Kyle G. Howell

Unintegrated lentivirus DNA persistence and accessibility to expression in nondividing cells: analysis with class I integrase mutants.

ABSTRACT The circumstances under which unintegrated lentivirus DNA can persist and be a functional template for transcription and protein expression are not clear. We constructed and validated the first class I (nonpleiotropic) integrase (IN) mutants for a non-human lentivirus (feline immunodeficiency virus [FIV]) and analyzed both these and known class I human immunodeficiency virus type 1 IN mutants. The FIV IN mutants (D66V and D66V/D118A) had class I properties: Gag/Pol precursor expression, proteolytic processing, particle formation, and reverse transcriptase (RT) production were normal, while the transduction of dividing fibroblasts was prevented and integration was blocked. When injected into rat retinas, the wild-type (WT) vector produced extensive, persistent transgene expression, compared with only rare positive neuronal cells for the IN mutant vector. In contrast, both WT and mutant vectors produced entirely equivalent, effective transduction levels of primary rat neurons (retinal ganglion cells). By testing the hypothesis that the unexpected retinal neuron transduction was related to cell cycle status, we found that when fibroblasts were growth arrested, transduction and internally promoted transgene expression were not inhibited at all by the class I FIV or HIV-1 IN mutations. Cells were then transduced under aphidicolin arrest and were released from the block 48 h later. Vector expression was stable and durable during repeated passaging in WT vector-transduced cells, while the release of cells transduced with equivalent RT units of class I IN mutant FIV or HIV vector resulted in a steady decline of expression, from 97 to 0% of cells by day 10. Southern blot and PCR analyses showed a lack of integration, irrespective of cell cycle, for the class I mutants and an increase in one- and two-long terminal repeat circular and linear unintegrated DNAs in growth-arrested cells. We conclude that if cell division is prevented, unintegrated FIV and HIV-1 vector DNAs can produce high-level internally promoted transgene expression equivalent to WT vectors. The expression correlates with the unintegrated DNA levels. These observations may facilitate the study of the roles of IN and other preintegration complex components in preintegration phases of infection by (i) providing an alternative way to monitor unintegrated nuclear cDNA forms, (ii) restricting ascertainment to the transcriptionally functional subset of unintegrated DNA, (iii) enabling analysis in individual, nondividing cells, and (iv) uncoupling other potential functions of IN from integration.

Interleukin 6 induces monocyte chemoattractant protein-1 expression in myeloma cells

Interleukin 6 (IL-6) is known to play an important role in the biology of the malignant plasma cells in multiple myeloma. In an effort to better understand IL-6 stimulated myeloma cell growth, we have performed gene expression profiling to identify IL-6 early response genes. Using the KAS-6/1 IL-6-dependent human myeloma cell line, IL-6 stimulation dramatically induced expression of monocyte chemoattractant protein-1 (MCP-1) mRNA. To verify this result, we used reverse transcriptase PCR and RNAse protection assays and demonstrated using both assays that MCP-1 is indeed an IL-6 responsive gene in a variety of IL-6-responsive myeloma cell lines. Moreover, we also demonstrated IL-6 stimulated MCP-1 secretion by the myeloma cell lines as well as by fresh patient tumor cells. Lastly, we present evidence that fresh patient tumor cells express mRNA for the MCP-1 receptor, CCR2, as do myeloma cell lines along with a second MCP-1 receptor, CCR11. Although MM cell chemotaxis in response to MCP-1 was only minimal, we were able to demonstrate that MCP-1 stimulated activation of MAPK. Because of the important role that this chemokine plays in both angiogenesis and bone homeostasis, and the ability of MCP-1 to activate myeloma cells, these results suggest a new mechanism by which IL-6 may contribute to disease pathogenesis.

