Trehalose reduces retinal degeneration, neuroinflammation and storage burden caused by a lysosomal hydrolase deficiency

<p>The accumulation of undegraded molecular material leads to progressive neurodegeneration in a number of lysosomal storage disorders (LSDs) that are caused by functional deficiencies of lysosomal hydrolases. To determine whether inducing macroautophagy/autophagy via small-molecule therapy would be effective for neuropathic LSDs due to enzyme deficiency, we treated a mouse model of mucopolysaccharidosis IIIB (MPS IIIB), a storage disorder caused by deficiency of the enzyme NAGLU (alpha-N-acetylglucosaminidase [Sanfilippo disease IIIB]), with the autophagy-inducing compound trehalose. Treated <i>naglu</i>^{–/ –} mice lived longer, displayed less hyperactivity and anxiety, retained their vision (and retinal photoreceptors), and showed reduced inflammation in the brain and retina. Treated mice also showed improved clearance of autophagic vacuoles in neuronal and glial cells, accompanied by activation of the TFEB transcriptional network that controls lysosomal biogenesis and autophagic flux. Therefore, small-molecule-induced autophagy enhancement can improve the neurological symptoms associated with a lysosomal enzyme deficiency and could provide a viable therapeutic approach to neuropathic LSDs.</p> <p><b>Abbreviations</b>: ANOVA: analysis of variance; <i>Atg7</i>: autophagy related 7; AV: autophagic vacuoles; CD68: cd68 antigen; ERG: electroretinogram; ERT: enzyme replacement therapy; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary acidic protein; GNAT2: guanine nucleotide binding protein, alpha transducing 2; HSCT: hematopoietic stem cell transplantation; INL: inner nuclear layer; LC3: microtubule-associated protein 1 light chain 3 alpha; MPS: mucopolysaccharidoses; NAGLU: alpha-N-acetylglucosaminidase (Sanfilippo disease IIIB); ONL: outer nuclear layer; PBS: phosphate-buffered saline; PRKCA/PKCα: protein kinase C, alpha; S1BF: somatosensory cortex; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TFEB: transcription factor EB; VMP/VPL: ventral posterior nuclei of the thalamus</p

An Allelic Series of Mice Reveals a Role for RERE in the Development of Multiple Organs Affected in Chromosome 1p36 Deletions

<div><p>Individuals with terminal and interstitial deletions of chromosome 1p36 have a spectrum of defects that includes eye anomalies, postnatal growth deficiency, structural brain anomalies, seizures, cognitive impairment, delayed motor development, behavior problems, hearing loss, cardiovascular malformations, cardiomyopathy, and renal anomalies. The proximal 1p36 genes that contribute to these defects have not been clearly delineated. The arginine-glutamic acid dipeptide (RE) repeats gene (<i>RERE</i>) is located in this region and encodes a nuclear receptor coregulator that plays a critical role in embryonic development as a positive regulator of retinoic acid signaling. <i>Rere</i>-null mice die of cardiac failure between E9.5 and E11.5. This limits their usefulness in studying the role of RERE in the latter stages of development and into adulthood. To overcome this limitation, we created an allelic series of RERE-deficient mice using an <i>Rere</i>-null allele, <i>om</i>, and a novel hypomorphic <i>Rere</i> allele, <i>eyes3</i> (c.578T>C, p.Val193Ala), which we identified in an N-ethyl-N-nitrosourea (ENU)-based screen for autosomal recessive phenotypes. Analyses of these mice revealed microphthalmia, postnatal growth deficiency, brain hypoplasia, decreased numbers of neuronal nuclear antigen (NeuN)-positive hippocampal neurons, hearing loss, cardiovascular malformations–aortic arch anomalies, double outlet right ventricle, and transposition of the great arteries, and perimembranous ventricular septal defects–spontaneous development of cardiac fibrosis and renal agenesis. These findings suggest that RERE plays a critical role in the development and function of multiple organs including the eye, brain, inner ear, heart and kidney. It follows that haploinsufficiency of <i>RERE</i> may contribute–alone or in conjunction with other genetic, environmental, or stochastic factors–to the development of many of the phenotypes seen in individuals with terminal and interstitial deletions that include the proximal region of chromosome 1p36.</p> </div

<i>Rere</i>^om/eyes3 mice have a high rate of perinatal mortality.

