Optical clearing methods facilitate deep biological imaging by mitigating light scattering in situ. Multi-scale high-resolution imaging requires preservation of tissue integrity for accurate signal reconstruction. However, existing clearing reagents contain chemical components that could compromise tissue structure, preventing reproducible anatomical and fluorescence signal stability. We developed ScaleS, a sorbitol-based optical clearing method that provides stable tissue preservation for immunochemical labeling and three-dimensional (3D) signal rendering. ScaleS permitted optical reconstructions of aged and diseased brain in Alzheimer's disease models, including mapping of 3D networks of amyloid plaques, neurons and microglia, and multi-scale tracking of single plaques by successive fluorescence and electron microscopy. Human clinical samples from Alzheimer's disease patients analyzed via reversible optical re-sectioning illuminated plaque pathogenesis in the z axis. Comparative benchmarking of contemporary clearing agents showed superior signal and structure preservation by ScaleS. These findings suggest that ScaleS is a simple and reproducible method for accurate visualization of biological tissue.

Unfolded Pael receptor (Pael-R) is a substrate of the E3 ubiquitin ligase Parkin. Accumulation of Pael-R in the endoplasmic reticulum (ER) of dopaminergic neurons induces ER stress leading to neurodegeneration. Here, we show that CHIP, Hsp70, Parkin, and Pael-R formed a complex in vitro and in vivo. The amount of CHIP in the complex was increased during ER stress. CHIP promoted the dissociation of Hsp70 from Parkin and Pael-R, thus facilitating Parkin-mediated Pael-R ubiquitination. Moreover, CHIP enhanced Parkin-mediated in vitro ubiquitination of Pael-R in the absence of Hsp70. Furthermore, CHIP enhanced the ability of Parkin to inhibit cell death induced by Pael-R. Taken together, these results indicate that CHIP is a mammalian E4-like molecule that positively regulates Parkin E3 activity.

Amyloid-␤ protein (A␤) aggregates in the brain to form senile plaques. By using thioflavin T, a dye that specifically binds to fibrillar structures, we found that metals such as Zn(II) and Cu(II) normally inhibit amyloid ␤-aggregation. Another method for detecting A␤, which does not distinguish the types of aggregates, showed that these metals induce a non-␤-sheeted aggregation, as reported previously. Secondary structural analysis and microscopic studies revealed that metals induced A␤ to make non-fibrillar aggregates by disrupting ␤-sheet formation. These non-fibrillar A␤ aggregates displayed much weaker Congo Red birefringence, and in separate cell culture experiments, were less toxic than self ␤-aggregates, as demonstrated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. The toxicity of soluble A␤ was enhanced in the presence of Cu(II), which suggests the previously hypothesized role of A␤ in generating oxidative stress. Finally, under an acidic condition, similar to that in the inflammation associated with senile plaques, ␤-aggregation was robustly facilitated at one specific concentration of Zn(II) in the presence of heparin. However, because a higher concentration of Zn(II) virtually abolished this abnormal phenomenon, and at normal pH any concentrations strongly inhibit ␤-aggregation and its associated cytotoxicity, including its anti-oxidative nature we suggest that Zn(II) has an overall protective effect against ␤-amyloid toxicity. Amyloid-␤ protein (A␤)1 is one of the main components of senile plaques, a pathological hallmark of Alzheimer's disease (AD) (1, 2). Although A␤ is undisputedly associated with the pathology of AD, it is still an open question as to what specific aspects of A␤ and its processing are the important variables in the pathophysiology of the disease. For example, fibrillar A␤, but not non-amyloidogenic, amorphous aggregates of A␤, was reported to cause neuronal cell death in primary rat hippocampal cultures (3), and soluble monomeric species of A␤ are relatively nontoxic as compared with fibrillar A␤ (4). Thus, these in vitro studies suggest that the degree of ␤-aggregation is particularly important for neurotoxicity to occur (5-8). However, many controversial results from in vivo studies have been reported concerning the pathological role of plaque formation in AD. Irizarry et al. (9) reported that transgenic (TG) mice expressing human A␤ failed to exhibit neuronal loss despite depositing substantial amounts of A␤. On the other hand, TG mice that express Swedish mutant amyloid precursor protein (APP) formed plaques that were detected by both an anti-APP antibody and a ␤-sheet specific dye (10). Moreover, these APP TG mice also displayed memory deficits. Taken together, these results indicate that, although the plaque assembly process may require further investigation, amyloid ␤-aggregation certainly is an essential event in the pathogenesis of AD.Based on these lines of evidence, the search for a compound that interrupts ␤-aggregation and thus protects agai...

Mutations in the EPM2A gene encoding a dual-specificity phosphatase (laforin) cause Lafora disease (LD), a progressive and invariably fatal epilepsy with periodic acid-Schiff-positive (PAS+) cytoplasmic inclusions (Lafora bodies) in the central nervous system. To study the pathology of LD and the functions of laforin, we disrupted the Epm2a gene in mice. At two months of age, homozygous null mutants developed widespread degeneration of neurons, most of which occurred in the absence of Lafora bodies. Dying neurons characteristically exhibit swelling in the endoplasmic reticulum, Golgi networks and mitochondria in the absence of apoptotic bodies or fragmentation of DNA. As Lafora bodies become more prominent at 4-12 months, organelles and nuclei are disrupted. The Lafora bodies, present both in neuronal and non-neural tissues, are positive for ubiquitin and advanced glycation end-products only in neurons, suggesting different pathological consequence for Lafora inclusions in neuronal tissues. Neuronal degeneration and Lafora inclusion bodies predate the onset of impaired behavioral responses, ataxia, spontaneous myoclonic seizures and EEG epileptiform activity. Our results suggest that LD is a primary neurodegenerative disorder that may utilize a non-apoptotic mechanism of cell death.

No abstract