Maternal protein malnutrition throughout pregnancy and lactation compromises brain development in late gestation and after birth, affecting structural, biochemical, and pathway dynamics with lasting consequences for motor and cognitive function. However, the importance of nutrition during the preimplantation period for brain development is unknown. We have previously shown that maternal low-protein diet (LPD) confined to the preimplantation period (Emb-LPD) in mice, with normal nutrition thereafter, is sufficient to induce cardiometabolic and locomotory behavioral abnormalities in adult offspring. Here, using a range of in vivo and in vitro techniques, we report that Emb-LPD and sustained LPD reduce neural stem cell (NSC) and progenitor cell numbers at E12.5, E14.5, and E17.5 through suppressed proliferation rates in both ganglionic eminences and cortex of the fetal brain. Moreover, Emb-LPD causes remaining NSCs to up-regulate the neuronal differentiation rate beyond control levels, whereas in LPD, apoptosis increases to possibly temper neuron formation. Furthermore, Emb-LPD adult offspring maintain the increase in neuron proportion in the cortex, display increased cortex thickness, and exhibit short-term memory deficit analyzed by the novel-object recognition assay. Last, we identify altered expression of fragile X family genes as a potential molecular mechanism for adverse programming of brain development. Collectively, these data demonstrate that poor maternal nutrition from conception is sufficient to cause abnormal brain development and adult memory loss.

Autism spectrum disorders (ASDs) are a heterogeneous group of neurodevelopmental disorders of genetic and environmental etiologies. Some ASD cases are syndromic: associated with clinically defined patterns of somatic abnormalities and a neurobehavioral phenotype (e.g., Fragile X syndrome). Many cases, however, are idiopathic or non-syndromic. Such disorders present themselves during the early postnatal period when language, speech, and personality start to develop. ASDs manifest by deficits in social communication and interaction, restricted and repetitive patterns of behavior across multiple contexts, sensory abnormalities across multiple modalities and comorbidities, such as epilepsy among many others. ASDs are disorders of connectivity, as synaptic dysfunction is common to both syndromic and idiopathic forms. While multiple theories have been proposed, particularly in idiopathic ASDs, none address why certain brain areas (e.g., frontotemporal) appear more vulnerable than others or identify factors that may affect phenotypic specificity. In this hypothesis article, we identify possible routes leading to, and the consequences of, altered connectivity and review the evidence of central and peripheral synaptic dysfunction in ASDs. We postulate that phenotypic specificity could arise from aberrant experience-dependent plasticity mechanisms in frontal brain areas and peripheral sensory networks and propose why the vulnerability of these areas could be part of a model to unify preexisting pathophysiological theories.

Tissue inhibitor of metalloproteinase-3 (TIMP-3) is a component of the extracellular environment, where it mediates diverse processes including matrix regulation/turnover, inflammation and angiogenesis. Rare TIMP-3 risk alleles and mutations are directly linked with retinopathies such as age-related macular degeneration (AMD) and Sorsby fundus dystrophy, and potentially, through indirect mechanisms, with Alzheimer's disease. Insights into TIMP-3 activities may be gleaned from studying Sorsby-linked mutations. However, recent findings do not fully support the prevailing hypothesis that a gain of function through the dimerisation of mutated TIMP-3 is responsible for retinopathy. Findings from Alzheimer's patients suggest a hitherto poorly studied relationship between TIMP-3 and the Alzheimer's-linked amyloid-beta (Aβ) proteins that warrant further scrutiny. This may also have implications for understanding AMD as aged/diseased retinae contain high levels of Aβ. Findings from TIMP-3 knockout and mutant knock-in mice have not led to new treatments, particularly as the latter does not satisfactorily recapitulate the Sorsby phenotype. However, recent advances in stem cell and in vitro approaches offer novel insights into understanding TIMP-3 pathology in the retina-brain axis, which has so far not been collectively examined. We propose that TIMP-3 activities could extend beyond its hitherto supposed functions to cause age-related changes and disease in these organs.

Background Deletions in the Xq22.3–Xq23 region, inclusive of COL4A5 , have been associated with a contiguous gene deletion syndrome characterised by A lport syndrome with intellectual disability ( M ental retardation), M idface hypoplasia and E lliptocytosis (AMME). The extrarenal biological and clinical significance of neighbouring genes to the Alport locus has been largely speculative. We sought to discover a genetic cause for two half-brothers presenting with nephrocalcinosis, early speech and language delay and midface hypoplasia with submucous cleft palate and bifid uvula. Methods Whole exome sequencing was undertaken on maternal half-siblings. In-house genomic analysis included extraction of all shared variants on the X chromosome in keeping with X-linked inheritance. Patient-specific mutants were transfected into three cell lines and microscopically visualised to assess the nuclear expression pattern of the mutant protein. Results In the affected half-brothers, we identified a hemizygous novel non-synonymous variant of unknown significance in AMMECR1 (c.G530A; p.G177D), a gene residing in the AMME disease locus. Transfected cell lines with the p.G177D mutation showed aberrant nuclear localisation patterns when compared with the wild type. Blood films revealed the presence of elliptocytes in the older brother. Conclusions Our study shows that a single missense mutation in AMMECR1 causes a phenotype of midface hypoplasia, mild intellectual disability and the presence of elliptocytes, previously reported as part of a contiguous gene deletion syndrome. Functional analysis confirms mutant-specific protein dysfunction. We conclude that AMMECR1 is a critical gene in the pathogenesis of AMME, causing midface hypoplasia and elliptocytosis and contributing to early speech and language delay, infantile hypotonia and hearing loss, and may play a role in dysmorphism, nephrocalcinosis and submucous cleft palate.

