Quantitative Proteomic Analysis Reveals apoE4-Dependent Phosphorylation of the Actin-Regulating Protein VASP.

Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer's disease. While neurons generally produce a minority of the apoE in the central nervous system, neuronal expression of apoE increases dramatically in response to stress and is sufficient to drive pathology. Currently, the molecular mechanisms of how apoE4 expression may regulate pathology are not fully understood. Here, we expand upon our previous studies measuring the impact of apoE4 on protein abundance to include the analysis of protein phosphorylation and ubiquitylation signaling in isogenic Neuro-2a cells expressing apoE3 or apoE4. ApoE4 expression resulted in a dramatic increase in vasodilator-stimulated phosphoprotein (VASP) S235 phosphorylation in a protein kinase A (PKA)-dependent manner. This phosphorylation disrupted VASP interactions with numerous actin cytoskeletal and microtubular proteins. Reduction of VASP S235 phosphorylation via PKA inhibition resulted in a significant increase in filopodia formation and neurite outgrowth in apoE4-expressing cells, exceeding levels observed in apoE3-expressing cells. Our results highlight the pronounced and diverse impact of apoE4 on multiple modes of protein regulation and identify protein targets to restore apoE4-related cytoskeletal defects.

Functional Impairment in Miro Degradation and Mitophagy Is a Shared Feature in Familial and Sporadic Parkinson's Disease.

Mitochondrial movements are tightly controlled to maintain energy homeostasis and prevent oxidative stress. Miro is an outer mitochondrial membrane protein that anchors mitochondria to microtubule motors and is removed to stop mitochondrial motility as an early step in the clearance of dysfunctional mitochondria. Here, using human induced pluripotent stem cell (iPSC)-derived neurons and other complementary models, we build on a previous connection of Parkinson's disease (PD)-linked PINK1 and Parkin to Miro by showing that a third PD-related protein, LRRK2, promotes Miro removal by forming a complex with Miro. Pathogenic LRRK2G2019S disrupts this function, delaying the arrest of damaged mitochondria and consequently slowing the initiation of mitophagy. Remarkably, partial reduction of Miro levels in LRRK2G2019S human neuron and Drosophila PD models rescues neurodegeneration. Miro degradation and mitochondrial motility are also impaired in sporadic PD patients. We reveal that prolonged retention of Miro, and the downstream consequences that ensue, may constitute a central component of PD pathogenesis.

Focal Epithelial Hyperplasia (Heck's Disease) in a 57-Year-Old Brazilian Patient: A Case Report and Literature Review.

Focal epithelial hyperplasia (FEH), or Heck's disease, is a rare disease of the oral mucosa associated with infection by some subtypes of human papilloma virus, especially subtypes 13 or 32. The disease is predominantly found in children and adolescents with indigenous heritage, but other ethnic groups can be affected worldwide. To the best of the authors' knowledge, it has not been reported in Brazil's elderly population. This article describes a case of FEH in a 57-year-old Brazilian patient presenting since childhood, with multiple lesions in the lips, buccal mucosa and tongue. The solitary tongue lesion underwent excisional biopsy and the histopathological analysis showed parakeratosis, acanthosis, rete pegs with a club-shaped appearance, koilocytosis and the presence of mitosoid cells. These microscopic findings in conjunction with clinical presentation were sufficient to establish the accurate diagnosis of FEH. Polymerase chain reaction (PCR) was performed, but no one human papillomavirus (HPV) subtype could be identified. Clinicians must be aware of this rare oral disease, which can even affect elderly patients, as we described here. Treatment may be indicated in selected cases due to esthetic and/or functional problems.

Stage-specific role for shh in dopaminergic differentiation of human embryonic stem cells induced by stromal cells.

