An initial top-down proteomic analysis of the standard cuprizone mouse model of multiple sclerosis

An initial proteomic analysis of the cuprizone mouse model to characterise the breadth of toxicity by assessing cortex, skeletal muscle, spleen and peripheral blood mononuclear cells. Cuprizone treated vs. control mice for an initial characterisation. Select tissues from each group were pooled, analysed in triplicate using two-dimensional gel electrophoresis (2DE) and deep imaging and altered protein species identified using liquid chromatography tandem mass spectrometry (LC/MS/MS). Forty-three proteins were found to be uniquely detectable or undetectable in the cuprizone treatment group across the tissues analysed. Protein species identified in the cortex may potentially be linked to axonal damage in this model, and those in the spleen and peripheral blood mononuclear cells to the minimal peripheral immune cell infiltration into the central nervous system during cuprizone mediated demyelination. Primary oligodendrocytosis has been observed in type III lesions in multiple sclerosis. However, the underlying mechanisms are poorly understood. Cuprizone treatment results in oligodendrocyte apoptosis and secondary demyelination. This initial analysis identified proteins likely related to axonal damage; these may link primary oligodendrocytosis and secondary axonal damage. Furthermore, this appears to be the first study of the cuprizone model to also identify alterations in the proteomes of skeletal muscle, spleen and peripheral blood mononuclear cells. Notably, protein disulphide isomerase was not detected in the cuprizone cohort; its absence has been linked to reduced major histocompatibility class I assembly and reduced antigen presentation. Overall, the results suggest that, like experimental autoimmune encephalomyelitis, results from the standard cuprizone model should be carefully considered relative to clinical multiple sclerosis.     

Inhibition of Neuronal Degenerin/Epithelial Na+ Channels by the Multiple Sclerosis Drug 4-Aminopyridine

The voltage-gated K⁺ (Kv) channel blocker 4-aminopyridine (4-AP) is used to target symptoms of the neuroinflammatory disease multiple sclerosis (MS). By blocking Kv channels, 4-AP facilitates action potential conduction and neurotransmitter release in presynaptic neurons, lessening the effects of demyelination. Because they conduct inward Na⁺ and Ca²⁺ currents that contribute to axonal degeneration in response to inflammatory conditions, acid-sensing ion channels (ASICs) contribute to the pathology of MS. Consequently, ASICs are emerging as disease-modifying targets in MS. Surprisingly, as first demonstrated here, 4-AP inhibits neuronal degenerin/epithelial Na⁺ (Deg/ENaC) channels, including ASIC and BLINaC. This effect is specific for 4-AP compared with its heterocyclic base, pyridine, and the related derivative, 4-methylpyridine; and akin to the actions of 4-AP on the structurally unrelated Kv channels, dose- and voltage-dependent. 4-AP has differential actions on distinct ASICs, strongly inhibiting ASIC1a channels expressed in central neurons but being without effect on ASIC3, which is enriched in peripheral sensory neurons. The voltage dependence of the 4-AP block and the single binding site for this inhibitor are consistent with 4-AP binding in the pore of Deg/ENaC channels as it does Kv channels, suggesting a similar mechanism of inhibition in these two classes of channels. These findings argue that effects on both Kv and Deg/ENaC channels should be considered when evaluating the actions of 4-AP. Importantly, the current results are consistent with 4-AP influencing the symptoms of MS as well as the course of the disease because of inhibitory actions on Kv and ASIC channels, respectively.     

Astrocyte–neuron interactions in neurological disorders

Astrocytes have long been considered as just providing trophic support for neurons in the central nervous system, but recently several studies have highlighted their importance in many functions such as neurotransmission, metabolite and electrolyte homeostasis, cell signaling, inflammation, and synapse modulation. Astrocytes are, in fact, part of a bidirectional crosstalk with neurons. Moreover, increasing evidence is stressing the emerging role of astrocyte dysfunction in the pathophysiology of neurological disorders, including neurodegenerative disease, stroke, epilepsy, migraine, and neuroinflammatory diseases.     

