Deregulation of subcellular biometal homeostasis through loss of the metal transporter, Zip7, in a childhood neurodegenerative disorder

Abstract

Background: Aberrant biometal metabolism is a key feature of neurodegenerative disorders including Alzheimer's and Parkinson's diseases. Metal modulating compounds are promising therapeutics for neurodegeneration, but their mechanism of action remains poorly understood. Neuronal ceroid lipofuscinoses (NCLs), caused by mutations in CLN genes, are fatal childhood neurodegenerative lysosomal storage diseases without a cure. We previously showed biometal accumulation in ovine and murine models of the CLN6 variant NCL, but the mechanism is unknown. This study extended the concept that alteration of biometal functions is involved in pathology in these disorders, and investigated molecular mechanisms underlying impaired biometal trafficking in CLN6 disease.

Results: We observed significant region-specific biometal accumulation and deregulation of metal trafficking pathways prior to disease onset in CLN6 affected sheep. Substantial progressive loss of the ER/Golgi-resident Zn transporter, Zip7, which colocalized with the disease-associated protein, CLN6, may contribute to the subcellular deregulation of biometal homeostasis in NCLs. Importantly, the metal-complex, ZnII(atsm), induced Zip7 upregulation, promoted Zn redistribution and restored Zn-dependent functions in primary mouse Cln6 deficient neurons and astrocytes.

Conclusions: This study demonstrates the central role of the metal transporter, Zip7, in the aberrant biometal metabolism of CLN6 variants of NCL and further highlights the key contribution of deregulated biometal trafficking to the pathology of neurodegenerative diseases. Importantly, our results suggest that ZnII(atsm) may be a candidate for therapeutic trials for NCLs.

Figure 1

Lysosomal dysfunction and increased α-synuclein concentrations in CLN6 sheep. (A) Cathepsin D activity was measured in homogenates (1 μg) isolated from the occipital lobe of 3, 7 and 14 month old control or CLN6 affected Merino sheep (N = 3 per group) using a fluorometric Cathepsin D activity assay. (B-D) Densitometry and representative immunoblots of homogenates (5–40 μg) isolated from the occipital lobe of 3, 7 and 14 month old control or CLN6 affected sheep probed with antibodies directed against V-ATPase (B), α-synuclein (C) or ATP13a2 (D). GAPDH was used as a loading control. Quantification was performed in ImageJ and concentrations are expressed relative to those in control sheep at each age. Data are mean + SEM. **p < 0.01, ***p < 0.001 by Student’s t test. C, control.

Figure 2

Altered biometal trafficking pathways are associated with loss of CLN6 in sheep. (A-F) Densitometry and representative immunoblots of homogenates (5–40 μg) isolated from the occipital lobe of 3, 7 and 14 month old control or CLN6 affected sheep (N = 3 per group) probed with antibodies directed against CLN6 or a range of metal transporters or metal binding proteins. GAPDH, β-tubulin, total Akt or total ERK, as appropriate, were used as loading controls. Quantification was performed in ImageJ and levels are expressed relative to those in control sheep at each age. Data are mean + SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test. C, control. (G-H) Normalized Zip7 protein levels in Merino sheep occipital lobe were plotted against normalized levels of CLN6 (G) or MT (H) to determine correlations between these proteins. Linear regression analysis was performed in GraphPad Prism.

Figure 3

Biometals accumulate in subcellular fractions that display Zip7 loss in presymptomatic CLN6 sheep occipital lobe. Sucrose density gradient fractions from 3 month old sheep occipital lobe were analyzed for metal content by ICP-MS. Data are expressed as the mean + SEM of Zn (A) and Cu (B) concentrations in CLN6 brains (white bars throughout) and control brains (black bars throughout) from 3 individual sheep per genotype. (C) Representative immunoblot of Zip7 protein present in fractions from control and CLN6 affected sheep brains.

Figure 4

Cln6 cortical neurons display reduced Cln6 transcripts and Zip7 staining with increased labile Zn accumulation. (A)Cln6 mRNA expression in primary murine cortical neurons was measured using qRT-PCR. Expression values were normalized to tubulin using the delta Ct method. All data are mean + SEM. (B) Zip7 expression was assessed by immunofluorescence. Zip7 was labeled with rabbit Zip7 antibodies and AlexaFluor conjugated anti-rabbit antibodies. Nuclei were stained with DAPI. Quantitative analysis of Zip7 perinuclear distribution was performed using the ArrayScan reader in conjunction with the compartmental analysis software on >1000 cells per genotype. (C) Labile Zn in control and Cln6 cortical neurons was measured by FluoZin-3 fluorescence. (D) Zip7 immunofluorescence images are representative of 3 experiments performed on triplicate coverslips. Scale bars represent 10 μm.

Figure 5

Zip7 co-localizes with CLN6. Primary mouse cortical neurons were reacted with rabbit primary anti-CLN6 and goat primary anti-Zip7 antibodies. Anti-goat AlexaFluor-488 and anti-rabbit AlexaFluor-568 dye labeled secondary antibodies were used to reveal Zip7 and CLN6 expression, respectively. (A) DAPI, (B) Zip7 and (C) CLN6 expression in primary cortical neurons was visualized by confocal microscopy using the Zeiss Meta confocal scanning laser microscope using a magnification of 40×. (D) Overlay images indicate colocalization in punctate structures throughout the cells. Scale bars correspond to 20 μm.

Figure 6

Delivery of bioavailable Zn restores Zn-dependent phenotypes through upregulation of Zip7. (A) Labile Zn in control (black bars throughout) and Cln6 (white bars throughout) cortical neurons after 1 h Zn^II(atsm) treatment was measured by FluoZin-3 fluorescence (N = 3). (B)MT1A mRNA expression after 1 h Zn^II(atsm) treatment in cortical neurons was assessed using qRT-PCR (N = 3). (C) Neurite length in control and Cln6 primary cortical neurons treated for 1 h with Zn^II(atsm) was determined by tubulin immunofluorescence. Images (>1,000 cells/well from 4 experiments) were taken using the ArrayScan High Content Platform and analysis was performed using Neuronal Profiling software (refer to Additional file 1). (D) ALP activity in control and Cln6 primary mouse astrocytes was determined after 1 h Zn^II(atsm) treatment (N = 3). (E) Zn^II(atsm)-dependent regulation of P-GSK and Zip7 levels after 4 h treatment was assessed in primary mouse astrocytes by western blotting. Images are from different lanes on the same gel. (F-G) Normalized Zip7 protein levels in Merino sheep occipital lobe were plotted against normalized levels of Zn (F) and P-GSK (G) to determine correlations between these proteins (N = 3 sheep per genotype). Linear regression analysis was performed in GraphPad Prism. (H-J) Zip7 knockdown results in hyperphosphorylation of GSK3. 100nM negative control siRNA (-) or Zip7 siRNA (+) was transfected into mouse NIH 3T3 cells. Zip7 (H) and P-GSK3 (I) protein expression was determined by Western blotting and normalized to expression of GAPDH. Data are expressed as mean + SEM values. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test. (J) Images are representative of 3 independent experiments.