Diet plays a significant role in the pathogenesis of inflammatory bowel disease (IBD). A recent epidemiological study has shown an inverse relationship between nutritional manganese (Mn) status and IBD patients. Mn is an essential micronutrient required for normal cell function and physiological processes. To date, the roles of Mn in intestinal homeostasis remain unknown and the contribution of Mn to IBD has yet to be explored. Here, we provide evidence that Mn is critical for the maintenance of the intestinal barrier and that Mn deficiency exacerbates dextran sulfate sodium (DSS)-induced colitis in mice. Specifically, when treated with DSS, Mn-deficient mice showed increased morbidity, weight loss, and colon injury, with a concomitant increase in inflammatory cytokine levels and oxidative and DNA damage. Even without DSS treatment, dietary Mn deficiency alone increased intestinal permeability by impairing intestinal tight junctions. In contrast, mice fed a Mn-supplemented diet showed slightly increased tolerance to DSS-induced experimental colitis, as judged by the colon length. Despite the well-appreciated roles of intestinal microbiota in driving inflammation in IBD, the gut microbiome composition was not altered by changes in dietary Mn. We conclude that Mn is necessary for proper maintenance of the intestinal barrier and provides protection against DSS-induced colon injury. K E Y W O R D Scolitis, gut microbiota, inflammatory bowel disease, manganese, tight junction 2930 | CHOI et al. | RNA isolation and RT-PCR

Deficiencies of the transmembrane iron-transporting protein ferroportin (FPN1) cause the iron misdistribution that underlies ferroportin disease, anemia of inflammation, and several other human diseases and conditions. A small molecule natural product, hinokitiol, was recently shown to serve as a surrogate transmembrane iron transporter that can restore hemoglobinization in zebrafish deficient in other iron transporting proteins and can increase gut iron absorption in FPN1-deficient flatiron mice. However, whether hinokitiol can restore normal iron physiology in FPN1-deficient animals or primary cells from patients and the mechanisms underlying such targeted activities remain unknown. Here, we show that hinokitiol redistributes iron from the liver to red blood cells in flatiron mice, thereby increasing hemoglobin and hematocrit. Mechanistic studies confirm that hinokitiol functions as a surrogate transmembrane iron transporter to release iron trapped within liver macrophages, that hinokitiol-Fe complexes transfer iron to transferrin, and that the resulting transferrin-Fe complexes drive red blood cell maturation in a transferrin-receptor–dependent manner. We also show in FPN1-deficient primary macrophages derived from patients with ferroportin disease that hinokitiol moves labile iron from inside to outside cells and decreases intracellular ferritin levels. The mobilization of nonlabile iron is accompanied by reductions in intracellular ferritin, consistent with the activation of regulated ferritin proteolysis. These findings collectively provide foundational support for the translation of small molecule iron transporters into therapies for human diseases caused by iron misdistribution.

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Humans age and the ageing process affects cells in all areas of the human body, including nerve cells within the brain. With advancing age there is also a rise in the probability of developing a neurodegenerative disorder such as, e.g., amyotrophic lateral sclerosis, Huntington's disease, Parkinson's disease, or Alzheimer's disease. In all these age-related neurodegenerative disorders, distinct neuron populations within specific brain regions are primarily affected. For example, Parkinson's disease is characterized by a slowly progressive degeneration of dopaminergic neurons in the substantia nigra whereas the entorhinal cortex is first affected in Alzheimer's disease. In patients suffering from Huntington's disease, neurons in both striatum and cortex undergo substantial cell loss and in amyotrophic lateral sclerosis the neurodegeneration arises from the spinal cord and the motor cortex. For the investigation of the differences in neuronal vulnerability, it is important to examine the protein expression pattern in these specific neural populations. By this, conclusions about the origination process of these diseases can be achieved. In order to obtain this objective, specific isolation of distinct neurons from the surrounding brain tissue is indispensable. However, discrimination as well as isolation of distinct types of neurons can be challenging, due to the brain tissue's complexity. With traditional methods such as the homogenization of tissue samples, a specific isolation of single neuron populations is not feasible because homogenization results into a mixture containing all cell types. Laser microdissection can overcome this technical limitation. First, this method enables visualization of tissues via a microscopic unit and therefore an enhanced discrimination of different brain cells. Second, a laser device guarantees a contact-free and consequently a contamination-free separation of distinct neurons from the surrounding brain tissue. In the following, we present a detailed protocol that includes a workflow for the isolation and analysis of neurons from freshly frozen post mortem human brain tissue samples. During this procedure, the brain tissue is sectioned, stained, laser microdissected, and ultimately analyzed by high-performance liquid chromatography-mass spectrometry.

