Recent experimental and clinical retrospective studies support the view that reduction of brain cholesterol protects against Alzheimer's disease (AD). However, genetic and pharmacological evidence indicates that low brain cholesterol leads to neurodegeneration. This apparent contradiction prompted us to analyze the role of neuronal cholesterol in amyloid peptide generation in experimental systems that closely resemble physiological and pathological situations. We show that, in the hippocampus of control human and transgenic mice, only a small pool of endogenous APP and its β-secretase, BACE 1, are found in the same membrane environment. Much higher levels of BACE 1–APP colocalization is found in hippocampal membranes from AD patients or in rodent hippocampal neurons with a moderate reduction of membrane cholesterol. Their increased colocalization is associated with elevated production of amyloid peptide. These results suggest that loss of neuronal membrane cholesterol contributes to excessive amyloidogenesis in AD and pave the way for the identification of the cause of cholesterol loss and for the development of specific therapeutic strategies.

Axon specification triggers the polarization of neurons and requires the localized destabilization of filamentous actin. Here we show that plasma membrane ganglioside sialidase (PMGS) asymmetrically accumulates at the tip of one neurite of the unpolarized rat neuron, inducing actin instability. Suppressing PMGS activity blocks axonal generation, whereas stimulating it accelerates the formation of a single (not several) axon. PMGS induces axon specification by enhancing TrkA activity locally, which triggers phosphatidylinositol-3-kinase (PI3K)- and Rac1-dependent inhibition of RhoA signaling and the consequent actin depolymerization in one neurite only. Thus, spatial restriction of an actin-regulating molecular machinery, in this case a membrane enzymatic activity, before polarization is enough to determine axonal fate.

The serine protease plasmin can efficiently degrade amyloid peptide in vitro, and is found at low levels in the hippocampus of patients with Alzheimer's disease (AD). The cause of such paucity remains unknown. We show here that the levels of total brain plasminogen and plasminogen-binding molecules are normal in these brain samples, yet plasminogen membrane binding is greatly reduced. Biochemical analysis reveals that the membranes of these brains have a mild, still significant, cholesterol reduction compared to age-matched controls, and anomalous raft microdomains. This was reflected by the loss of raft-enriched proteins, including plasminogen-binding and -activating molecules. Using hippocampal neurons in culture, we demonstrate that removal of a similar amount of membrane cholesterol is sufficient to induce raft disorganization, leading to reduced plasminogen membrane binding and low plasmin activity. These results suggest that brain raft alterations may contribute to AD by rendering the plasminogen system inefficient.

Polo-like kinases (Plks) are characterized by the presence of a specific domain, known as the polo box (PBD), involved in protein-protein interactions. Plk1 to Plk4 are involved in centrosome biology as well as the regulation of mitosis, cytokinesis, and cell cycle checkpoints in response to genotoxic stress. We have analyzed here the new member of the vertebrate family, Plk5, a protein that lacks the kinase domain in humans. Plk5 does not seem to have a role in cell cycle progression; in fact, it is downregulated in proliferating cells and accumulates in quiescent cells. This protein is mostly expressed in the brain of both mice and humans, and it modulates the formation of neuritic processes upon stimulation of the brain-derived neurotrophic factor (BDNF)/nerve growth factor (NGF)-Ras pathway in neurons. The human PLK5 gene is significantly silenced in astrocytoma and glioblastoma multiforme by promoter hypermethylation, suggesting a tumor suppressor function for this gene. Indeed, overexpression of Plk5 has potent apoptotic effects in these tumor cells. Thus, Plk5 seems to have evolved as a kinase-deficient PBD-containing protein with nervous system-specific functions and tumor suppressor activity in brain cancer.

Ubiquitous occurrence in Nature, abundant presence at strategically important places such as the cell surface and dynamic shifts in their profile by diverse molecular switches qualifies the glycans to serve as versatile biochemical signals. However, their exceptional structural complexity often prevents one noting how simple the rules of objective-driven assembly of glycan-encoded messages are. This review is intended to provide a tutorial for a broad readership. The principles of why carbohydrates meet all demands to be the coding section of an information transfer system, and this at unsurpassed high density, are explained. Despite appearing to be a random assortment of sugars and their substitutions, seemingly subtle structural variations in glycan chains by a sophisticated enzymatic machinery have emerged to account for their specific biological meaning. Acting as ‘readers’ of glycan-encoded information, carbohydrate-specific receptors (lectins) are a means to turn the glycans’ potential to serve as signals into a multitude of (patho)physiologically relevant responses. Once the far-reaching significance of this type of functional pairing has become clear, the various modes of spatial presentation of glycans and of carbohydrate recognition domains in lectins can be explored and rationalized. These discoveries are continuously revealing the intricacies of mutually adaptable routes to achieve essential selectivity and specificity. Equipped with these insights, readers will gain a fundamental understanding why carbohydrates form the third alphabet of life, joining the ranks of nucleotides and amino acids, and will also become aware of the importance of cellular communication via glycan–lectin recognition.