HH Domain of Alzheimer’s Disease Aβ Provides Structural Basis for Neuronal Binding in PC12 and Mouse Cortical/Hippocampal Neurons

A key question in understanding AD is whether extracellular Aβ deposition of parenchymal amyloid plaques or intraneuronal Aβ accumulation initiates the AD process. Amyloid precursor protein (APP) is endocytosed from the cell surface into endosomes where it is cleaved to produce soluble Aβ which is then released into the brain interstitial fluid. Intraneuronal Aβ accumulation is hypothesized to predominate from the neuronal uptake of this soluble extracellular Aβ rather than from ER/Golgi processing of APP. We demonstrate that substitution of the two adjacent histidine residues of Aβ40 results in a significant decrease in its binding with PC12 cells and mouse cortical/hippocampal neurons. These substitutions also result in a dramatic enhancement of both thioflavin-T positive fibril formation and binding to preformed Aβ fibrils while maintaining its plaque-binding ability in AD transgenic mice. Hence, alteration of the histidine domain of Aβ prevented neuronal binding and drove Aβ to enhanced fibril formation and subsequent amyloid plaque deposition - a potential mechanism for removing toxic species of Aβ. Substitution or even masking of these Aβ histidine residues might provide a new therapeutic direction for minimizing neuronal uptake and subsequent neuronal degeneration and maximizing targeting to amyloid plaques.

Role of mutations in the cellular internalization of amyloidogenic light chains into cardiomyocytes

Light chain (AL) amyloidosis is characterized by the misfolding of immunoglobulin light chains, accumulating as amyloid fibrils in vital organs. Multiple reports have indicated that amyloidogenic light chains internalize into a variety of cell types, but these studies used urine-derived proteins without indicating any protein sequence information. As a result, the role of somatic mutations in amyloidogenic protein internalization has not been yet studied. We characterized the internalization of AL-09, an AL amyloidosis protein into mouse cardiomyocytes. We also characterized the internalization of the germline protein κI O18/O8, devoid of somatic mutations, and three AL-09 restorative mutations (I34N, Q42K, and H87Y) previously characterized for their role in protein structure, stability, and amyloid formation kinetics. All proteins shared a common internalization pathway into lysosomal compartments. The proteins caused different degrees of lysosomal expansion. Oregon green (OG) labeled AL-09 showed the most rapid internalization, while OG-Q42K presented the slowest rate of internalization.

Characterization of monoclonal antibodies against the glaucoma-associated protein myocilin

Although the glaucoma-associated protein myocilin has been the focus of intensive research, its biological function is still unknown. One of the limiting factors has been the lack of well-characterized antibodies, particularly monoclonal antibodies. We describe the development of six monoclonal antibodies specific to myocilin and characterize their suitability in Western blot and immunohistochemical applications. Three of the six monoclonal antibodies recognize the N-terminus of myocilin (amino acids 33-214), two antibodies recognize the middle third of the protein (amino acids 215-368), and one antibody recognizes the C-terminus (amino acids 369-504). Isotyping revealed that all antibodies are of the IgG1 kappa class except one, which is IgG2b kappa. Purified myocilin monoclonal antibodies were able to recognize myocilin in human aqueous humor separated on denatured/reduced and native gels, and human trabecular meshwork lysate by Western blot. Myocilin was also detected by immunohistochemistry in trabecular meshwork, ciliary body, iris, cornea, sclera, choroid, and retinal pigment epithelial cells.

Cell Damage in Light Chain Amyloidosis FIBRIL INTERNALIZATION, TOXICITY AND CELL-MEDIATED SEEDING

Light chain (AL) amyloidosis is an incurable human disease characterized by the misfolding, aggregation, and systemic deposition of amyloid composed of immunoglobulin light chains (LC). This work describes our studies on potential mechanisms of AL cytotoxicity. We have studied the internalization of AL soluble proteins and amyloid fibrils into human AC16 cardiomyocytes by using real time live cell image analysis. Our results show how external amyloid aggregates rapidly surround the cells and act as a recruitment point for soluble protein, triggering the amyloid fibril elongation. Soluble protein and external aggregates are internalized into AC16 cells via macropinocytosis. AL amyloid fibrils are shown to be highly cytotoxic at low concentrations. Additionally, caspase assays revealed soluble protein induces apoptosis, demonstrating different cytotoxic mechanisms between soluble protein and amyloid aggregates. This study emphasizes the complex immunoglobulin light chain-cell interactions that result in fibril internalization, protein recruitment, and cytotoxicity that may occur in AL amyloidosis.

