localization-of-the-locus-coeruleus-in-the-mouse-brain

Localization of the Locus Coeruleus in the Mouse Brain

The locus coeruleus (LC) is a major hub of norepinephrine producing neurons that modulate a number of physiological functions. Structural or functional ...

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Metals and other trace elements play an important role in many physiological processes in all biological systems. Characterization of precise metal concentrations, their spatial distribution, and chemical speciation in individual cells and cell compartments will provide much needed information to explore the metallome in health and disease. Synchrotron-based X-ray fluorescent microscopy (SXRF) is the ideal tool to quantitatively measure trace elements with high sensitivity at high resolution. SXRF is based on the intrinsic fluorescent properties of each element and is therefore element specific. Recent advances in synchrotron technology and optimization of sample preparation have made it possible to image metals in mammalian tissue with submicron resolution. In combination with correlative methods, SXRF can now, for example, determine the amount and oxidation state of trace elements in intra-cellular compartments and identify cell-specific changes in the metal ion content during development or disease progression.

Quantitative imaging of metals in tissues.

Cu-ATPase ATP7B (Wilson's disease protein) transports copper into the trans-Golgi network for biosynthetic incorporation into ceruloplasmin and sequesters excess copper to endocytic vesicles for further export out of the cell. The activity and intracellular location of ATP7B are regulated by copper levels; the trafficking of ATP7B between cellular compartments is coupled to changes in the level of protein phosphorylation. Neither the nature of the kinase(s) phosphorylating ATP7B nor the location of phosphorylation sites is known. We demonstrate that the membrane-bound ATP7B is phosphorylated by an ATP-dependent, GTP-independent kinase that can be either soluble or membrane-associated. Mg(2+) or Mn(2+) is necessary for kinase activity. We further show that the recombinant N-terminal domain of ATP7B (N-ATP7B) is a specific target for a kinase-mediated phosphorylation in vitro and in cells. Although exogenous addition of copper is not required for kinase activity, copper binding to N-ATP7B markedly alters the exposure of loops connecting the metal-binding subdomains (MBDs) to proteolysis and facilitates phosphorylation by 25-30%. MBD1-2 and MBD4-5 linkers become protected, while MBD2-3 and MBD3-4 regions remain exposed. A significant, 5-fold increase in the level of phosphorylation is also observed for the ATP7B variant that lacks the 29 kDa N-terminal fragment (mostly likely comprised of MBD1-3). Analysis of phosphorylated peptides by two-dimensional gel electrophoresis and mass spectrometry points to the loop connecting MBD3 and MBD4 as a region of phosphorylation. Altogether, the results suggest a mechanism in which kinase-mediated phosphorylation of ATP7B is controlled by a conformational state of N-ATP7B.

The loop connecting metal-binding domains 3 and 4 of ATP7B is a target of a kinase-mediated phosphorylation.

Wilson disease (WD) is a severe hepato-neurologic disorder that affects primarily children and young adults. WD is caused by mutations in ATP7B and subsequent copper overload. However, copper levels alone do not predict severity of the disease. We demonstrate that temporal and spatial distribution of copper in hepatocytes may play an important role in WD pathology. High resolution synchrotron-based x-ray fluorescence imaging in situ indicates that copper does not continuously accumulate in Atp7b(-/-) hepatocytes, but reaches a limit at 90-300 fmol. The lack of further accumulation is associated with the loss of copper transporter Ctr1 from the plasma membrane and the appearance of copper-loaded lymphocytes and extracellular copper deposits. The WD progression is characterized by changes in subcellular copper localization and transcriptome remodeling. The synchrotron-based x-ray fluorescence imaging and mRNA profiling both point to the key role of nucleus in the initial response to copper overload and suggest time-dependent sequestration of copper in deposits as a protective mechanism. The metabolic pathways, up-regulated in response to copper, show compartmentalization that parallels changes in subcellular copper concentration. In contrast, significant down-regulation of lipid metabolism is observed at all stages of WD irrespective of copper distribution. These observations suggest new stage-specific as well as general biomarkers for WD. The model for the dynamic role of copper in WD is proposed.

Wilson disease at a single cell level: intracellular copper trafficking activates compartment-specific responses in hepatocytes.

