David R. Hinton

Optic-Nerve Degeneration in Alzheimer's Disease

Alzheimers disease is a dementing disorder of unknown cause in which there is degeneration of neuronal subpopulations in the central nervous system. In postmortem studies, we found widespread axonal degeneration in the optic nerves of 8 of 10 patients with Alzheimers disease. The retinas of four of the patients were also examined histologically, and three had a reduction in the number of ganglion cells and in the thickness of the nerve-fiber layer. There was no retinal neurofibrillary degeneration or amyloid angiopathy, which are typically seen in the brains of patients with Alzheimers disease. The changes we observed in the patients with Alzheimers disease were clearly distinguishable from the findings in 10 age-matched controls and represent a sensory-system degeneration that occurs in Alzheimers disease. Study of the retina in patients with this disease may be helpful diagnostically, and isolation of the affected ganglion cells may facilitate molecular analysis of the disorder.

MDR1 Gene Expression in Brain of Patients with Medically Intractable Epilepsy

Summary: Why some patients with seizures are successfully treated with antiepileptic drugs (AEDs) and others prove medically intractable is not known. Inadequate intraparenchymal drug concentration is a possible mechanism of resistance to AEDs. The multiple drug resistance gene (MDRI) encodes P‐glycoprotein, an energy‐dependent efflux pump that exports planar hydrophobic molecules from the cell. If P‐glycoprotein is expressed in brain of some patients with intractable epilepsy and AEDs are exported by P‐glycoprotein, lower intraparenchymal drug concentrations could contribute to lack of drug response in such patients. Eleven of 19 brain specimens removed from patients during operation for intractable epilepsy had MDR1 mRNA levels > 10 times greater than those in normal brain, as determined by quantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) method. Immunohistochemistry for P‐glycoprotein from 14 of the patients showed increased staining in capillary endothelium in samples from epileptic patients as compared with staining in normal brain samples. In epileptic brain specimens with high MDR1 mRNA levels, expression of P‐glycoprotein in astrocytes also was identified. Last, steady‐state intracellular phenytoin (PHT) concentrations in MDR1 expressing neuroectodermal cells was one fourth that in MDR1‐negative cells. MDR1 expression is increased in brain of some patients with medically intractable epilepsy, suggesting that the patients’ lack of response to medication may be caused by inadequate accumulation of AED in brain.

DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration

Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell degeneration. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA, and that conditional ablation of Dicer1, but not seven other miRNA-processing enzymes, induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA, and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness.

DICER1 Loss and Alu RNA Induce Age-Related Macular Degeneration via the NLRP3 Inflammasome and MyD88

Alu RNA accumulation due to DICER1 deficiency in the retinal pigmented epithelium (RPE) is implicated in geographic atrophy (GA), an advanced form of age-related macular degeneration that causes blindness in millions of individuals. The mechanism of Alu RNA-induced cytotoxicity is unknown. Here we show that DICER1 deficit or Alu RNA exposure activates the NLRP3 inflammasome and triggers TLR-independent MyD88 signaling via IL18 in the RPE. Genetic or pharmacological inhibition of inflammasome components (NLRP3, Pycard, Caspase-1), MyD88, or IL18 prevents RPE degeneration induced by DICER1 loss or Alu RNA exposure. These findings, coupled with our observation that human GA RPE contains elevated amounts of NLRP3, PYCARD, and IL18 and evidence of increased Caspase-1 and MyD88 activation, provide a rationale for targeting this pathway in GA. Our findings also reveal a function of the inflammasome outside the immune system and an immunomodulatory action of mobile elements.

The unfolded protein response regulator GRP78/BiP is required for endoplasmic reticulum integrity and stress-induced autophagy in mammalian cells

In mammalian cells, endoplasmic reticulum (ER) stress has recently been shown to induce autophagy and the induction requires the unfolded protein response (UPR) signaling pathways. However, little is known whether autophagy regulates UPR pathways and how specific UPR targets might control autophagy. Here, we demonstrated that although ER stress-induced autophagy was suppressed by class III phosphatidylinositol-3′-kinase (PI3KC3) inhibitor 3-methyladenine (3-MA), wortmannin and knockdown of Beclin1 using small interfering RNA (siRNA), only 3-MA suppressed UPR activation. We discovered that the UPR regulator and ER chaperone GRP78/BiP is required for stress-induced autophagy. In cells in which GRP78 expression was knocked down by siRNA, despite spontaneous activation of UPR pathways and LC3 conversion, autophagosome formation induced by ER stress as well as by nutrition starvation was inhibited. GRP78 knockdown did not disrupt PI3KC3–Beclin1 association. However, electron microscopic analysis of the intracellular organelle structure reveals that the ER, a putative membrane source for generating autophagosomal double membrane, was massively expanded and disorganized in cells in which GRP78 was knocked down. ER expansion is known to be dependent on the UPR transcription factor XBP-1. Simultaneous knockdown of GRP78 and XBP-1 recovered normal levels of stress-induced autophagosome formation. Thus, these studies uncover 3-MA as an inhibitor of UPR activation and establish GRP78 as a novel obligatory component of autophagy in mammalian cells.