Alzheimer's disease amyloid β-protein mutations and deletions that define neuronal binding/internalization as early stage nonfibrillar/fibrillar aggregates and late stage fibrils.

Accumulation of amyloid β-protein (Aβ) in neurons has been demonstrated to precede its formation as amyloid plaques in the extracellular space in Alzheimers disease (AD) patients. Consequently, intraneuronal Aβ accumulation is thought to be a critical first step in the fatal cascade of events that leads to neuronal degeneration in AD. Understanding the structural basis of neuronal binding and uptake of Aβ might lead to potential therapeutic targets that could block this binding and the subsequent neurodegeneration that leads to the pathogenesis of AD. Previously, we demonstrated that mutation of the two adjacent histidine residues of Aβ40 (H13,14G) resulted in a significant decrease in its level of binding to PC12 cells and mouse cortical/hippocampal neurons. We now demonstrate that the weakened neuronal binding follows the mutation order of H13G < H14G < H13,14G, which suggests that the primary domain for neuronal binding of Aβ40 involves histidine at position 13. A novel APP mutation (E693Δ) that produced a variant Aβ lacking glutamate 22 (E22Δ) in Japanese pedigrees was recently identified to have AD-type dementia without amyloid plaque formation but with extensive intraneuronal Aβ in transfected cells and transgenic mice expressing this deletion. Deletion of glutamate 22 of Aβ40 resulted in a 6-fold enhancement of PC12 neuronal binding that was not decreased by the H13G mutation. The dose-dependent enhanced binding of E22Δ explains the high level of intraneuronal Aβ seen in this pedigree. Fluorescence anisotropy experiments at room temperature showed very rapid aggregation with increased tyrosine rigidity of Aβ39E22Δ, Aβ41E22Δ, and Aβ42 but not Aβ40. This rigidity was decreased but not eliminated by prior treatment with dimethyl sulfoxide. Surprisingly, all peptides showed an aggregated state when evaluated by transmission electron microscopy, with Aβ39E22Δ having early stage fibrils, which was also verified by atomic force microscopy. This aggregation was not affected by centrifugation or pretreatment with organic solvents. The enhanced neuronal binding of Aβ, therefore, results from aggregate binding to neurons, which requires H13 for Aβ40 but not for E22Δ or Aβ42. These latter proteins display increased tyrosine rigidity that likely masks the H13 residue, or alternatively, the H13 residue is not required for neuronal binding of these proteins as it is for Aβ40. Late state fibrils also showed enhanced neuronal binding for E22Δ but not Aβ40 with subsequent intraneuronal accumulation in lysosomes. This suggests that there are multiple pathways of binding/internalization for the different Aβ proteins and their aggregation states or fibrillar structure.

MYOCILIN LEVELS IN PRIMARY OPEN-ANGLE GLAUCOMA AND PSEUDOEXFOLIATION GLAUCOMA HUMAN AQUEOUS HUMOR

PurposeTo determine the concentration of myocilin in primary open-angle glaucoma (POAG) and pseudoexfoliation glaucoma (PEXG) aqueous humor. MethodsAqueous humor was collected during surgery from patients with POAG, PEXG, and elective cataract removal (control). Volume-equivalent aqueous samples were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gradient gels. Quantification of myocilin levels was performed using Western blots probed with 2 independent N-terminal polyclonal anti-myocilin antibodies (AB1 and AB2) followed by densitometry. Myocilin levels in aqueous humor were quantified by plotting the densitometry readings of the aqueous samples against a recombinant myocilin standard curve. Total protein concentration was determined by Bradford protein assay. Transforming growth factor &bgr; 2 levels were assessed by enzyme-linked immunosorbent assay. ResultsMyocilin levels are significantly elevated in human POAG aqueous humor when compared with control aqueous humor (AB1: 0.66±0.53 ng/&mgr;L vs. 0.23±0.20 ng/&mgr;L, P<0.001; AB2: 0.98±0.59 ng/&mgr;L vs. 0.65±0.5 ng/&mgr;L, P<0.03; mean±SD). Myocilin makes up a larger percent of the total protein in POAG aqueous humor compared with control aqueous (AB1: 0.26±0.20% vs. 0.10±0.20%, P<0.001; AB2: 0.43±0.32% vs. 0.28±0.18%, P<0.05). In contrast to POAG, myocilin levels were not elevated in PEXG aqueous humor when compared with control aqueous humor. No correlation between myocilin and transforming growth factor &bgr; 2 levels was observed. ConclusionsMyocilin is elevated in POAG, but not in PEXG aqueous humor.

