Cargo hold and delivery: Ankyrins, spectrins, and their functional patterning of neurons

The highly polarized, typically very long, and nonmitotic nature of neurons present them with unique challenges in the maintenance of their homeostasis. This architectural complexity serves a rich and tightly controlled set of functions that enables their fast communication with neighboring cells and endows them with exquisite plasticity. The submembrane neuronal cytoskeleton occupies a pivotal position in orchestrating the structural patterning that determines local and long-range subcellular specialization, membrane dynamics, and a wide range of signaling events. At its center is the partnership between ankyrins and spectrins, which self-assemble with both remarkable long-range regularity and micro- and nanoscale specificity to precisely position and stabilize cell adhesion molecules, membrane transporters, ion channels, and other cytoskeletal proteins. To accomplish these generally conserved, but often functionally divergent and spatially diverse, roles these partners use a combinatorial program of a couple of dozens interacting family members, whose code is not fully unraveled. In a departure from their scaffolding roles, ankyrins and spectrins also enable the delivery of material to the plasma membrane by facilitating intracellular transport. Thus, it is unsurprising that deficits in ankyrins and spectrins underlie several neurodevelopmental, neurodegenerative, and psychiatric disorders. Here, I summarize key aspects of the biology of spectrins and ankyrins in the mammalian neuron and provide a snapshot of the latest advances in decoding their roles in the nervous system

CNF1 Improves Astrocytic Ability to Support Neuronal Growth and Differentiation In vitro

Modulation of cerebral Rho GTPases activity in mice brain by intracerebral administration of Cytotoxic Necrotizing Factor 1 (CNF1) leads to enhanced neurotransmission and synaptic plasticity and improves learning and memory. To gain more insight into the interactions between CNF1 and neuronal cells, we used primary neuronal and astrocytic cultures from rat embryonic brain to study CNF1 effects on neuronal differentiation, focusing on dendritic tree growth and synapse formation, which are strictly modulated by Rho GTPases. CNF1 profoundly remodeled the cytoskeleton of hippocampal and cortical neurons, which showed philopodia-like, actin-positive projections, thickened and poorly branched dendrites, and a decrease in synapse number. CNF1 removal, however, restored dendritic tree development and synapse formation, suggesting that the toxin can reversibly block neuronal differentiation. On differentiated neurons, CNF1 had a similar effacing effect on synapses. Therefore, a direct interaction with CNF1 is apparently deleterious for neurons. Since astrocytes play a pivotal role in neuronal differentiation and synaptic regulation, we wondered if the beneficial in vivo effect could be mediated by astrocytes. Primary astrocytes from embryonic cortex were treated with CNF1 for 48 hours and used as a substrate for growing hippocampal neurons. Such neurons showed an increased development of neurites, in respect to age-matched controls, with a wider dendritic tree and a richer content in synapses. In CNF1-exposed astrocytes, the production of interleukin 1β, known to reduce dendrite development and complexity in neuronal cultures, was decreased. These results demonstrate that astrocytes, under the influence of CNF1, increase their supporting activity on neuronal growth and differentiation, possibly related to the diminished levels of interleukin 1β. These observations suggest that the enhanced synaptic plasticity and improved learning and memory described in CNF1-injected mice are probably mediated by astrocytes

INCREASED OSMIOPHILIA OF GLYCOSAMINOGLYCAN-LIKE STRUCTURES AFTER FIXATION WITH CETYLPYRIDINIUM CHLORIDE IN HUMAN COLONIC MUCOSA

The effects on tissue ultrastructural appearance of fixation with cetylpyridinium chloride (CPC)-additioned formaldehyde were studied at the ultrastructural level in human colonic mucosa. We found that CK did not impair tissue morphology and induced a remarkable osmiophilia in glycosaminoglycan-like structures, which did not require further staining

Astrocyte modulation of in vitro beta-amyloid neurotoxicity.

