APOL1 toxin, innate immunity, and kidney injury.

The discovery that two common APOL1 alleles were strongly associated with nondiabetic kidney diseases in African descent populations led to hope for improved diagnosis and treatment. Unfortunately, we still do not have a clear understanding of the biological function played by APOL1 in podocytes or other kidney cells, nor how the renal risk alleles initiate the development of nephropathies. Important clues for APOL1 function may be gleaned from the natural defense mechanism of APOL1 against trypanosome infections and from similar proteins (e.g., diphtheria toxin, mammalian Bcl-2 family members). This review provides an update on the biological functions for circulating (trypanosome resistance) and intracellular (emerging role for autophagy) APOL1. Further, we introduce a multimer model for APOL1 in kidney cells that reconciles the gain-of-function variants with the recessive inheritance pattern of APOL1 renal risk alleles.

Multidimensional analysis of cortical interneuron synaptic features reveals underlying synaptic heterogeneity.

Cortical interneurons represent a diverse set of neuronal subtypes characterized in part by their striking degree of synaptic specificity. However, little is known about the extent of synaptic diversity because of the lack of unbiased methods to extract synaptic features among interneuron subtypes. Here, we develop an approach to aggregate image features from fluorescent confocal images of interneuron synapses and their post-synaptic targets, in order to characterize the heterogeneity of synapses at fine scale. We started by training a model that recognizes pre- and post-synaptic compartments and then determines the target of each genetically-identified interneuron synapse in vitro and in vivo. Our model extracts hundreds of spatial and intensity features from each analyzed synapse, constructing a multidimensional data set, consisting of millions of synapses, which allowed us to perform an unsupervised analysis on this dataset, uncovering novel synaptic subgroups. The subgroups were spatially distributed in a highly structured manner that revealed the local underlying topology of the postsynaptic environment. Dendrite-targeting subgroups were clustered onto subdomains of the dendrite along the proximal to distal axis. Soma-targeting subgroups were enriched onto different postsynaptic cell types. We also find that the two main subclasses of interneurons, basket cells and somatostatin interneurons, utilize distinct strategies to enact inhibitory coverage. Thus, our analysis of multidimensional synaptic features establishes a conceptual framework for studying interneuron synaptic diversity.

St18 specifies globus pallidus projection neuron identity in MGE lineage.

The medial ganglionic eminence (MGE) produces both locally-projecting interneurons, which migrate long distances to structures such as the cortex as well as projection neurons that occupy subcortical nuclei. Little is known about what regulates the migratory behavior and axonal projections of these two broad classes of neurons. We find that St18 regulates the migration and morphology of MGE neurons in vitro. Further, genetic loss-of-function of St18 in mice reveals a reduction in projection neurons of the globus pallidus pars externa. St18 functions by influencing cell fate in MGE lineages as we observe a large expansion of nascent cortical interneurons at the expense of putative GPe neurons in St18 null embryos. Downstream of St18, we identified Cbx7, a component of Polycomb repressor complex 1, and find that it is essential for projection neuron-like migration but not morphology. Thus, we identify St18 as a key regulator of projection neuron vs. interneuron identity.

Vascular-derived SPARC and SerpinE1 regulate interneuron tangential migration and accelerate functional maturation of human stem cell-derived interneurons.

Cortical interneurons establish inhibitory microcircuits throughout the neocortex and their dysfunction has been implicated in epilepsy and neuropsychiatric diseases. Developmentally, interneurons migrate from a distal progenitor domain in order to populate the neocortex - a process that occurs at a slower rate in humans than in mice. In this study, we sought to identify factors that regulate the rate of interneuron maturation across the two species. Using embryonic mouse development as a model system, we found that the process of initiating interneuron migration is regulated by blood vessels of the medial ganglionic eminence (MGE), an interneuron progenitor domain. We identified two endothelial cell-derived paracrine factors, SPARC and SerpinE1, that enhance interneuron migration in mouse MGE explants and organotypic cultures. Moreover, pre-treatment of human stem cell-derived interneurons (hSC-interneurons) with SPARC and SerpinE1 prior to transplantation into neonatal mouse cortex enhanced their migration and morphological elaboration in the host cortex. Further, SPARC and SerpinE1-treated hSC-interneurons also exhibited more mature electrophysiological characteristics compared to controls. Overall, our studies suggest a critical role for CNS vasculature in regulating interneuron developmental maturation in both mice and humans.

Non-canonical Wnt Signaling through Ryk Regulates the Generation of Somatostatin- and Parvalbumin-Expressing Cortical Interneurons.

GABAergic interneurons have many important functions in cortical circuitry, a reflection of their cell diversity. The developmental origins of this diversity are poorly understood. Here, we identify rostral-caudal regionality in Wnt exposure within the interneuron progenitor zone delineating the specification of the two main interneuron subclasses. Caudally situated medial ganglionic eminence (MGE) progenitors receive high levels of Wnt signaling and give rise to somatostatin (SST)-expressing cortical interneurons. By contrast, parvalbumin (PV)-expressing basket cells originate mostly from the rostral MGE, where Wnt signaling is attenuated. Interestingly, rather than canonical signaling through ß-catenin, signaling via the non-canonical Wnt receptor Ryk regulates interneuron cell-fate specification in vivo and in vitro. Indeed, gain of function of Ryk intracellular domain signaling regulates SST and PV fate in a dose-dependent manner, suggesting that Ryk signaling acts in a graded fashion. These data reveal an important role for non-canonical Wnt-Ryk signaling in establishing the correct ratios of cortical interneuron subtypes.