There is increasing evidence for the crucial role of metals in the pathology of Alzheimer's disease. Both the aggregation and neurotoxicity of amyloid-β are dependent on the presence of copper. This study investigated the ability of the copper-complexing drug tetrathiomolybdate to reduce amyloid-β pathology and spatial memory impairment in both a prevention and a treatment paradigm in the Tg2576 mouse model of Alzheimer's disease. Tetrathiomolybdate treatment lowered brain copper and reduced amyloid-β levels in the prevention paradigm, but not in the treatment paradigm. Our data suggests that controlled lowering of systemic copper may achieve anti-amyloid effects if initiated early in the disease process.

A copper-lowering strategy attenuates amyloid pathology in a transgenic mouse model of Alzheimer's disease.

Alzheimer's disease (AD) causes progressive, age-dependent cortical and hippocampal dysfunction leading to abnormal intellectual capacity and memory. We propose a novel protective treatment for AD pathology with phytic acid (inositol hexakisphosphate), a phytochemical found in food grains and a key signaling molecule in mammalian cells. We evaluated the protective and beneficial effects of phytic acid against amyloid-β (Aβ) pathology in MC65 cells and the Tg2576 mouse model. In MC65 cells, 48-72-hour treatment with phytic acid provided complete protection against amyloid precursor protein-C-terminal fragment-induced cytotoxicity by attenuating levels of increased intracellular calcium, hydrogen peroxide, superoxide, Aβ oligomers, and moderately upregulated the expression of autophagy (beclin-1) protein. In a tolerance paradigm, wild type mice were treated with 2% phytic acid in drinking water for 70 days. Phytic acid was well tolerated. Ceruloplasmin activity, brain copper and iron levels, and brain superoxide dismutase and ATP levels were unaffected by the treatment. There was a significant increase in brain levels of cytochrome oxidase and a decrease in lipid peroxidation with phytic acid administration. In a treatment paradigm, 12-month old Tg2576 and wild type mice were treated with 2% phytic acid or vehicle for 6 months. Brain levels of copper, iron, and zinc were unaffected. The effects of phytic acid were modest on the expression of AβPP trafficking-associated protein AP180, autophagy-associated proteins (beclin-1, LC3B), sirtuin 1, the ratio of phosphorylated AMP-activated protein kinase (PAMPK) to AMPK, soluble Aβ1-40, and insoluble Aβ1-42. These results suggest that phytic acid may provide a viable treatment option for AD.

Phytic acid as a potential treatment for alzheimer's pathology: evidence from animal and in vitro models.

The effect of gender on systemic and brain levels of copper is relatively understudied. We examined gender effects in mice and human subjects. We observed a trend to higher serum copper levels in female compared to male LaFerla "triple transgenic" (1399 ± 233 versus 804 ± 436 ng/mL, P = 0.06) mice, and significantly higher brain copper levels in female- versus male wild-type mice (5.2 ± 0.2 versus 4.18 ± 0.3 ng/mg wet wt, P = 0.03). Plasma copper was significantly correlated with brain copper in mice (R2 = 0.218; P = 0.038). Among human subjects with AD, both plasma copper (1284 ± 118 versus 853 ± 81 ng/mL, P = 0.005) and cerebrospinal fluid copper (12.8 ± 1 versus 10.4 ± 0.7 ng/mL, P = 0.01) were elevated in women compared to men. Among healthy control subjects, plasma copper (1008 ± 51 versus 836 ± 41 ng/mL; P = 0.01) was higher in women than in men, but there was no difference in cerebrospinal fluid copper. We conclude that gender differences in copper status may influence copper-mediated pathological events in the brain.

Gender effects on plasma and brain copper.

Copper plays an important role in numerous biological processes across all living systems predominantly because of its versatile redox behavior. Cellular copper homeostasis is tightly regulated and disturbances lead to severe disorders such as Wilson disease and Menkes disease. Age-related changes of copper metabolism have been implicated in other neurodegenerative disorders such as Alzheimer disease. The role of copper in these diseases has been a topic of mostly bioinorganic research efforts for more than a decade, metal-protein interactions have been characterized, and cellular copper pathways have been described. Despite these efforts, crucial aspects of how copper is associated with Alzheimer disease, for example, are still only poorly understood. To take metal-related disease research to the next level, emerging multidimensional imaging techniques are now revealing the copper metallome as the basis to better understand disease mechanisms. This review describes how recent advances in X-ray fluorescence microscopy and fluorescent copper probes have started to contribute to this field, specifically in Wilson disease and Alzheimer disease. It furthermore provides an overview of current developments and future applications in X-ray microscopic methods.

