid peroxide level in the paraplegic patients was higher than that in healthy subjects (p < 0.05). These findings suggest decreased endogenous blood protection against oxygen derived free radicals in these paraplegic patients.

Morphometric and neurophysiological analysis of skeletal muscle in paraplegic patients with traumatic cord lesion.

Morphometric and electrophoretic properties of soleus and medialis gastrocnemius fibres from paraplegic patients were studied 1 to 10 months following complete traumatic cord transection (spinal cord level C5-T1). In the short term of paraplegia (1-6 months) gastrocnemius medialis and soleus muscles showed predominant atrophy of IIA fibre types. In long term paraplegia (8-10 months) atrophy and reduction of type 1 fibres, with presence of high percentages of type IIB fibres, were seen in both studied muscles. The consistence in both muscles of IIB intermediate fibres in long term paraplegia, seems to indicate the initial stage of a mechanism of fibre transformation reflecting the adaptative capacity of the paretic muscle to spasticity. Electrophysiological studies of the H-reflex and the H/M ratio values reveal an increase of the H-reflex excitability in the soleus and gastrocnemius muscles during a 1 to 10 months follow-up.

Thyrotoxic neuropathy (Basedow's paraplegia).

Polyneuropathy is a rare but possible manifestation of severe hyperthyroidism. The patient reported here developed a polyneuropathy affecting mostly leg muscles (Basedow's paraplegia) during the course of severe thyrotoxicosis. The polyneuropathy was confirmed with sequential electrophysiological studies of nerves and muscles and by muscle biopsy. The involvement of the proximal leg muscle is also interpreted as a neuropathic or nerve-mediated process rather than a concomitant thyrotoxic myopathy.