Loss of NCB5OR in the cerebellum disturbs iron pathways, potentiates behavioral abnormalities, and exacerbates harmaline-induced tremor in mice

Abstract

Iron dyshomeostasis has been implicated in many diseases, including a number of neurological conditions. Cytosolic NADH cytochrome b5 oxidoreductase (NCB5OR) is ubiquitously expressed in animal tissues and is capable of reducing ferric iron in vitro. We previously reported that global gene ablation of NCB5OR resulted in early-onset diabetes and altered iron homeostasis in mice. To further investigate the specific effects of NCB5OR deficiency on neural tissue without contributions from known phenotypes, we generated a conditional knockout (CKO) mouse that lacks NCB5OR only in the cerebellum and midbrain. Assessment of molecular markers in the cerebellum of CKO mice revealed changes in pathways associated with cellular and mitochondrial iron homeostasis. 59Fe pulse-feeding experiments revealed cerebellum-specific increased or decreased uptake of iron by 7 weeks and 16 weeks of age, respectively. Additionally, we characterized behavioral changes associated with loss of NCB5OR in the cerebellum and midbrain in the context of dietary iron deprivation-evoked generalized iron deficiency. Locomotor activity was reduced and complex motor task execution was altered in CKO mice treated with an iron deficient diet. A sucrose preference test revealed that the reward response was intact in CKO mice, but that iron deficient diet consumption altered sucrose preference in all mice. Detailed gait analysis revealed locomotor changes in CKO mice associated with dysfunctional proprioception and locomotor activation independent of dietary iron deficiency. Finally, we demonstrate that loss of NCB5OR in the cerebellum and midbrain exacerbated harmaline-induced tremor activity. Our findings suggest an essential role for NCB5OR in maintaining both iron homeostasis and the proper functioning of various locomotor pathways in the mouse cerebellum and midbrain.

NCB5OR Deficiency in the Cerebellum and Midbrain Leads to Dehydration and Alterations in Thirst Response, Fasted Feeding Behavior, and Voluntary Exercise in Mice

“…Rather, male CKO mice have decreased locomotor activity during times in which peak activity is expected (dark cycle). This observation is consistent with our previous study detailing altered locomotor behavior in CKO mice [10]. However, the finding is novel in that these data indicate male CKO mice might possess an atypical disposition to voluntary wheel running: an activity normally reveled by mice.…”

“…All mice were of C57BL/6J background. The generation of CKO mice was achieved as previously described [10]. Deletion of NCB5OR in the cerebellum was confirmed through qPCR analysis of functional NCB5OR transcripts, of which the loss of sequences corresponding to exon 3 results in a premature "stop" codon and a highly truncated protein product of 89 residues.…”

“…CKO and WT mice underwent gait analysis using a DigiGait apparatus (Mouse Specifics) and were tested on the Rota-rod as described in more detail elsewhere [10]. Briefly, DigiGait analysis was conducted under walking conditions at a speed of 10 cm/s, and Rota-rod measurements were collected at an accelerating speed of 4 to 40 rpm for no longer than 5 min.…”

“…Mice globally deficient for NCB5OR experience early-onset diabetes, growth retardation, decreased serum leptin levels, and increased food consumption despite maintaining a significantly reduced body weight relative to wild-type (WT) littermates [6][7][8][9]. We recently reported altered iron homeostasis, decreased iron-positive Purkinje cells, altered locomotor activity, and an increased response to harmaline-induced tremor in mice lacking NCB5OR in the cerebellum and the midbrain (conditional knockout or CKO mice) [10].…”

Crystal structures of the naturally fused CS and cytochrome b 5 reductase (b 5R) domains of Ncb5or reveal an expanded CS fold, extensive CS–b 5R interactions and productive binding of the NAD(P)+ nicotinamide ring

“…Cyb5R3 plays key roles in methemoglobin reduction (Kitao et al, 1974) and fatty-acid desaturation (Holloway, 1971), while much less is known about Cyb5R1 and Cyb5R2. The fourth isoform (Cyb5R4), known as Ncb5or (NADH cytochrome b 5 oxidoreductase; Zhu et al, 1999), is a flavohemoprotein that is implicated in diabetes, neurological diseases and iron homeostasis (Xie et al, 2004;Zhu et al, 2004;Wang et al, 2011;Stroh et al, 2016Stroh et al, , 2018. Ncb5or differs from Cyb5R3 in several notable ways.…”