Trace elements profile in the blood of Huntington’ disease patients

Huntington’ disease (HD) is an autosomal dominant neurodegenerative disease characterized by progressive motor, psychiatric, and cognitive deterioration. HD is, together with spinocerebellar ataxias, spinobulbar muscular atrophy and dentatorubral-pallido- luysian atrophy, one of the nine disorders caused by an expansion of glutamine residues in the causative protein where the polyglutamine expansion cause aberrant protein folding. Since an excessive metal’s accumulation in organs may induce protein misfolding and oxidative stress, we have studied the blood concentration of essential (Cr, Co, Cu, Fe, Mn, Mo, Ni, Se, Zn) and nonessential (As, Cd, Sb, Sn, V) trace elements in HD patients.We found increased levels of the essential elements iron, chromium, selenium and zinc and of the nonessential element arsenic in the blood of HD patients.Since alteration in metals homeostasis may contribute to the pathogenesis of neurodegenerative disease and could eventually constitute a target for therapy, we may suggest the utilize of the blood metal profile as a further in vivo tool to study and characterize Huntington disease.     

Age-Dependent Impairment of Mitochondrial Function in Primate Brain

Abstract: It has been hypothesized that some of the functional impairments associated with aging are the result of increasing oxidative damage to mitochondrial DNA that produces defects in oxidative phosphorylation. To test this hypothesis, we examined the enzymes that catalyze oxidative phosphorylation in crude mitochondrial preparations from frontoparietal cortex of 20 rhesus monkeys (5-34 years old). Samples were assayed for complex I, complex II-III, complex IV, complex V, and citrate synthase activities. When enzyme activities were corrected for citrate synthase activities (to account for variable degrees of mitochondrial enrichment), linear regression analysis demonstrated a significant negative correlation of the activities of complex I (p < 0.002) and complex IV (p < 0.03) with age but no significant change in complex II-III or complex V activities. Relative to animals 6.9 ± 0.9 years old (n = 7), the citrate synthase-corrected activity of complex I was reduced by 17% in animals 22.5 ± 0.9 years old (n = 6) (p < 0.05) and by 22% in animals 30.7 ± 0.9 years old (n = 7) (p < 0.01). Similar age-related reductions in the activities of complexes I and IV were obtained when enzyme activities were corrected for complex II-III activity. These findings show an age-associated progressive impairment of mitochondrial complex I and complex IV activities in cerebral cortices of primates.     

Altered Protein Tyrosine Phosphorylation in Alzheimer''s Disease

The activity of protein tyrosine kinase was determined in extracts from Alzheimer's disease brains and age- and postmortem time-matched control brains at autopsy using the synthetic peptide substrate poly(Glu4Tyr1). The specific activity of protein tyrosine kinases in the particulate fraction decreased roughly twofold (p less than 0.02) in Alzheimer's disease frontal cortex relative to unaffected control cortex. Cytosolic protein tyrosine kinase activity in Alzheimer's disease tissue was not significantly different from that in control tissue. In contrast to reduced particulate protein tyrosine kinase activity, analysis of Western blots of cytosolic and particulate fractions revealed increases in cytosolic antiphosphotyrosine immunoreactive polypeptides with molecular masses of 55 and 60 kDa. Quantitative immunohistochemistry and morphometry of frontal cortex sections with the antiphosphotyrosine antibody indicated increased antiphosphotyrosine staining in the neurons, although the number of antiphosphotyrosine-positive neurons per square millimeter decreased. Also, increased antiphosphotyrosine staining was observed in the hippocampal neurons. These results suggest that altered protein tyrosine kinases and protein tyrosine phosphorylation are involved in the pathology of Alzheimer's disease.     

