Ratneshwar Lal

Amyloid β protein forms ion channels: implications for Alzheimer’s disease pathophysiology

Amyloid β protein (AβP) is the major constituent of senile plaques associated with Alzheimers disease (AD). However, its mechanistic role in AD pathogenesis is poorly understood. Globular and non‐fibrillar AβPs are continuously released during normal metabolism. Using techniques of atomic force microscopy, laser confocal microscopy, electrical recording, and biochemical assays, we have examined the molecular conformations of reconstituted globular AβPs as well as their real‐time and acute effects on neuritic degeneration. Atomic force microscopy (AFM) of AβP1–42 shows globular structures that do not form fibers in physiological‐buffered solution for up to 8 h of continuous imaging. AFM of AβP1–42 reconstituted in a planar lipid bilayer reveals multimeric channel‐like structures. Consistent with these AFM resolved channel‐like structures, biochemical analysis demonstrates that predominantly monomeric AβPs in solution form stable tetramers and hexamers after incorporation into lipid membranes. Electrophysiological recordings demonstrate the presence of multiple single channel currents of different sizes. At the cellular level, AβP1–42 allows calcium uptake and induces neuritic abnormality in a dose‐ and time‐dependent fashion. At physiological nanomolar concentrations, rapid neuritic degeneration was observed within minutes;at micromolar concentrations, neuronal death was observed within 3–4 h. These effects are prevented by zinc (an AβP channel blocker) and by the removal of extracellular calcium, but are not prevented by antagonists of putative AβP cell surface receptors. Thus, AβP channels may provide a direct pathway for calcium‐dependent AβP toxicity in AD.—Lin, H., Bhatia, R., Lal, R. Amyloid β‐protein forms ion channels: implications for Alzheimers disease pathophysiology. FASEB J. 15, 2433–2444 (2001)

Fresh and globular amyloid β protein (1–42) induces rapid cellular degeneration: evidence for AβP channel-mediated cellular toxicity

Amyloid β peptides (AβP) deposit as plaques in vascular and parenchymal areas of Alzheimers disease (AD) tissues and Downs syndrome patients. Although neuronal toxicity is a feature of late stages of AD, vascular pathology appears to be a feature of all stages of AD. Globular and nonfibrillar AβPs are continuously released during normal cellular metabolism, form calcium‐permeable channels, and alter cellular calcium level. We used atomic force microscopy, laser confocal microscopy, and calcium imaging to examine the real‐time and acute effects of fresh and globular AβP1–42, AβP1–40, and AβP25–35 on cultured endothelial cells. AβPs induced morphological changes that were observed within minutes after AβP treatment and led to eventual cellular degeneration. Cellular morphological changes were most sensitive to AβP1–42.AβP1–42‐induced morphological changes were observed at nanomolar concentrations and were accompanied by an elevated cellular calcium level. Morphological changes were prevented by anti‐AβP antibody, AβP‐channel antagonist zinc, and the removal of extracellular calcium, but not by tachykinin neuropeptide, voltage‐sensitive calcium channel blocker cadmium, or antioxidants DTT and Trolox. Thus, nanomolar fresh and globular AβP1–42 induces rapid cellular degeneration by elevating intracellular calcium, most likely via calcium‐permeable AβP channels and not by its interaction with membrane receptors or by activating oxidative pathways. Such rapid degeneration also suggests that the plaques, and especially fibrillar AβPs, may not have a direct causative role in AD pathogenic cascades.—Bhatia, R., Lin H., Lal, R. Fresh and globular amyloid β protein (1–42) induces rapid cellular degeneration: evidence for Aβ P channel‐mediated cellular toxicity FASEB J. 14, 1233–1243 (2000)