In Alzheimer's disease brain, beta-amyloid (Abeta) deposition is accompanied by astrocyte activation, whose role in the pathogenesis of the disease is still unclear. To explore the subject, we compared Abeta neurotoxicity in pure hippocampal cultures and neuronal-astrocytic cocultures, where astrocytes conditioned neurons but were not in contact with them or Abeta. In the presence of astrocytes, neurons were protected from Abeta neurotoxicity. Neuritic dystrophy was reduced, synapses were partially preserved, and apoptosis was contrasted. The protection disappeared when astrocytes were also treated with Abeta, suggesting that Abeta-astrocyte interaction is deleterious for neurons. This was supported by comparing Abeta neurotoxicity in pure neurons and neurons grown on astrocytes. In this case, where astrocytes were also in contact with Abeta, neuritic damage was enhanced and expression of synaptic vesicle proteins decreased. Our results suggest that astrocytes can protect neurons from Abeta neurotoxicity, but when they interact with Abeta, the protection is undermined and neurotoxicity enhanced. Copyright 2004 Wiley-Liss, Inc

Ultrastructural immunolocalization of laminin and fibronectin in human large intestinal mucosa.

The ultrastructural expression of laminin and fibronectin was studied in human colonic mucosa with a pre-embedding immunoperoxidase technique. Both antigens were present in epithelial basement membrane. The lamina densa was uniformly and homogeneously immunostained by both fibronectin and laminin, while the lamina rara showed discrete, positive sites occasionally connecting the lamina densa with the epithelial plasma membrane. In the lamina reticularis, fibronectin was diffusely positive, while laminin showed a discontinuous positivity only in the uppermost region in vicinity of the lamina densa. Collagen fibrils were regularly decorated by fibronectin-positive material. Basement membranes in the capillary wall showed a similar pattern of reactivity both with laminin and fibronectin. These results point out that laminin as well as fibronectin are integral components of intestinal mucosa basement membranes

Use of CNF1 to treat neuroinflammation and astrogliosis in CNS diseases

We have previously reported that the Rho-activating Cytotoxic Necrotizing Factor 1 (CNF1) enhances cognitive performances, counteracts inflammatory pain, and triggers adult visual cortex functional plasticity in experimental animal models. CNF1 is a 113.8 Kda protein, produced by several Escherichia coli strains, which selectively and permanently activate the Rho GTPases, proteins primarily involved in the actin cytoskeleton regulation. In this work, we firstly demonstrate that CNF1 can specifically act on astrocytes, the most abundant type of glial cells in the central nervous system (CNS), which are involved in the induction of neuroinflammation. CNF1-treated astrocytes exert a supportive role for neurogenesis and reduce the production of the pro-inflammatory cytokine IL-1beta in vitro and also in vivo. In fact, in transgenic mice for ApoE4, a genetic risk factor for diseases with lipid metabolism and transport dysregulation and alteration of immune regulation (such as cerebral ischemia, brain infarction, arteriosclerosis, cerebral amyloid angiopathy, Alzheimer's disease etc.), CNF1 reduces hippocampal astrogliosis, brings ATP and IL-1beta to levels comparable to those of control ApoE3 animals, and decreases the beta amyloid accumulation. Reactive astrogliosis and inflammatory changes are also involved in all forms of seizures and, interestingly, CNF1 induces, even at sensorimotor cortex level, suppression of epileptiform phenomena in the DBA2/J mouse model of \u201cpetit mal\u201d (Absence seizures). CNF1 can thus represent a unique pharmacological agent for treatment of those CNS disorders characterized by neuroinflammation and astrogliosis

Endocrine cells in intestinal metaplasia of the stomach

In this study we have investigated the mucin profile and the endocrine cell population in gastric endoscopic biopsies from 22 patients affected by chronic gastritis and intestinal metaplasia and in five surgical specimens of stomachs removed because of intestinal-type carcinoma (4) or peptic ulcer (1). High iron diamine-Alcian blue (HID-Ab) staining and peptide immunocytochemistry (peroxidase anti-peroxidase technique) were used. Forty-one foci of intestinal metaplasia were detected, 15 produced sulphomucins and 26 sialomucins. Of the endocrine cells investigated, gastrin and somatostatin cells were the most frequently observed, while cholecystokinin, glucose-dependent insulinotropic peptide-, secretin- and enteroglucagon-containing cells were also found in the metaplastic areas, but less frequently. No significant correlation was found between the type of mucin and the types of endocrine cells present, the latter usually resembling those normally found in the small intestine. On the basis of these results we conclude that intestinal metaplasia involves mucin- and peptide-producing cells of the stomach in a variable manner, with no correlation between the two

