Jaline Melo Oliveira

Analysis of Gene Expression Pattern and Neuroanatomical Correlates for SLC20A2 (PiT-2) Shows a Molecular Network with Potential Impact in Idiopathic Basal Ganglia Calcification (“Fahr’s Disease”)

Familial idiopathic basal ganglia calcification (FIBGC), also known as “Fahr’s disease,” is a neuropsychiatric disorder with motor and cognitive symptoms. It is characterized pathologically by bilateral calcification most commonly in the basal ganglia and also in other brain regions such as the thalamus and cerebellum. A recent report by Wang et al. (2012) discovered multiple families with FIBGC carrying mutations in the SLC20A2 gene, encoding the inorganic phosphate transporter PiT-2, which segregated in an autosomal dominant pattern. To understand further the role of SLC20A2 in FIBGC brain pathology, here we described the gene expression pattern across the whole brain for SLC20A2, using the Allen Institute Human Brain Atlas database. Microarray analysis provided evidence that the neuroanatomical pattern of expression for SLC20A2 is highest in the regions most commonly affected in FIBGC. Neuroanatomical regions that demonstrated high correlation or anti-correlation with SLC20A2 expression also showed a molecular network with potential to explain the limited neuroanatomical distribution of calcifications in IBGC. Lastly, these co-expression networks suggest additional further candidate genes for FIBGC.

New Studies on Knockout Mouse for the SLC20A2 Gene Show Much More Than Brain Calcifications

To the editor We read with great interest the recent contribution by Jensen et al. (2015) and Wallingford et al. (2016a, b), reporting additional phenotyping data for SLC20A2 knockout (KO) mouse, originally described in 2013 (Jensen et al. 2013). The SLC20A2 KO was evaluated as a model for human primary familial brain calcification (PFBC) and appears to correlate well with the loss of function mutants found in PFBC patients, where SLC20A2 represents ~40-50 % of inherited cases (Ferreira et al. 2014). The reported similarities between SLC20A2 KO mouse and human pathologies was striking. Now, Jensen et al. (2015) show significantly elevated inorganic phosphate levels (Pi) in cerebrospinal fluid (CSF) from the SLC20A2 KO, confirming PiT-2 regulates Pi in the CSF. Separately, Wallingford et al. (2016a, b) described several additional central and peripheral phenotypes in SLC20A2 −/− mouse, including: elevated phosphate in CSF and CSF producing tissues (choroid plexus, ependymal, arteriolar smooth muscle cells), calcified optic nerve tissue, and hydrocephalus. Both studies confirm the importance of SLC20A2 in Pi homeostasis and clearance from the CSF generating tissues, increasing the vulnerability of smooth muscle cells to glymphatic pathwayassociated arteriolar calcification. They also report that heterozygous mice would present calcifications, as they get older, mimicking the patient profile. PFBC is characterized by symmetric calcification in the basal ganglia and other brain regions, including a wide spectrum of neuropsychiatric symptoms such as parkinsonism, psychosis, seizures, and chronic headache. The usage of Bprimary^ alludes to the lack of hormonal, infectious, or traumatic causes that could lead to brain calcifications (Sobrido et al. 2014). PFBC is inherited in an autosomal dominant manner and, thus far, four genes have been identified. Two have been linked to phosphate metabolism (SLC20A2, XPR1), while the other two were initially associated with blood brain barrier (BBB) integrity and pericytes maintenance (PDGFB, PDGFRB)(Tadic et al. 2015). The Wallingford et al. (2016a, b) analysis challenges previous interpretations linking brain calcification to BBB integrity, suggesting that brain calcifications are found even with normal BBB functioning, through a two-hit mechanism whereby increased CSF Pi leads to calcification in arteriolar smooth muscle cells due to an enhanced vulnerability caused by SLC20A2 deficiency. This idea is further supported by the recent questioning of the BBB hypothesis as a potential cause for PFBC. Part of the original group who linked PDFGB BBB deficiency with calcification in PDGFB KO mice found that calcification-prone brain regions had a more intact blood– brain barrier (BBB) and higher pericyte coverage compared to non-calcification-prone brain regions (Vanlandewijck et al. 2015). On the other hand, a patient reported with brain calcification, and a novelPDGFBmutation presented with electron microscopy of skin biopsy showing capillary basal membrane abnormalities consistent with microangiopathy (Biancheri et al. 2016). Large scale phenotyping of this very same SLC20A2KO is available in public repositories (http://www.mousephenotype. * J. R. M. Oliveira joao.ricardo@ufpe.br

Combined Genome-Wide CSF Aβ-42’s Associations and Simple Network Properties Highlight New Risk Factors for Alzheimer’s Disease