The retinal pigment epithelium (RPE) plays a key role in the pathogenesis of several blinding retinopathies. Alterations to RPE structure and function are reported in Age-related Macular Degeneration, Stargardt and Best disease as well as pattern dystrophies. However, the precise role of RPE cells in disease aetiology remains incompletely understood. Many studies into RPE pathobiology have utilised animal models, which only recapitulate limited disease features. Some studies are also difficult to carry out in animals as the ocular space remains largely inaccessible to powerful microscopes. In contrast, in-vitro models provide an attractive alternative to investigating pathogenic RPE changes associated with age and disease. In this article we describe the step-by-step approach required to establish an experimentally versatile in-vitro culture model of the outer retina incorporating the RPE monolayer and supportive Bruch’s membrane (BrM). We show that confluent monolayers of the spontaneously arisen human ARPE-19 cell-line cultured under optimal conditions reproduce key features of native RPE. These models can be used to study dynamic, intracellular and extracellular pathogenic changes using the latest developments in microscopy and imaging technology. We also discuss how RPE cells from human foetal and stem-cell derived sources can be incorporated alongside sophisticated BrM substitutes to replicate the aged/diseased outer retina in a dish. The work presented here will enable users to rapidly establish a realistic in-vitro model of the outer retina that is amenable to a high degree of experimental manipulation which will also serve as an attractive alternative to using animals. This in-vitro model therefore has the benefit of achieving the 3Rs objective of reducing and replacing the use of animals in research. As well as recapitulating salient structural and physiological features of native RPE, other advantages of this model include its simplicity, rapid set-up time and unlimited scope for detailed single-cell resolution and matrix studies.

BackgroundThe objective of this study was to investigate the expression and localisation of thymosin β4 (Tβ4) in the developing human heart. Tβ4 is a cardioprotective protein which may have therapeutic potential. While Tβ4 is an endogenously produced protein with known importance during development, its role within the developing human heart is not fully understood. Elucidating the localisation of Tβ4 within the developing heart will help in understanding its role during cardiac development and is crucial for understanding its potential for cardioprotection and repair in the adult heart.MethodsExpression of Tβ4 mRNA in the early fetal human heart was assessed by PCR using both ventricular and atrial tissue. Fluorescence immunohistochemistry was used to assess the localisation of Tβ4 in sections of early fetal human heart. Co-staining with CD31, an endothelial cell marker, and with myosin heavy chain, a cardiomyocyte marker, was used to determine whether Tβ4 is localised to these cell types within the early fetal human heart.ResultsTβ4 mRNA was found to be expressed in both the atria and the ventricles of the early fetal human heart. Tβ4 protein was found to be primarily localised to CD31-expressing endothelial cells and the endocardium as well as being present in the epicardium. Tβ4-associated fluorescence was greater in the compact layer of the myocardial wall and the interventricular septum than in the trabecular layer of the myocardium.ConclusionsThe data presented illustrates expression of Tβ4 in the developing human heart and demonstrates for the first time that Tβ4 in the human heart is primarily localised to endothelial cells of the cardiac microvasculature and coronary vessels as-well as to the endothelial-like cells of the endocardium and to the epicardium.

Sorsby fundus dystrophy (SFD) is a rare autosomal dominant disease of the macula that leads to bilateral loss of central vision and is caused by mutations in the TIMP3 gene. However, the mechanisms by which TIMP3 mutations cause SFD are poorly understood. Here, we generated human induced pluripotent stem cell‐derived retinal pigmented epithelial (hiPSC‐RPE) cells from three SFD patients carrying TIMP3 p.(Ser204Cys) and three non‐affected controls to study disease‐related structural and functional differences in the RPE. SFD‐hiPSC‐RPE exhibited characteristic RPE structure and physiology but showed significantly reduced transepithelial electrical resistance associated with enriched expression of cytoskeletal remodelling proteins. SFD‐hiPSC‐RPE exhibited basolateral accumulation of TIMP3 monomers, despite no change in TIMP3 gene expression. TIMP3 dimers were observed in both SFD and control hiPSC‐RPE, suggesting that mutant TIMP3 dimerisation does not drive SFD pathology. Furthermore, mutant TIMP3 retained matrix metalloproteinase activity. Proteomic profiling showed increased expression of ECM proteins, endothelial cell interactions and angiogenesis‐related pathways in SFD‐hiPSC‐RPE. By contrast, there were no changes in VEGF secretion. However, SFD‐hiPSC‐RPE secreted higher levels of monocyte chemoattractant protein 1, PDGF and angiogenin. Our findings provide a proof‐of‐concept that SFD patient‐derived hiPSC‐RPE mimic mature RPE cells and support the hypothesis that excess accumulation of mutant TIMP3, rather than an absence or deficiency of functional TIMP3, drives ECM and angiogenesis‐related changes in SFD. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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