AMYLOID BETA PROTEIN-(1-42) FORMS CALCIUM-PERMEABLE, ZN2+-SENSITIVE CHANNEL

Amyloid β protein (AβP) forms senile plaques in the brain of the patients with Alzheimer’s disease. The early-onset AD has been correlated with an increased level of 42-residue AβP (AβP1–42). However, very little is known about the role of AβP1–42 in such pathology. We have examined the activity of AβP1–42 reconstituted in phospholipid vesicles. Vesicles reconstituted with AβP show strong immunofluorescence labeling with an antibody raised against an extracellular domain of AβP suggesting the incorporation of AβP peptide in the vesicular membrane. Vesicles reconstituted with AβP showed a significant level of 45Ca2+ uptake. The 45Ca2+ uptake was inhibited by (i) a monoclonal antibody raised against the N-terminal region of AβP, (ii) Tris, and (iii) Zn2+. However, reducing agents Trolox and dithiothreitol did not inhibit the 45Ca2+uptake, indicating that the oxidation of AβP or its surrounding lipid molecules is not directly involved in the AβP-mediated Ca2+ uptake. An atomic force microscope was used to image the structure and physical properties of these vesicles. Vesicles ranged from 0.5 to 1 μm in diameter. The stiffness of the AβP-containing vesicles was significantly higher in the presence of calcium. The stiffness change was prevented in the presence of zinc, Tris, and anti-AβP antibody but not in the presence of Trolox and dithiothreitol. Thus the stiffness change is consistent with the vesicular uptake of Ca2+. These findings provide biochemical and structural evidence that AβP1–42 forms calcium-permeable channels and thus may induce cellular toxicity by regulating the calcium homeostasis in Alzheimer’s disease.

Fresh and nonfibrillar amyloid β protein(1–40) induces rapid cellular degeneration in aged human fibroblasts: evidence for AβP-channel-mediated cellular toxicity

Alzheimers disease (AD) is primarily nonfamilial or sporadic (SAD) in origin, although several genetic linkages are reported. Tissues from AD patients contain fibrillar plaques made of 39 to 43 amino acid‐long amyloid beta peptide (AβP), although the mechanisms of AβP toxicity are poorly understood. AβP1–40 is the most prevalent AβP present in the neuronal and non‐neuronal tissues from SAD patients. AβP1–40 toxicity has been examined mainly after prolonged incubation and correlates with the age and fibrillar morphology of AβP1–40. Globular and nonfibrillar AβPs are released continually during normal cellular metabolism; they elevate cellular Ca2+ and form cation‐permeable channels. However, their role in cellular toxicity is poorly understood. We have used an integrated atomic force and light fluorescence microscopy (AFM‐LFM), laser confocal microscopy, and calcium imaging to examine real‐time and acute effect of fresh and globular AβP1–40 on cultured, aged human, AD‐free fibroblasts. AFM images show that freshly prepared AβP1–40 in phosphate‐buffered saline (PBS) are globular and do not form fiber for an extended time period. AβP1–40 induced rapid structural modifications, including cytoskeletal reorganization, retraction of cellular processes, and loss of cell‐cell contacts, within minutes of incubation. This led to eventual cellular degeneration. AβP1–40‐induced degeneration was prevented by anti‐AβP antibody, zinc, and Tris, but not by tachykinin neuropeptides. In Ca2+‐free extracellular medium, AβP1–40 did not induce cellular degeneration. In the presence of extracellular Ca2+, AβP1–40 induced a sustained increase in the cellular Ca2+. Thus, short‐term and acute AβP1–40 toxicity is mediated by Ca2+ uptake, most likely via calcium‐permeable AβP pores. Such rapid degeneration does not require fibrillar plaques, suggesting that the plaques may not have any causative role.—Zhu, Y. J., Lin, H., Lal, R. Fresh and nonfibrillar amyloid β protein(1–40) induces rapid cellular degeneration in aged human fibroblasts: evidence for AβP‐channel‐mediated cellular toxicity. FASEB J. 14, 1244–1254 (2000)