Blockade of chloride intracellular ion channel 1 stimulates Abeta phagocytosis

In amyloid-\u3b2 (A\u3b2)-stimulated microglial cells, blockade of chloride intracellular ion channel 1 (CLIC1) reverts the increase in tumor necrosis factor-\u3b1 and nitric oxide (NO) production and results in neuroprotection of cocultured neurons. This effect could be of therapeutic efficacy in Alzheimer's disease (AD), where microglial activation may contribute to neurodegeneration, but it could reduce A\u3b2 phagocytosis, which could facilitate amyloid plaque removal. Here, we analyzed the CLIC1 blockade effect on A\u3b2-stimulated mononuclear phagocytosis. In the microglial cell line BV-2, A\u3b225-35 treatment enhanced fluorescent bead phagocytosis, which persisted also in the presence of IAA-94, a CLIC1 channel blocker. The same result was obtained in rat primary microglia and in BV2 cells, where CLIC1 expression had been knocked down with a plasmid producing small interfering RNAs. To address specifically the issue of A\u3b2 phagocytosis, we treated BV-2 cells with biotinylated A\u3b21-42 and measured intracellular amyloid by morphometric analysis. IAA-94-treated cells showed an increased A\u3b2 phagocytosis after 24 hr and efficient degradation of ingested material after 72 hr. In addition, we tested A\u3b21-42 phagocytosis in adult rat peritoneal macrophages. Also, these cells actively phagocytosed A\u3b21-42 in the presence of IAA-94. However, the increased expression of inducible NO synthase (iNOS), stimulated by A\u3b2, was reverted by IAA-94. In parallel, a decrease in NO release was detected. These results suggest that blockade of CLIC1 stimulates A\u3b2 phagocytosis in mononuclear phagocytes while inhibiting the induction of iNOS and further point to CLIC1 as a possible therapeutic target in AD

Blockade of chloride intracellular ion channel 1 stimulates a beta phagocytosis

In amyloid-beta (A beta)-stimulated microglial cells, blockade of chloride intracellular ion channel 1 (CLIC1) reverts the increase in tumor necrosis factor-a and nitric oxide (NO) production and results in neuroprotection of cocultured neurons. This effect could be of therapeutic efficacy in Alzheimer's disease (AD), where microglial activation may contribute to neurodegeneration, but it could reduce A beta phagocytosis, which could facilitate amyloid plaque removal. Here, we analyzed the CLIC1 blockade effect on A beta-stimulated mononuclear phagocytosis. In the microglial cell line BV-2, A beta 25-35 treatment enhanced fluorescent bead phagocytosis, which persisted also in the presence of IAA-94, a CLIC1 channel blocker. The same result was obtained in rat primary microglia and in BV2 cells, where CLIC1 expression had been knocked down with a plasmid producing small interfering RNAs. To address specifically the issue of A beta phagocytosis, we treated BV-2 cells with biotinylated A beta 1-42 and measured intracellular amyloid by morphometric analysis. IAA-94-treated cells showed an increased A beta phagocytosis after 24 hr and efficient degradation of ingested material after 72 hr. In addition, we tested A beta 1-42 phagocytosis in adult rat peritoneal macrophages. Also, these cells actively phagocytosed A beta 1-42 in the presence of IAA-94. However, the increased expression of inducible NO synthase (NOS), stimulated by A beta, was reverted by IAA-94. In parallel, a decrease in NO release was detected. These results suggest that blockade of CLIC1 stimulates A beta phagocytosis in mononuclear phagocytes while inhibiting the induction of NOS and further point to CLIC1 as a possible therapeutic target in AD. (C) 2008 Wiley-Liss, Inc