Retinal pathology in Alzheimer's disease. I. Ganglion cell loss in foveal/parafoveal retina

Morphometric analysis of the numbers of neurons in the ganglion cell layer (GCL) of the central retina (fovea/foveola/parafoveal retina) in eyes from 9 Alzheimers disease (AD) and 11 age-matched control cases revealed an overall decrease of 25% in total numbers of neurons in AD as compared with control eyes. Detailed analyses of GCL neurons at various eccentricities from the foveola showed that the greatest decrease in neuronal density (43% decrease) occurred in the central 0-0.5 mm (foveal region), while at 0.5-1 mm and at 1-1.5 mm eccentricities, neuronal loss amounted to 24 and 26%, respectively. The temporal region of the central retina appeared most severely affected, with up to 52% decrease in neuronal density near the foveola (central 0-0.5 mm eccentricity). There was close agreement between fellow eyes analyzed separately for three AD and three control cases. Analysis of neuronal sizes showed that all sizes of neurons were similarly affected in AD. In the GCL of control retinas, neurons decreased with age (coefficient of correlation = -0.67), while in AD retinas no such relationship was evident. Since in the central 0-2 mm region of the retina 97% of neurons in the GCL are ganglion cells (while the remaining 3% consist of displaced amacrine cells), these results demonstrate extensive ganglion cell loss in the central retina in AD.

PROTEIN KINASE C INHIBITORS INDUCE APOPTOSIS IN HUMAN MALIGNANT GLIOMA CELL LINES

Previous work has demonstrated the importance of the protein kinase C (PKC) system in regulating glioma growth, and has led to clinical trials utilizing PKC inhibitors as adjuncts in the therapy of patients harboring malignant gliomas. This study was performed to explore the possibility that inhibition of PKC in gliomas was triggering an apoptosis signal. Glioma cell lines were treated with PKC inhibitors staurosporine (10 nM), and tamoxifen (10 μM). DNA from cells treated with each of these drugs exhibited a ‘ladder’ pattern of oligonucleosome‐sized fragments characteristic of apoptosis, thus suggesting that in glioma cells, these drugs may be cytocidal in action.

Retinal pathology in Alzheimer's disease. II. Regional neuron loss and glial changes in GCL.

Detailed analyses of neuronal and astrocyte cell numbers in the ganglion cell layer (GCL) of whole-mounted peripheral retinas from 16 Alzheimers disease (AD) and 11 control eyes (11 and 9 cases, respectively) demonstrate extensive neuronal loss throughout the entire retina in AD as compared to control eyes. The observed neuronal loss is most pronounced in the superior and inferior quadrants, ranging between 40 and 49% throughout the midperipheral regions, and reaching 50-59% in the far peripheral inferior retina, while the overall neuronal loss throughout the entire retina amounts to 36.4% (p < 0.004). Although the 16% increase in astrocyte numbers is not significant, the ratio of astrocytes to neurons is significantly higher (82%; p < 0.0008) in AD as compared to normal retina (0.238 +/- 0.070 vs. 0.131 +/- 0.042). These results are strengthened by the close agreement (within +/- 15% of respective means) found between fellow eyes. Analysis of glial fibrillary acidic protein immunoreactivity (GFAP-ir) in sections of retinas from an additional 12 AD and 19 control cases show increased GFAP-ir with more extensive labeling of astrocytes in the GCL as well as increased labeling of Müller cell end-feet and radial processes in AD as compared to control retinas. The extensive loss of neurons documented in these retinas, accompanied by an increased astrocyte/neuron ratio, provides further support for the substantial involvement of the retina in AD.

A protocol for the culture and differentiation of highly polarized human retinal pigment epithelial cells.

We provide our detailed, standardized in vitro protocol for the culture and differentiation of human retinal pigment epithelial (RPE) cells into a highly polarized and functional monolayer. Disruption of the polarized RPE function plays an important role in the pathogenesis of common blinding disorders of the retina. The availability of this polarized RPE monolayer allows for reproducible evaluation of RPE function, modeling of RPE dysfunction in retinal disease and in vitro evaluation of new therapies. The protocol, which takes approximately 6 weeks to complete, describes the culture of RPE from human fetal donor eyes, the differentiation of these cells into a polarized monolayer with high transepithelial resistance and morphologic characteristics that mimic the RPE monolayer in vivo. By modifying the procedure for initial isolation of pure RPE cells and the culture conditions used in existing protocols, we have established a standardized protocol that provides highly reproducible RPE monolayers from the same donor eye.

Oxygen Metabolism Causes Chromosome Breaks and Is Associated with the Neuronal Apoptosis Observed in DNA Double-Strand Break Repair Mutants

Cells deficient in a major DNA double-strand break repair pathway (nonhomologous DNA end joining [NHEJ]) have increased spontaneous chromosome breaks; however, the source of these chromosome breaks has remained undefined. Here, we show that the observed spontaneous chromosome breaks are partially suppressed by reducing the cellular oxygen tension. Conversely, elevating the level of reactive oxygen species by overexpressing the antioxidant enzyme superoxide dismutase 1 (SOD1), in a transgenic mouse, increases chromosome breakage. The effect of SOD1 can also be modulated by cellular oxygen tension. The elevated chromosome breakage correlates histologically with a significant increase in the amount of neuronal cell death in Ku86(-/-) SOD1 transgenic embryos over that seen in Ku86(-/-) embryos. Therefore, oxygen metabolism is a major source of the genomic instability observed in NHEJ-deficient cells and, presumably, in all cells.