The abnormal deposition of amyloid-β protein in the brain plays an important role in Alzheimer’s disease (AD), being considered a potential clinical biomarker. To investigate genetic associations with amyloid-β we used biomarker data and genome-wide variants from individuals with AD and mild cognitive impairment in the Alzheimer’s Disease Neuroimaging Initiative (ADNI) database. We used a standard linear model and retested the associations with a mixed linear model to correct the residual sample structure. Both methods’ results showed two identical significant SNPs associated with the A β-42 levels in CSF (rs2075650 at intron region TOMM40 with p-value ≥ 1 × 10–16 and rs439401 in the intergenic region of LOC100129500 and APOC1 with p-value ≥ 1 × 10-9) and highlighted APOC1 and TOMM40, which are well-known genes previously associated with AD. Extending our analysis, we considered possible candidate genes mapped to SNPs with p-value ≥ 1 × 10-6 to explore gene-set enrichment e gene-gene network analysis, which reveals genes related to synaptic transmission, transmission of nerve impulses, cell-cell signaling and neurological processes. These genes require fine mapping and replication studies to allow more detailed understanding of how they may contribute to the genetic architecture of AD.

Improving Significance in Association Studies: a New Perspective for Association Studies Submitted to the Journal of Molecular Neuroscience

Dear Editor, The Journal of Molecular Neuroscience has a long tradition of accepting various formats of studies concerning neurosciences, with a great variety of methodological approaches. Amongst them, we find case-control studies to be one of the most frequently occurring formats. Case-control studies are appropriate to evaluate the influence of genetic variants, often linked to complex disorders. A significant example is the positive association between the e4 APOE allele and Alzheimer’s disease, widely replicated since the beginning of the 90s (Ridge et al 2013). However, wewant to keep supporting such studies but with a more stringent selection. This will avoid redundant attempts to analyze complex disorders with unsuitable approaches such as limited number of SNPs and/or small populations. Basic recommendations include:

Erratum to: MiR-9-5p Down-Regulates PiT2, but not PiT1 in Human Embryonic Kidney 293 Cells

PiT1 (SLC20A1) and PiT2 (SLC20A2) are members of the mammalian type-III inorganic phosphate transporters and recent studies linked SLC20A2 mutations with primary brain calcifications. MicroRNAs (miRNAs) are endogenous noncoding regulatory RNAs and MicroRNA-9 (miR-9) modulates neurogenesis but is also involved with different types of cancer. We evaluated possible interactions between miR-9 and the phosphate transporters (PiT1 and PiT2). SLC20A2, platelet-derived growth factor receptor beta (PDGFRB) and Fibrillin-2 (FBN2) showed binding sites with high affinity for mir-9, In silico. miR-9 mimic was transfected into HEK293 cells and expression was confirmed by RT-qPCR. Overexpression of miR-9 in these cells caused a significant reduction in PiT2 and FBN2. PDGFRB appeared to be decreased, but was not significantly down-regulated. PiT1 showed no significant difference relative to controls. The down-regulation of PiT2 protein by miR-9 was confirmed by western blotting. In conclusion, we showed that miR-9 can down-regulate PiT2, in HEK293 cells. The online version of the original article can be found at http://dx.doi. org/10.1007/s12031-017-0906-0 * D. P. Bezerra darlenepaiva@gmail.com * M. Keasey keasey@etsu.edu * J. R. M. Oliveira joao.ricardo@ufpe.br 1 Keizo Asami Laboratory, Federal University of Pernambuco, Recife, PE, Brazil 2 Neuropsychiatry Department, Federal University of Pernambuco, Recife, PE, Brazil 3 Department of Biomedical Sciences – Quillen College of Medicine, East Tennessee State University, Johnson City, TN, USA J Mol Neurosci (2017) 62:34 DOI 10.1007/s12031-017-0913-1

MiR-9-5p Down-Regulates PiT2, but not PiT1 in Human Embryonic Kidney 293 Cells

Inorganic phosphate (Pi) is an essential component for structure and metabolism. PiT1 (SLC20A1) and PiT2 (SLC20A2) are members of the mammalian type-III inorganic phosphate transporters. SLC20A2 missense variants are associated with primary brain calcification. MicroRNAs (miRNAs) are endogenous noncoding regulatory RNAs, which play important roles in post-transcriptional gene regulation. MicroRNA-9 (miR-9) acts at different stages of neurogenesis, is deeply rooted in gene networks controlling the regulation of neural progenitor proliferation, and is also linked with cancers outside the nervous system. We evaluated possible interactions between miR-9 and the phosphate transporters (PiT1 and PiT2). SLC20A2, platelet-derived growth factor receptor beta (PDGFRB) and Fibrillin-2 (FBN2) showed binding sites with high affinity for mir-9, in silico. miR-9 mimic was transfected into HEK293 cells and expression confirmed by RT-qPCR. Overexpression of miR-9 in these cells caused a significant reduction in PiT2 and FBN2. PDGFRB appeared to be decreased, but was not significantly down-regulated in our hands. PiT1 showed no significant difference relative to controls. The down-regulation of PiT2 protein by miR-9 was confirmed by western blotting. In conclusion, we showed miR-9 can down-regulate PiT2, in HEK293 cells.