*<p><i>p</i> = 0.1,</p>**<p><i>p</i><0.0001.</p

Cardiovascular malformations are seen in <i>Rere</i>^om/eyes3 embryos on a C57BL6 background.

<p>doi:10.1371/journal.pone.0057460.t002</p

<i>Rere</i>^om/eyes3 embryos on a C57BL6background have cardiovascular malformations.

<p>Transverse sections of <i>Rere</i>^om/eyes3 embryos and wild-type littermate controls at E15.5. A–B) A wild-type embryo with a normal left-sided descending aorta (white arrow), a pulmonary artery that emerges from the right ventricle (red arrow), and an ascending aorta that emerges from the left ventricle (yellow arrow). C) An <i>Rere</i>^om/eyes3 embryo with a right-sided aorta (white arrow) with an aberrant left subclavian artery (not shown) and double outlet right ventricle in which ascending aorta emerges from the right ventricle (yellow arrow). In this embryo, the left pulmonary artery (blue arrow) originates abnormally from the ductus arteriosus. D–F) An <i>Rere</i>^om/eyes3 embryo with transposition of the great arteries in which the ascending aorta emerges from the right ventricle (yellow arrow) and the ductus arteriosus and left and right pulmonary arteries (blue arrows) emerge from the left ventricle. The red arrow in panel E points to the region of the left ventricle below the valve leading to the pulmonary artery. This embryo also has a periventricular septal defect (black arrow). G–I) An <i>Rere</i>^om/eyes3 embryo with double outlet right ventricle in which the ascending aorta (yellow arrow) emerges from the right ventricle. This embryo had two separate periventricular septal defects (black arrows in panel H and I). LV = Left ventricle, RV = right ventricle, red scale bars = 0.5 mm.</p

A hypomorphic mutation in <i>Rere</i> is responsible for microphthalmia in <i>eyes3</i> mice.

<p>A) <i>eyes3</i> mice were identified in an ENU screen for recessive mutations based on microphthalmia and were also found to have decreased body size and occasional unilateral renal agenesis. B) Sequencing of the <i>Rere</i> gene in <i>eyes3</i> mice revealed a homozygous missense mutation, c.578T>C, which codes for a single amino acid change in a highly conserved residue in RERE’s BAH domain (p.Val193Ala). The locations of the previously described <i>om</i> allele and the <i>eyes3</i> allele <i>Rere</i> are shown along with chromatograms from wild-type (top), <i>Rere</i>^om/om (bottom left) and <i>Rere</i>^eyes3/eyes3 (bottom right) embryos. C) No discernible RERE protein is identified by western blot from <i>Rere</i>^om/om embryos at E10.5 suggesting that the <i>om</i> allele is a null allele. In contrast, the expression of RERE protein is not affected in <i>Rere</i>^eyes3/eyes3 embryos at E10.5. This suggests that the deleterious effect of the p.Val193Ala change is caused primarily by reduced RERE function rather than decreased RERE protein expression. D) In contrast to <i>Rere</i>^om/om embryos that die between E9.5 and E11.5, some <i>Rere</i>^om/eyes3 mice live into adulthood but have microphthalmia which is more severe than that seen in <i>Rere</i>^eyes3/eyes3 mice. These results suggest that the <i>eyes3</i> allele is a hypomorphic allele of <i>Rere</i>.</p

<i>Rere</i>^om/eyes3 embryos exhibit postnatal growth deficiency.

<p>A) The sizes of <i>Rere</i>^om/eyes3 embryos were comparable to wild-type embryos at E17.5 and E18.5. B) There was no significant difference in embryo weights between <i>Rere</i>^om/eyes3 embryos and their wild-type littermates at E17.5 and E18.5 (n≥4). C) Body weight was measured to evaluate the somatic growth of <i>Rere</i>^om/eyes3 mice between birth and 12 weeks of age. The body weights of <i>Rere</i>^om/eyes3 mice were significantly reduced after 1 week of age and plateaued at 6 weeks of age while wild-type, <i>Rere</i>^+/eyes3 and <i>Rere</i>^+/om mice continued to gain weight (n = 5–10 for each genotype. *<i> = p</i><0.01, ** = <i>p</i><0.03).</p

Cardiac fibrosis and renal agenesis in <i>Rere</i>^om/eyes3 mice.