Manganese (Mn), primarily acquired through diet, is required for brain function and development. Epidemiological studies have found an association between both low and high levels of Mn and impaired neurodevelopment in children. Recent genetic studies have revealed that patients with congenital Mn deficiency display severe psychomotor disability and cerebral and cerebellar atrophy. Although the impact of Mn on gene expression is beginning to be appreciated, Mn-dependent gene expression remains to be explored in vertebrate animals. The goal of this study was to use a mouse model to define the impact of a low-Mn diet on brain metal levels and gene expression. We interrogated gene expression changes in the Mn-deficient mouse brain at the genome-wide scale by RNA-seq analysis of the cerebellum of mice fed low or normal Mn diets. A total of 137 genes were differentially expressed in Mn-deficient cerebellums compared with Mn-adequate cerebellums (Padj < 0.05). Mn-deficient mice displayed downregulation of key pathways involved with “focal adhesion,” “neuroactive ligand-receptor interaction,” and “cytokine-cytokine receptor interaction” and upregulation of “herpes simplex virus 1 infection,” “spliceosome,” and “FoxO signaling pathway.” Reactome pathway analysis identified upregulation of the splicing-related pathways and transcription-related pathways, as well as downregulation of “metabolism of carbohydrate,” and “extracellular matrix organization,” and “fatty acid metabolism” reactomes. The recurrent identifications of splicing-related pathways suggest that Mn deficiency leads to upregulation of splicing machineries and downregulation of diverse biological pathways.

Objectives Neurodegeneration with brain iron accumulation (NBIA) is a clinically and genetically heterogeneous group of neurodegenerative diseases characterized by an abnormal accumulation of brain iron and progressive degeneration of the nervous system. β-propeller protein-associated neurodegeneration (BPAN) (OMIM #300,894) is a recently identified subtype of NBIA. BPAN is caused by de novo mutations in the WD repeat domain 45 (WDR45) gene. WDR45 deficiency in BPAN patients and animal models has shown defects in autophagic flux, suggesting a role for WDR45 in autophagy. How WDR45 deficiency leads to brain iron overload remains unclear. The goal of the present study is to identify the pathogenic mechanisms of WDR45 deficiency that cause iron overload and neurodegeneration. Methods To elucidate the role of WDR45 in dopaminergic neuronal cells, we generated a WDR45-knockout (KO) SH-SY5Y cell line by CRISPR/Cas9-mediated genome editing. To identify mechanisms underlying iron homeostasis and transport, we examined two cellular iron acquisition pathways in these cells using radioactive isotope 59Fe: 1) the canonical transferrin-bound iron (TBI) uptake pathway and 2) the nontransferrin-bound iron (NTBI) pathway. Results Loss of WDR45 increased total iron levels with a concomitant increase in the iron storage protein ferritin in neuronal cells. Specifically, WDR45-KO cells preferentially took up NTBI compared to wild-type cells. Concordant with these functional data, the level of divalent metal transporter-1 (DMT1) expression was upregulated in WDR45-KO cells, providing a causal link to iron overload in WDR45 deficiency. In addition, loss of WDR45 led to defects in autophagic flux and impaired ferritinophagy, a lysosomal process that promotes ferritin degradation, suggesting that iron overload is driven by impaired ferritinophagy. Interestingly, WDR45 deficiency increased iron accumulation in the mitochondria, impaired mitochondrial function, and in turn, elevated reactive oxygen species generation. Conclusions Our study provides the first evidence that WDR45 deficiency alters cellular iron acquisition pathways thereby leading to iron accumulation in neuronal cells. These findings will serve as a basis for developing therapeutic strategies for patients with NBIA. Funding Sources NIH, NBIA Disorder Association.