Integrated multiple patch-clamp array chip via lateral cell trapping junctions

We present an integrated multiple patch-clamp array chip by utilizing lateral cell trapping junctions. The intersectional design of a microfluidic network provides multiple cell addressing and manipulation sites for efficient electrophysiological measurements at a number of patch sites. The patch pores consist of openings in the sidewall of a main fluidic channel, and a membrane patch is drawn into a smaller horizontal channel. This device geometry not only minimizes capacitive coupling between the cell reservoir and the patch channel, but also allows simultaneous optical and electrical measurements of ion channel proteins. Evidence of the hydrodynamic placement of mammalian cells at the patch sites as well as measurements of patch sealing resistance is presented. Device fabrication is based on micromolding of polydimethylsiloxane, thus allowing inexpensive mass production of disposable high-throughput biochips.

Microtubule-dependent oligomerization of tau. Implications for physiological tau function and tauopathies.

The accumulation of abnormal tau filaments is a pathological hallmark of many neurodegenerative diseases. In 1998, genetic analyses revealed a direct linkage between structural and regulatory mutations in the tau gene and the neurodegenerative disease, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). Importantly, the FTDP-17 phenotype is transmitted in a dominant rather than a recessive manner. However, the underlying molecular mechanisms causing disease remain uncertain. The most common molecular mechanism generating dominant phenotypes is the loss of function of a multimeric complex containing both mutant and wild-type subunits. Therefore, we sought to determine whether tau might normally function as a multimer. We co-incubated 35S-radiolabeled tau and biotinylated tau with taxol stabilized microtubules, at very low molar ratios of tau to tubulin. Subsequent covalent cross-linking followed by affinity-precipitation of the biotinylated tau revealed the formation of microtubule-dependent tau oligomers. We next used atomic force microscopy to independently assess this conclusion. Our results are consistent with the hypothesis that tau forms oligomers upon binding to microtubules. In addition to providing insights into normal tau action, our findings lead us to propose that one mechanism by which mutations in tau may cause cell death is through the formation of tau complexes containing mutant tau molecules in association with wild-type tau. These wild-type-mutant tau complexes may possess altered biological and/or biophysical properties that promote onset of the FTDP-17 phenotype, including neuronal cell death by either altering normal tau-mediated regulation of microtubule-dependent cellular functions and/or promoting the formation of pathological tau aggregates.

Imaging real-time aggregation of amyloid beta protein (1-42) by atomic force microscopy.

Amyloid beta protein (AbetaP) is the major fibrillar constituent of senile plaques. However, no causative role for AbetaP-fibers in Alzheimers disease (AD) pathology is established. Globular AbetaPs are continuously released during normal cellular metabolism at pico- to nano-molar concentration. We used atomic force microscopy (AFM) to examine aggregation of freshly prepared AbetaP(1-42) and to examine the role of AbetaP concentration, imaging medium (air, water, or PBS) and agonists/antagonists on AbetaP-fibrillogenesis. At even very high and non-physiological AbetaP concentrations, 24-48 h of real-time AFM imaging (a) in water show only multiple layers of globular aggregates and no fibrils and (b) in PBS show mainly the globular structures and some short fibrils. On-line addition of Zn, an agonist for AbetaP-fibrillogenesis, induced a slow but non-fibrillar aggregation of globular AbetaPs. EDTA, a chelator of Zn and calcium (a modulator of AbetaP-mediated toxicity) induced a reversible change in the Zn-mediated aggregation. These results strongly suggest that no AbetaP-fibers are formed for the physiologically relevant concentration and thus the plaque-associated fibers may not account for the AD pathophysiology.