Stromal cells have been used to induce dopaminergic differentiation of mouse, primate, and human embryonic stem cells (hESCs), but the mechanism that governs this induction is unknown. In this manuscript, we show that medium conditioned by the stromal cell line PA6 (PA6-CM) can induce dopaminergic differentiation in neural stem cells (NSCs) derived from hESCs but not directly from hESCs, indicating that soluble factors produced by PA6 cells act at the NSC stage to specify a dopaminergic fate. To identify such soluble factors, we analyzed the transcriptomes of PA6 cells, NSCs, and dopaminergic populations induced by PA6-CM from hESC-derived NSCs. We focused our analysis on growth factors expressed by PA6 and receptors expressed by NSCs, and generated a list of growth factors and receptors that are differentially expressed. Some of the growth factor/receptor pairs are categorized into the Shh, Wnt5A, TGFbeta, and IGF pathways. The expression of genes activated by these pathways in dopaminergic populations was analyzed to confirm that these signals were likely candidates for specifying dopaminergic fate. Results were verified for Shh by using perturbation agents such as cyclopamine to show that Shh is indeed one of the active agents in PA6-CM, and by showing that Shh and FGF8 can substitute for PA6-CM at the NSC induction stage. We conclude that PA6-CM can induce dopaminergic differentiation in hESCs in a stage-specific manner. Shh is likely an important soluble dopaminergic inducing factor secreted by stromal cells and acts after the neural fate determination.

Swiss Cheese, a protein involved in progressive neurodegeneration, acts as a noncanonical regulatory subunit for PKA-C3.

The Drosophila Swiss Cheese (SWS) protein and its vertebrate ortholog Neuropathy Target Esterase (NTE) are required for neuronal survival and glial integrity. In humans, NTE is the target of organophosphorous compounds which cause a paralyzing axonal degeneration and recently mutations in NTE have been shown to cause a Hereditary Spastic Paraplegia called NTE-related Motor-Neuron Disorder. SWS and NTE are concentrated in the endoplasmic reticulum and both have been shown to have an esterase function against an artificial substrate. However, the functional mechanisms and the pathways in which SWS/NTE are involved in are still widely unknown. Here, we show that SWS interacts specifically with the C3 catalytic subunit of cAMP activated protein kinase (PKA-C3), which together with orthologs in mouse (Pkare) and human (PrKX) forms a novel class of catalytic subunits of unknown function. This interaction requires a domain of SWS which shows homology to regulatory subunits of PKA and, like conventional regulatory subunits, the binding of SWS to the PKA-C3 inhibits its function. Consistent with this result, expression of additional PKA-C3 induces degeneration and enhances the neurodegenerative phenotype in sws mutants. We also show that the complex formation with the membrane-bound SWS tethers PKA-C3 to membranes. We therefore propose a model in which SWS acts as a noncanonical subunit for PKA-C3, whereby the complex formation regulates the localization and kinase activity of PKA-C3, and that disruption of this regulation can induce neurodegeneration.

Human embryonic stem cells have enhanced repair of multiple forms of DNA damage.

Embryonic stem cells need to maintain genomic integrity so that they can retain the ability to differentiate into multiple cell types without propagating DNA errors. Previous studies have suggested that mechanisms of genome surveillance, including DNA repair, are superior in mouse embryonic stem cells compared with various differentiated murine cells. Using single-cell gel electrophoresis (comet assay) we found that human embryonic stem cells (BG01, I6) have more efficient repair of different types of DNA damage (generated from H2O2, UV-C, ionizing radiation, or psoralen) than human primary fibroblasts (WI-38, hs27) and, with the exception of UV-C damage, HeLa cells. Microarray gene expression analysis showed that mRNA levels of several DNA repair genes are elevated in human embryonic stem cells compared with their differentiated forms (embryoid bodies). These data suggest that genomic maintenance pathways are enhanced in human embryonic stem cells, relative to differentiated human cells.

Loss of Swiss cheese/neuropathy target esterase activity causes disruption of phosphatidylcholine homeostasis and neuronal and glial death in adult Drosophila.