APOL1 risk allele RNA contributes to renal toxicity by activating protein kinase R.

APOL1 risk alleles associate with chronic kidney disease in African Americans, but the mechanisms remain to be fully understood. We show that APOL1 risk alleles activate protein kinase R (PKR) in cultured cells and transgenic mice. This effect is preserved when a premature stop codon is introduced to APOL1 risk alleles, suggesting that APOL1 RNA but not protein is required for the effect. Podocyte expression of APOL1 risk allele RNA, but not protein, in transgenic mice induces glomerular injury and proteinuria. Structural analysis of the APOL1 RNA shows that the risk variants possess secondary structure serving as a scaffold for tandem PKR binding and activation. These findings provide a mechanism by which APOL1 variants damage podocytes and suggest novel therapeutic strategies.

Transgenic expression of human APOL1 risk variants in podocytes induces kidney disease in mice.

African Americans have a heightened risk of developing chronic and end-stage kidney disease, an association that is largely attributed to two common genetic variants, termed G1 and G2, in the APOL1 gene. Direct evidence demonstrating that these APOL1 risk alleles are pathogenic is still lacking because the APOL1 gene is present in only some primates and humans; thus it has been challenging to demonstrate experimental proof of causality of these risk alleles for renal disease. Here we generated mice with podocyte-specific inducible expression of the APOL1 reference allele (termed G0) or each of the risk-conferring alleles (G1 or G2). We show that mice with podocyte-specific expression of either APOL1 risk allele, but not of the G0 allele, develop functional (albuminuria and azotemia), structural (foot-process effacement and glomerulosclerosis) and molecular (gene-expression) changes that closely resemble human kidney disease. Disease development was cell-type specific and likely reversible, and the severity correlated with the level of expression of the risk allele. We further found that expression of the risk-variant APOL1 alleles interferes with endosomal trafficking and blocks autophagic flux, which ultimately leads to inflammatory-mediated podocyte death and glomerular scarring. In summary, this is the first demonstration that the expression of APOL1 risk alleles is causal for altered podocyte function and glomerular disease in vivo.

APOL1 Kidney Disease Risk Variants: An Evolving Landscape.

Apolipoprotein L1 (APOL1) genetic variants account for much of the excess risk of chronic and end-stage kidney disease, which results in a significant global health disparity for persons of African ancestry. We estimate the lifetime risk of kidney disease in APOL1 dual-risk allele individuals to be at least 15%. Experimental evidence suggests a direct role of APOL1 in pore formation, cellular injury, and programmed cell death in renal injury. The APOL1 BH3 motif, often associated with cell death, is unlikely to play a role in APOL1-induced cytotoxicity because it is not conserved within the APOL family and is dispensable for cell death in vitro. We discuss two models for APOL1 trypanolytic activity: one involving lysosome permeabilization and another involving colloid-osmotic swelling of the cell body, as well as their relevance to human pathophysiology. Experimental evidence from human cell culture models suggests that both mechanisms may be operative. A systems biology approach whereby APOL1-associated perturbations in gene and protein expression in affected individuals are correlated with molecular pathways may be productive to elucidate APOL1 function in vivo.

Integrated Design of Antibodies for Systems Biology Using Ab Designer.

In the current era of large-scale biology, systems biology has evolved as a powerful approach to identify complex interactions within biological systems. In addition to high throughput identification and quantification techniques, methods based on high-quality mono-specific antibodies remain an essential element of the approach. To assist the large-scale design and production of peptide-directed antibodies for systems biology studies, we developed a fully integrated online application, AbDesigner (http://helixweb.nih.gov/AbDesigner/), to help researchers select optimal peptide immunogens for antibody generation against relatively disordered regions of target proteins. Here we describe AbDesigner in terms of its features, comparing it to other software tools, and use it to design three antibodies against kidney disease-related proteins in human, viz. nephrin, podocin, and apolipoprotein L1.

Endocytosis of albumin by podocytes elicits an inflammatory response and induces apoptotic cell death.

The presence of albuminuria is strongly associated with progression of chronic kidney disease. While albuminuria has been shown to injure renal proximal tubular cells, the effects of albumin on podocytes have been less well studied. We have addressed the hypothesis that exposure of podocytes to albumin initiates an injury response. We studied transformed human-urine derived podocytes-like epithelial cells (HUPECS, or podocytes). Upon differentiation, these cells retain certain characteristics of differentiated podocytes, including expression of synaptopodin, CD2AP, and nestin. We exposed podocytes to recombinant human albumin, which lacks lipids and proteins that bind serum albumin; this reagent allowed a direct examination of the effects of albumin. Podocytes endocytosed fluoresceinated albumin and this process was inhibited at 4°C, suggesting an energy-dependent process. Exposure to albumin at concentrations of 5 and 10 mg/ml was associated with increased cell death in a dose-dependent manner. The mechanism of cell death may involve apoptosis, as caspase 3/7 were activated and the pan-caspase inhibitor z-VAD reduced cell death. Albumin exposure also increased nuclear factor (NF)-κB activation and increased transcription and release of interleukin (IL-) 1ß, tumor necrosis factor (TNF), and IL-6. We extended these findings to an in vivo model. Glomeruli isolated from mice with nephrotic syndrome also had increased expression of IL-1ß and TNF RNA. These data suggest that while podocyte injury begets albuminuria, albumin in the glomerular ultrafiltrate may also beget podocyte injury. Thus, an additional mechanism by which anti-proteinuric therapies are beneficial in the treatment of glomerular diseases may be a reduction in injury to the podocyte by albumin.