Imaging molecular structure and physiological function of gap junctions and hemijunctions by multimodal atomic force microscopy

Gap junctions are specialized plasma membrane structures that join neighboring cells via specialized intercellular ion channels (hemichannels) and provide a direct pathway for cell‐cell communication. They presumably mediate regulation of growth, transmission of developmental signals, coordination of muscle contraction, and maintenance of metabolic homeostasis. Hemichannels are also present in the non‐junctional regions of the cell plasma membrane and they provide a direct pathway for communication between the cytoplasm and the extracellular region. Recent studies suggest that gap junctional communication is much more complex than previously anticipated, in terms of both its structure as well as its activity. While the mechanism of gap junction activity is being studied extensively, their quaternary structure, assembly, and conformational changes underlying gating of their activity as well as their physiological role are poorly understood because, due to their complex structure, these junctions are less amenable to existing techniques for high‐resolution three‐dimensional structure‐function analyses. Atomic Force Microscopy (AFM) images molecular structure of biological specimens in an aqueous environment, allows on‐line perturbations, and can be coupled with electrophysiological, biochemical, and other microscopic techniques. The present review examines the potential of AFM application for the study of the molecular structure of hydrated, native gap junctions and hemijunctions as well as their physiological functions. Special attention is paid to new, complementary, or provocative findings from AFM studies of both vertebrate and invertebrate gap junctions and hemijunctions. Microsc. Res. Tech. 52:273–288, 2001.

Multimodal atomic force microscopy: Biological imaging using atomic force microscopy combined with light fluorescence and confocal microscopies and electrophysiologic recording

Because the atomic force microscope (AFM) allows molecular resolution imaging of hydrated specimens, it provides a unique window to the microscopic biological world. A high signal‐to‐noise ratio in AFM images sets them apart from the images obtained from other techniques: One does not need extensive image analyses often required by other techniques to obtain high‐resolution information. AFM can provide molecular details on crystalline as well as amorphous materials. However, it is often limited in providing identity of the imaged structures, especially in a complex system such as a cellular membrane. AFMs application for biological imaging will rely on an unambiguous identification of imaged structures. For mixed macromolecules, it may be essential to make critical comparisons of the same structural features imaged with AFM and other techniques such as light fluorescence and confocal microscopies, electron microscopy and X‐ray diffraction, and biochemical, immunologic, and pharmacologic techniques and electrophysiologic recordings. Significantly, the simple design of AFM allows it to be integrated with other techniques for simultaneous multimodal imaging. Recent combined multimodal imaging include light fluorescence, confocal, and near‐field optical imaging as well as electrophysiologic recordings. Preliminary studies from such multimodal imaging include 1) an independent identification of macromolecules in a complex specimen using appropriately labeled markers such as fluorescent‐dye labeled antibodies or dark‐field microscopy; 2) imaging real‐time reorganization of surface features using laser confocal and AFM; 3) a direct correlation of structural features and ion transfer via pores in a membrane; and 4) macromolecular complexes such as receptor‐ligand and antigen‐antibody. These features of a multimodal imaging system will provide new and significant avenues for a direct real‐time structure‐function correlation studies of biological macromolecules.

Atomic force microscopy of the three-dimensional crystal of membrane protein, OmpC porin

Three-dimensional microcrystals of OmpC osmoporin were air-dried slowly and imaged in air with an atomic force microscope (AFM). The overall structural features in AFM images are in good agreement with the X-ray diffraction data of these OmpC osmoporin crystals: monoclinic P2(1) with the unit cell constants a=117.6 Å, b=110 Å, c=298.4 Å, beta=97 degrees. Such a good correspondence between X-ray diffraction and AFM data suggests that the slow and mild air-drying of these crystals did not induce any significant alterations in the crystal lattices as expected upon crystal dehydration. At the (100) crystal face, individual trimeric protein-detergent complexes were resolved. These results show the potential for studying the molecular structure of microcrystals of integral membrane proteins. This study also suggests that the crystal grew in a fashion of rapid two-dimensional expansion along the bc plane followed by a slow deposition along the a axis, perhaps as a rate-limiting nucleation process. Thus, AFM imaging of air-dried crystals would also be of considerable use in the early stages of a project to grow large three-dimensional crystals of membrane proteins suitable for high-resolution X-ray diffraction studies.