Recovery of Cholinergic Phenotype in the Injured Rat Neostriatum: Roles for Endogenous and Exogenous Nerve Growth Factor

Polyclonal antibodies against recombinant human nerve growth factor (rhNGF) potently inhibited PC12 neurite outgrowth, blocked high-affinity 125I-rhNGF binding but not its receptor, and cross-reacted with rat, mouse, and human nerve growth factor (NGF) but not with brain-derived neurotrophic factor, neurotrophin-3, ciliary neurotrophic factor, insulin-like growth factor, epidermal growth factor, or activin A. Immunocytochemistry revealed many NGF-positive neurons in the rat neostriatum. The NGF-positive neurons disappeared by 3 days after mechanical injury to the neostriatum and were replaced by intensely NGF- and glial fibrillary acidic protein-positive astrocytes. Enzyme-linked immunosorbent assay measurements revealed that the NGF content of the injured striatum was elevated by eightfold 3 days postinjury and by twofold 2 weeks later. The high-affinity choline uptake (HACU) into cholinergic nerve terminals was decreased by 23% at 2 and 4 weeks postinjury, yet choline acetyltransferase (ChAT) activity in these neurons was unchanged at 2 weeks and decreased by 14% at 4 weeks. Daily infusion of 1 microgram of rhNGF into the injury area did not alter the loss of HACU. However, this treatment elevated ChAT activity by 23-29% above intact neostriatal levels and by 53-65% relative to HACU at both survival times. Thus, lesion-induced increases in NGF levels within astrocytes are associated with maintenance of striatal ChAT activity at normal levels following cholinergic injury, even with decreases in HACU. Pharmacologic doses of rhNGF can further augment ChAT activity in damaged cholinergic neurons, showing the usefulness of exogenous NGF even when endogenous NGF is elevated in response to injury.     

Dexamethasone does not ameliorate gliosis in a mouse model of neurodegenerative disease

Prolonged neuroinflammation is a driving force for neurodegenerative disease, and agents against inflammatory responses are regarded as potential treatment strategies. Here we aimed to evaluate the prevention effects on gliosis by dexamethasone (DEX), an anti-inflammation drug. We used DEX to treat the nicastrin conditional knockout (cKO) mouse, a neurodegenerative mouse model. DEX (10 mg/kg) was given to 2.5-month-old nicastrin cKO mice, which have not started to display neurodegeneration and gliosis, for 2 months. Immunohistochemistry (IHC) and Western blotting techniques were used to detect changes in neuroinflammatory responses. We found that activation of glial fibrillary acidic protein (GFAP) positive or ionized calcium binding adapter molecule1 (Iba1) positive cells was not inhibited in nicastrin cKO mice treated with DEX as compared to those treated with saline. These data suggest that DEX does not prevent or ameliorate gliosis in a neurodegenerative mouse model when given prior to neuronal or synaptic loss.     

Early Diagnosis of Neurodegenerative Diseases – The Long Awaited Holy Grail and Bottleneck of Modern Brain Research – 19th HUPO BPP Workshop

The HUPO Brain Proteome Project (HUPO BPP) held its 19th workshop in Dortmund, Germany, from May 22 to 24, 2013. The focus of the spring workshop was on strategies and developments concerning early diagnosis of neurodegenerative diseases     

ATM-deficient human neural stem cells as an in vitro model system to study neurodegeneration

Loss of ATM kinase, a transducer of the DNA damage response and redox sensor, causes the neurodegenerative disorder ataxia-telangiectasia (A-T). While a great deal of progress has been made in elucidating the ATM-dependent DNA damage response (DDR) network, a key challenge remains in understanding the selective susceptibility of the nervous system to faulty DDR. Several factors appear implicated in the neurodegenerative phenotype in A-T, but which of them plays a crucial role remains unclear, especially since mouse models of A-T do not fully mirror the respective human syndrome. Therefore, a number of human neural stem cell (hNSC) systems have been developed to get an insight into the molecular mechanisms of neurodegeneration as consequence of ATM inactivation. Here we review the hNSC systems developed by us an others to model A-T.