Differential effect of amyloid beta peptides on mitochondrial axonal trafficking depends on their state of aggregation and binding to the plasma membrane

Inhibition of mitochondrial axonal trafficking by amyloid beta (Aβ) peptides has been implicated in early pathophysiology of Alzheimer’s Disease (AD). Yet, it remains unclear whether the loss of motility inevitably induces the loss of mitochondrial function, and whether restoration of axonal trafficking represents a valid therapeutic target. Moreover, while some investigations identify Aβ oligomers as the culprit of trafficking inhibition, others propose that fibrils play the detrimental role. We have examined the effect of a panel of Aβ peptides with different mutations found in familial AD on mitochondrial motility in primary cortical mouse neurons. Peptides with higher propensity to aggregate inhibit mitochondrial trafficking to a greater extent with fibrils inducing the strongest inhibition. Binding of Aβ peptides to the plasma membrane was sufficient to induce trafficking inhibition where peptides with reduced plasma membrane binding and internalization had lesser effect on mitochondrial motility. We also found that Aβ peptide with Icelandic mutation A673T affects axonal trafficking of mitochondria but has very low rates of plasma membrane binding and internalization in neurons, which could explain its relatively low toxicity. Inhibition of mitochondrial dynamics caused by Aβ peptides or fibrils did not instantly affect mitochondrial bioenergetic and function. Our results support a mechanism where inhibition of axonal trafficking is initiated at the plasma membrane by soluble low molecular weight Aβ species and is exacerbated by fibrils. Since trafficking inhibition does not coincide with the loss of mitochondrial function, restoration of axonal transport could be beneficial at early stages of AD progression. However, strategies designed to block Aβ aggregation or fibril formation alone without ensuring the efficient clearance of soluble Aβ may not be sufficient to alleviate the trafficking phenotype.

Unique molecular signatures of Alzheimer's disease amyloid β peptide mutations and deletion during aggregate/oligomer/fibril formation

The formation of amyloid β (Aβ) peptide aggregates, oligomers, and fibrils is a dynamic process; however, the kinetics of their formation is not well understood. This study compares the time course of aggregate/fibril formation by transmission electron microscopy (TEM) analyses with that of oligomer/fibril formation by Western blot analysis under native and denaturing conditions. Efforts to deaggregate/defibrillate these peptides by using hexafluoroisopropanol, ammonium hydroxide, or dimethylsulfoxide did not change the nondenaturing polyacrylamide gel electrophoresis (PAGE) footprints or drive the peptides to a monomeric species. Regardless of the pretreatment protocol, TEM analyses reveal that all Aβ peptides (Aβ40, Aβ42, Aβ39E22Δ [Osaka], Aβ40E22G [Arctic], Aβ40E22Q [Dutch], and Aβ40A2T [Icelandic]) immediately formed nonfibrillar, amorphous aggregates when first placed into solution with the Osaka mutation, quickly forming early‐stage fibrils. The extent of fibril formation for other Aβ peptides is time dependent, with the Arctic mutation forming fibrils at 1 hr, the Dutch and Icelandic at 4 hr, Aβ42 at 8 hr, and Aβ40 at 24 hr. In contrast, nondenaturing PAGE revealed unique footprints for the different Aβ species. The rapidity of aggregate formation and the rapid transition to fibrils, particularly for the Osaka deletion, suggest an important role for aggregates/fibrils of Aβ in the development of neuronal degeneration.