The Drosophila Swiss cheese (sws) mutant is characterized by progressive degeneration of the adult nervous system, glial hyperwrapping, and neuronal apoptosis. The Swiss cheese protein (SWS) shares 39% sequence identity with human neuropathy target esterase (NTE), and a brain-specific deletion of SWS/NTE in mice causes a similar pattern of progressive neuronal degeneration. NTE reacts with organophosphate compounds that cause a paralyzing axonal degeneration in humans and has been shown to degrade endoplasmic reticulum-associated phosphatidylcholine (PtdCho) in cultured mammalian cells. However, its function within the nervous system has remained unknown. Here, we show that both the fly and mouse SWS proteins can rescue the defects that arise in sws mutant flies, whereas a point mutation in the proposed active site cannot restore SWS function. Overexpression of catalytically active SWS caused formation of abnormal intracellular membraneous structures and cell death. Cell-specific expression revealed that not only neurons but also glia depend autonomously on SWS. In wild-type flies, endogenous SWS was detected by immmunohistochemistry in the endoplasmic reticulum (the primary site of PtdCho processing) of neurons and in some glia. sws mutant flies lacked NTE-like esterase activity and had increased levels of PtdCho. Conversely, overexpression of SWS resulted in increased esterase activity and reduced PtdCho. We conclude that SWS is essential for membrane lipid homeostasis and cell survival in both neurons and glia of the adult Drosophila brain and that NTE may play an analogous role in vertebrates.

Disruption of the MAP1B-related protein FUTSCH leads to changes in the neuronal cytoskeleton, axonal transport defects, and progressive neurodegeneration in Drosophila.

The elaboration of neuronal axons and dendrites is dependent on a functional cytoskeleton. Cytoskeletal components have been shown to play a major role in the maintenance of the nervous system through adulthood, and changes in neurofilaments and microtubule-associated proteins (MAPs) have been linked to a variety of neurodegenerative diseases. Here we show that Futsch, the fly homolog of MAP1B, is involved in progressive neurodegeneration. Although Futsch is widely expressed throughout the CNS, degeneration in futsch(olk) primarily occurs in the olfactory system and mushroom bodies. Consistent with the predicted function of Futsch, we find abnormalities in the microtubule network and defects in axonal transport. Degeneration in the adult brain is preceded by learning deficits, revealing a neuronal dysfunction before detectable levels of cell death. Futsch is negatively regulated by the Drosophila Fragile X mental retardation gene, and a mutation in this gene delays the onset of neurodegeneration in futsch(olk). A similar effect is obtained by expression of either fly or bovine tau, suggesting a certain degree of functional redundancy of MAPs. The futsch(olk) mutants exhibit several characteristics of human neurodegenerative diseases, providing an opportunity to study the role of MAPs in progressive neurodegeneration within an experimentally accessible, in vivo model system.

Glial and neuronal expression of polyglutamine proteins induce behavioral changes and aggregate formation in Drosophila.

Patients with polyglutamine expansion diseases, like Huntington's disease or several spinocerebellar ataxias, first present with neurological symptoms that can occur in the absence of neurodegeneration. Behavioral symptoms thus appear to be caused by neuronal dysfunction, rather than cell death. Pathogenesis in polyglutamine expansion diseases is largely viewed as a cell-autonomous process in neurons. It is likely, however, that this process is influenced by changes in glial physiology and, at least in the case of DRPLA glial inclusions and glial cell death, seems to be an important part in the pathogenesis. To investigate these aspects in a Drosophila model system, we expressed polyglutamine proteins in the adult nervous system. Glial-specific expression of a polyglutamine (Q)-expanded (n=78) and also a nonexpanded (n=27) truncated version of human ataxin-3 led to the formation of protein aggregates and glial cell death. Behavioral changes were observed prior to cell death. This reveals that glia is susceptible to the toxic action of polyglutamine proteins. Neuronal expression of the same constructs resulted in behavioral changes similar to those resulting from glial expression but did not cause neurodegeneration. Behavioral deficits were selective and affected two analyzed fly behaviors differently. Both glial and neuronal aggregates of Q78 and Q27 appeared early in pathogenesis and, at the electron microscopic resolution, had a fibrillary substructure. This shows that a nonexpanded stretch can cause similar histological and behavioral symptoms as the expanded stretch, however, with a significant delay.