<p>A) RERE nuclear staining was detected in the endocardium, myocardium, and epicardium in cardiac sections from 4 month-old wild-type mice. Lower panel (A’) is higher magnification of a boxed area in upper panel. B) Cardiac sections from 3 month-old wild-type (upper panel) and <i>Rere</i>^om/eyes3 (lower panel) mice. Masson’s trichrome staining revealed areas of interstitial fibrosis (black arrow) in sections from the <i>Rere</i>^om/eyes3 mouse that were not seen in those from the wild-type mouse. C) Renal agenesis in an <i>Rere</i>^om/eyes3 mouse at P0 with normal development of the adrenal gland (AG).</p

Decreased number of NeuN-positive neurons in the hippocampi of <i>Rere</i>^om/eyes3 embryos.

<p>A) RERE immunohistochemistry analyses of mid-sagittal sections of the brain at E18.5. Lower panel (A’) is higher magnification of the boxed area in the upper panel, which includes the hippocampus. RERE (red color) is primarily expressed in Ammon’s horn of the hippocampus at E18.5. Scale bar = 100 µm. B) Brain sections through the hippocampi of wild-type and <i>Rere</i>^om/eyes3 embryos were probed with anti-NeuN antibody (green color) at E18.5. Decreased numbers of NeuN-positive cells are seen in CA fields of the hippocampus from <i>Rere</i>^om/eyes3 embryos compared to those from wild-type embryos. Scale bar indicates 50 µm. C) NeuN-positive cells in Ammon’s horn, including CA1, CA2, and CA3, and the dentate gyrus were counted and normalized to the area of each region. The number of NeuN positive neurons per area was significantly decreased in the Ammon’s horns of <i>Rere</i>^om/eyes3 embryos when compared to wild-type embryos (analysis based on fifteen slides containing at least three sections for each of three or more embryos; * = <i>p</i><0.01). D) Immunohistochemical analyses of brain section from E18.5 embryos probed using either an anti-cleaved Caspase-3 antibody and an anti-Phospho-Histone H3 (pHH3) antibody. Cleaved Caspase-3-positive cells were not detected in <i>Rere</i>^om/eyes3 embryos. Phospho-Histone H3-positive cells were identified in the dentate gyri (outlined with a dashed yellow line) of embryos of each genotype. Scale bar = 100 µm. E) Phospho-Histone H3-positive cells were counted and normalized to the area of the dentate gyrus including subgranularzone (analysis based on fifteen slides containing at least three sections for each of three or more embryos). The number of Phospho-Histone H3-positive hippocampal cells per µm² was not significantly different between wild-type embryos and <i>Rere</i>^om/eyes3 embryos. CA = cornu ammonis; Cp = cortical plate; DG = dentate gyrus.</p

Brain hypoplasia in <i>Rere</i>^om/eyes3 embryos and mice.

<p>A) The brains of <i>Rere</i>^om/eyes3 embryos and mice appeared smaller than those of their wild-type litter mates at E17.5, E18.5 and P0 but no difference in overall morphology was observed between genotypes. B) Coronal brain sections from wild-type and <i>Rere</i>^om/eyes3 embryos at E18.5 were also comparable except for size. Representative examples of these sections are shown. Scale bar indicates 500 µm. C–D) The surface areas of the cerebral hemispheres (C) and cerebellum (D) were significantly reduced in <i>Rere</i>^om/eyes3 embryos and mice in comparison with those of wild-type embryos and mice between E17.5 and P0 (n≥5; * = <i>p</i><0.03). E) Whole brain weights were also significantly decreased in <i>Rere</i>^om/eyes3 embryos and mice in comparison with those of wild-type embryos and mice between E17.5 and P0 (n≥5; * = p<0.01).</p