Distinct physiological and behavioural functions for parental alleles of imprinted Grb10.

Imprinted genes, defined by their preferential expression of a single parental allele, represent a subset of the mammalian genome and often have key roles in embryonic development, but also postnatal functions including energy homeostasis and behaviour. When the two parental alleles are unequally represented within a social group (when there is sex bias in dispersal and/or variance in reproductive success), imprinted genes may evolve to modulate social behaviour, although so far no such instance is known. Predominantly expressed from the maternal allele during embryogenesis, Grb10 encodes an intracellular adaptor protein that can interact with several receptor tyrosine kinases and downstream signalling molecules. Here we demonstrate that within the brain Grb10 is expressed from the paternal allele from fetal life into adulthood and that ablation of this expression engenders increased social dominance specifically among other aspects of social behaviour, a finding supported by the observed increase in allogrooming by paternal Grb10-deficient animals. Grb10 is, therefore, the first example of an imprinted gene that regulates social behaviour. It is also currently alone in exhibiting imprinted expression from each of the parental alleles in a tissue-specific manner, as loss of the peripherally expressed maternal allele leads to significant fetal and placental overgrowth. Thus Grb10 is, so far, a unique imprinted gene, able to influence distinct physiological processes, fetal growth and adult behaviour, owing to actions of the two parental alleles in different tissues.

Glycosaminoglycan regulation by VEGFA and VEGFC of the glomerular microvascular endothelial cell glycocalyx in vitro.

Damage to endothelial glycocalyx impairs vascular barrier function and may contribute to progression of chronic vascular disease. An early indicator is microalbuminuria resulting from glomerular filtration barrier damage. We investigated the contributions of hyaluronic acid (HA) and chondroitin sulfate (CS) to glomerular microvascular endothelial cell (GEnC) glycocalyx and examined whether these are modified by vascular endothelial growth factors A and C (VEGFA and VEGFC). HA and CS were imaged on GEnCs and their resynthesis was examined. The effect of HA and CS on transendothelial electrical resistance (TEER) and labeled albumin flux across monolayers was assessed. Effects of VEGFA and VEGFC on production and charge characteristics of glycosaminoglycan (GAG) were examined via metabolic labeling and liquid chromatography. GAG shedding was quantified using Alcian Blue. NDST2 expression was examined using real-time PCR. GEnCs expressed HA and CS in the glycocalyx. CS contributed to the barrier to both ion (TEER) and protein flux across the monolayer; HA had only a limited effect. VEGFC promoted HA synthesis and increased the charge density of synthesized GAGs. In contrast, VEGFA induced shedding of charged GAGs. CS plays a role in restriction of macromolecular flux across GEnC monolayers, and VEGFA and VEGFC differentially regulate synthesis, charge, and shedding of GAGs in GEnCs. These observations have important implications for endothelial barrier regulation in glomerular and other microvascular beds.

Endothelial glycocalyx restoration by growth factors in diabetic nephropathy.

The endothelial glycocalyx (eGlx) constitutes the first barrier to protein in all blood vessels. This is particularly noteworthy in the renal glomerulus, an ultrafiltration barrier. Leakage of protein, such as albumin, across glomerular capillaries results in albumin in the urine (albuminuria). This is a hall mark of kidney disease and can reflect loss of blood vessel integrity in microvascular beds elsewhere. We discuss evidence demonstrating that targeted damage to the glomerular eGlx results in increased glomerular albumin permeability. EGlx is lost in diabetes and experimental models demonstrate loss from glomerular endothelial cells. Vascular endothelial growth factor (VEGF)A is upregulated in early diabetes, which is associated with albuminuria. Treatment with paracrine growth factors such as VEGFC, VEGF165b and angiopoietin-1 can modify VEGFA signalling, rescue albumin permeability and restore glomerular eGlx in models of diabetes. Manipulation of VEGF receptor 2 signalling, or a common eGlx biosynthesis pathway by these growth factors, may protect and restore the eGlx layer. This would help to direct future therapeutics in diabetic nephropathy.

VEGFC Reduces Glomerular Albumin Permeability and Protects Against Alterations in VEGF Receptor Expression in Diabetic Nephropathy.

Elevated levels of vascular endothelial growth factor (VEGF) A are thought to cause glomerular endothelial cell (GEnC) dysfunction and albuminuria in diabetic nephropathy. We hypothesized that VEGFC could counteract these effects of VEGFA to protect the glomerular filtration barrier and reduce albuminuria. Isolated glomeruli were stimulated ex vivo with VEGFC, which reduced VEGFA- and type 2 diabetes-induced glomerular albumin solute permeability (Ps'alb). VEGFC had no detrimental effect on glomerular function in vivo when overexpression was induced locally in podocytes (podVEGFC) in otherwise healthy mice. Further, these mice had reduced glomerular VEGFA mRNA expression, yet increased glomerular VEGF receptor heterodimerization, indicating differential signaling by VEGFC. In a model of type 1 diabetes, the induction of podVEGFC overexpression reduced the development of hypertrophy, albuminuria, loss of GEnC fenestrations and protected against altered VEGF receptor expression. In addition, VEGFC protected against raised Ps'alb by endothelial glycocalyx disruption in glomeruli. In summary, VEGFC reduced the development of diabetic nephropathy, prevented VEGF receptor alterations in the diabetic glomerulus, and promoted both glomerular protection and endothelial barrier function. These important findings highlight a novel pathway for future investigation in the treatment of diabetic nephropathy.

