Increased Zinc and Manganese in Parallel with Neurodegeneration, Synaptic Protein Changes and Activation of Akt/GSK3 Signaling in Ovine CLN6 Neuronal Ceroid Lipofuscinosis

Abstract

Mutations in the CLN6 gene cause a variant late infantile form of neuronal ceroid lipofuscinosis (NCL; Batten disease). CLN6 loss leads to disease clinically characterized by vision impairment, motor and cognitive dysfunction, and seizures. Accumulating evidence suggests that alterations in metal homeostasis and cellular signaling pathways are implicated in several neurodegenerative and developmental disorders, yet little is known about their role in the NCLs. To explore the disease mechanisms of CLN6 NCL, metal concentrations and expression of proteins implicated in cellular signaling pathways were assessed in brain tissue from South Hampshire and Merino CLN6 sheep. Analyses revealed increased zinc and manganese concentrations in affected sheep brain in those regions where neuroinflammation and neurodegeneration first occur. Synaptic proteins, the metal-binding protein metallothionein, and the Akt/GSK3 and ERK/MAPK cellular signaling pathways were also altered. These results demonstrate that altered metal concentrations, synaptic protein changes, and aberrant modulation of cellular signaling pathways are characteristic features in the CLN6 ovine form of NCL.

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

“…We reported biometal elevation in the brains of 2 CLN6 affected sheep models post-clinical onset [14], and in the brains and heart of presymptomatic Cln6 mice [15]. Here we show significantly increased concentrations of Cu and Zn in the brains of Merino CLN6 affected sheep prior to clinical onset.…”

“…For instance, the stabilization in the levels of Cu in the brainstem, frontal and parietal lobes observed at 7 months (Table 1) parallels a transient improvement in lysosomal function (Figure 1A). This improvement could represent the temporary efficacy of endogenous compensatory mechanisms such as increased MT expression (Figure 2F) that ultimately fail due to presence of persistent stressors, causing animals to succumb to disease by 14 months of age when metal concentrations rise as reported [14]. Moreover, homeostasis of multiple biometals is highly inter-related, and changes in one metal can greatly affect the absorption or trafficking of others in a cell- and tissue-specific manner [58].…”

A Murine Model of Variant Late Infantile Ceroid Lipofuscinosis Recapitulates Behavioral and Pathological Phenotypes of Human Disease

“…Protein-protein interaction studies have demonstrated binding of CLN6 to the collapsin response mediator protein-2 (CRMP-2), suggesting a role of CLN6 in axonal transport, elongation or maintenance [29]. Several recent studies in both mouse and sheep models have demonstrated that loss of CLN6 leads to a disruption in synaptic function and/or levels of essential synaptic proteins [30], [31]. Changes in cholesterol dynamics in CLN6 deficient cells have hinted a role of this protein in regulating structure and function of caveolae and lipid rafts, as well as protein sorting mechanisms [28].…”

X-ray fluorescence imaging reveals subcellular biometal disturbances in a childhood neurodegenerative disorder

“…We previously reported accumulation of zinc, copper, cobalt, manganese and iron in brain tissue homogenates from natural sheep and mouse models of NCLs, a group of inherited childhood neurodegenerative and lysosomal storage disorders 10, 11 . We observed early and progressive loss of the ER/Golgi resident biometal transporter Zip7, in the brains of sheep and primary cortical neurons from embryonic mice containing natural mutations in the Cln6 gene 14 .…”

“…ICP-MS was performed on cell pellets as previously described 11 . Cellular metal content was normalized to cellular protein content as measured by BCA assay (Thermo Fisher Scientific, VIC, Australia).…”