Opportunities in multidimensional trace metal imaging: taking copper-associated disease research to the next level.

Copper-transporting ATPase ATP7A is essential for mammalian copper homeostasis. Loss of ATP7A activity is associated with fatal Menkes disease and various other pathologies. In cells, ATP7A inactivation disrupts copper transport from the cytosol into the secretory pathway. Using fibroblasts from Menkes disease patients and mouse 3T3-L1 cells with a CRISPR/Cas9-inactivated ATP7A, we demonstrate that ATP7A dysfunction is also damaging to mitochondrial redox balance. In these cells, copper accumulates in nuclei, cytosol, and mitochondria, causing distinct changes in their redox environment. Quantitative imaging of live cells using GRX1-roGFP2 and HyPer sensors reveals highest glutathione oxidation and elevation of H2O2 in mitochondria, whereas the redox environment of nuclei and the cytosol is much less affected. Decreasing the H2O2 levels in mitochondria with MitoQ does not prevent glutathione oxidation; i.e. elevated copper and not H2O2 is a primary cause of glutathione oxidation. Redox misbalance does not significantly affect mitochondrion morphology or the activity of respiratory complex IV but markedly increases cell sensitivity to even mild glutathione depletion, resulting in loss of cell viability. Thus, ATP7A activity protects mitochondria from excessive copper entry, which is deleterious to redox buffers. Mitochondrial redox misbalance could significantly contribute to pathologies associated with ATP7A inactivation in tissues with paradoxical accumulation of copper (i.e. renal epithelia).

The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria.

The anterior pituitary is specialized for the synthesis, storage and release of peptide hormones. The activation of inactive peptide hormone precursors requires a specific set of proteases and other post-translational processing enzymes. High levels of peptidylglycine α-amidating monooxygenase (PAM), an essential peptide processing enzyme, occur in the anterior pituitary. PAM, which converts glycine-extended peptides into amidated products, requires copper and zinc to support its two catalytic activities and calcium for structure. We used X-ray fluorescence microscopy on rat pituitary sections and inductively coupled plasma mass spectrometry on subcellular fractions prepared from rat anterior pituitary to localize and quantify copper, zinc and calcium. X-ray fluorescence microscopy indicated that the calcium concentration in pituitary tissue was about 2.5 mM, 10-times more than zinc and 50-times more than copper. Although no higher than cytosolic levels, secretory granule levels of copper exceeded PAM levels by a factor of 10. Atp7a, which transports copper into the lumen of the secretory pathway, was enriched in endosomes and Golgi, not in secretory granules. If Atp7a transfers copper directly to PAM, this pH-dependent process is likely to occur in Golgi and endosomes.

Article	Year
Quantitative imaging of metals in tissues. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine\| PubMed ID: 19130257	2009
The loop connecting metal-binding domains 3 and 4 of ATP7B is a target of a kinase-mediated phosphorylation. Biochemistry\| PubMed ID: 19405516	2009
Wilson disease at a single cell level: intracellular copper trafficking activates compartment-specific responses in hepatocytes. The Journal of biological chemistry\| PubMed ID: 20647314	2010
A copper-lowering strategy attenuates amyloid pathology in a transgenic mouse model of Alzheimer's disease. Journal of Alzheimer's disease : JAD\| PubMed ID: 20693639	2010
Phytic acid as a potential treatment for alzheimer's pathology: evidence from animal and in vitro models. Journal of Alzheimer's disease : JAD\| PubMed ID: 20930278	2011
Gender effects on plasma and brain copper. International journal of Alzheimer's disease\| PubMed ID: 22028982	2011
Opportunities in multidimensional trace metal imaging: taking copper-associated disease research to the next level. Analytical and bioanalytical chemistry\| PubMed ID: 23079951	2013
The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria. The Journal of biological chemistry\| PubMed ID: 27226607	2016
Copper, zinc and calcium: imaging and quantification in anterior pituitary secretory granules. Metallomics : integrated biometal science\| PubMed ID: 27426256	2016

Martina Ralle

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