A local insult of okadaic acid in wild-type mice induces tau phosphorylation and protein aggregation in anatomically distinct brain regions.

In Alzheimer's disease (AD), the distribution and density of neurofibrillary tangles, a histological hallmark comprised predominately of phosphorylated tau protein, follows a distinct pattern through anatomically connected brain regions. Studies in transgenic mice engineered to regionally confine tau expression have suggested spreading of tau within neural networks. Furthermore, injection of protein lysates isolated from brains of transgenic mice or patients with tauopathies, including AD, were shown to behave like seeds, accelerating tau pathology and tangle formation in predisposed mice. However, it remains unclear how the initiation of primary aggregation events occurs and what triggers further dissemination throughout the neural system. To consolidate these findings, we pursued an alternative approach to assess the spreading of endogenous phosphorylated tau. To generate endogenous seeds, 130 nl of 100 µM protein phosphatase 2A inhibitor okadaic acid (OA) was injected unilaterally into the amygdala of 8-month-old C57Bl/6 wild-type mice. OA was detected in brain tissue by ELISA, and found to be restricted to the injected hemispheric quadrant, where it remained detectable a week post-injection. OA injection induced tau phosphorylation that was observed not only at the injection site but also in anatomically distinct areas across both hemispheres, including the cortex and hippocampus 24 h post-injection. An increase in insoluble tau was also observed in both hemispheres of injected brains by 7 days. Furthermore, thioflavin-S detected protein aggregation at the injection site and in the cortex of both injected and contralateral hemispheres. OA injection induced no thioflavin-positivity in tau knock-out mice. The data demonstrates that a local OA insult can rapidly initiate changes in protein phosphorylation, solubility and aggregation at anatomically distant sites. This model suggests that tau phosphorylation can be both a primary response to an insult, and a secondary response communicated to non-exposed brains regions. The study highlights the use of OA to assist in understanding the initiation of tau spreading in vivo.

Extracellular Vesicles Containing P301L Mutant Tau Accelerate Pathological Tau Phosphorylation and Oligomer Formation but Do Not Seed Mature Neurofibrillary Tangles in ALZ17 Mice.

In Alzheimer's disease, the distribution of neurofibrillary tangles, a histological hallmark comprised of phosphorylated forms of the protein tau, follows a distinct pattern through anatomically connected brain regions. The well-documented correlation between the severity of tau pathology and disease progression implies a prion-like seeding and spreading mechanism for tau. Experimentally, this has been addressed in transgenic mice by the injection of protein lysates isolated from brains of transgenic mice or patients with tauopathies, including AD, that were shown to behave like seeds, accelerating tau pathology and tangle formation in predisposed mice. More specifically, in vivo data suggest that brain lysates from mice harboring the P301S mutation of tau can seed protein aggregation when injected into the hippocampi of human wild-type tau transgenic ALZ17 mice. Here, we compared the seeding potential of lysates and extracellular vesicles enriched for exosomes (EVs) from wild-type and human P301L tau transgenic rTg4510 mouse brains. We show that transgenic EVs cause increased tau phosphorylation and soluble oligomer formation in a manner comparable to that of freely available proteins in brain lysates, a prerequisite for the formation of mature protein aggregates.

What we can learn from animal models about cerebral multi-morbidity.

Late-onset diseases such as Alzheimer's disease, Parkinson's disease, or frontotemporal lobar degeneration are considered to be protein-folding disorders, with the accumulation of protein deposits causing a gain-of-toxic function. Alzheimer's disease is characterized by two histological hallmark lesions: amyloid-ß-containing plaques and tau-containing neurofibrillary tangles. However, signature proteins, including α-synuclein, which are found in an aggregated fibrillar form in the Lewy bodies of Parkinson's disease brains, are also frequently found in Alzheimer's disease. This highlights the fact that, although specific aggregates form the basis for diagnosis, there is a high prevalence of clinical overlap between neuropathological lesions linked to different diseases, a finding known as cerebral co- or multi-morbidity. Furthermore, the proteins forming these lesions interact, and this interaction accelerates an ongoing degenerative process. Here, we review the contribution that transgenic animal models have made to a better mechanistic understanding of the causes and consequences of co- or multi-morbidity. We discuss selected vertebrate and invertebrate models as well as the insight gained from non-transgenic senescence-accelerated mouse-prone mice. This article is part of a series on 'Cerebral multi-morbidity of the aging brain'.