Spain ‡Institut f€ ur Physiologische Chemie, Tier€ arztliche Fakult€ at, Ludwig-Maximilians-Universit€ at, M€ unchen, GermanyAbstract Axon membrane glycoproteins are essential for neuronal differentiation, although the mechanisms underlying their polarized sorting and organization are poorly understood. We describe here that galectin-4 (Gal-4), a lectin highly expressed in gastrointestinal tissues and involved in epithelial glycoprotein transport, is expressed by hippocampal and cortical neurons where it is sorted to discrete segments of the axonal membrane in a microtubule-and sulfatide-dependent manner. Gal-4 knockdown retards axon growth, an effect that can be rescued by recombinant Gal-4 addition. This Gal-4 reduction, as inhibition of sulfatide synthesis does, lowers the presence and clustered organization of axon growth-promoting molecule NCAM L1 at the axon membrane. Furthermore, we find that Gal-4 interacts with L1 by specifically binding to LacNAc branch ends of L1 N-glycans. Impairing the maturation of these N-glycans precludes Gal-4/L1 association resulting in a failure of L1 membrane cluster organization. In all, Gal-4 sorts to axon plasma membrane segments by binding to sulfatide-containing microtubule-associated carriers and being bivalent, it organizes the transport of L1, and likely other axonal glycoproteins, by attaching them to the carriers through their LacNAc termini. This mechanism would underlie L1 functional organization on the plasma membrane, required for proper axon growth.

In order for neurons to perform their function, they must establish a highly polarized morphology characterized, in most of the cases, by a single axon and multiple dendrites. Herein we find that the evolutionarily conserved protein Kidins220 (kinase D-interacting substrate of 220-kDa), also known as ARMS (ankyrin repeat-rich membrane spanning), a downstream effector of protein kinase D and neurotrophin and ephrin receptors, regulates the establishment of neuronal polarity and development of dendrites. Kidins220/ARMS gain and loss of function experiments render severe phenotypic changes in the processes extended by hippocampal neurons in culture. Although Kidins220/ARMS early overexpression hinders neuronal development, its down-regulation by RNA interference results in the appearance of multiple longer axon-like extensions as well as aberrant dendritic arbors. We also find that Kidins220/ARMS interacts with tubulin and microtubule-regulating molecules whose role in neuronal morphogenesis is well established (microtubule-associated proteins 1b, 1a, and 2 and two members of the stathmin family). Importantly, neurons where Kidins220/ARMS has been knocked down register changes in the phosphorylation activity of MAP1b and stathmins. Altogether, our results indicate that Kidins220/ARMS is a key modulator of the activity of microtubuleregulating proteins known to actively regulate neuronal morphogenesis and suggest a mechanism by which it contributes to control neuronal development.Neuronal differentiation comprises several steps, among which the acquirement of a polarized axon-dendrite phenotype, with the corresponding asymmetrical distribution of proteins, is crucial. The morphological changes, followed by a neuron in order to polarize and form a single axon and multiple dendrites, are triggered by signaling cascades evoked by both intracellular and extracellular cues (1-4).Embryonic hippocampal neurons in culture constitute a model to study the mechanisms governing the establishment of polarity (2, 5, 6). These neurons undergo clear morphological changes during in vitro polarization. First, neurons attach to the plate and form lamellipodia and filopodia (stage 1). After several hours, they extend several minor immature neurites of apparent equivalent nature (stage 2) until one of these minor processes extends rapidly and becomes the axon (stage 3). The remaining neurites develop into dendrites (stage 4), after which neurons become morphologically and functionally mature (stage 5) (5, 6).During the early events of the establishment of polarity in this model, differences in local actin polymerization among the immature neurites play a crucial role in axonal determination (5, 7). In a similar manner, microtubule dynamics influence neuronal polarization, because local microtubule stabilization in one neurite specifies an axonal fate (8). Other known regulators of neuronal polarity and axon specification include proteins involved in polarized trafficking (4, 9, 10). However, how these different molecules are linked to t...

PNS axons have a high intrinsic regenerative ability, whereas most CNS axons show little regenerative response. We show that activation of Neu3 sialidase, also known as Neuraminidase-3, causing conversion of GD1a and GT1b to GM1 ganglioside, is an essential step in regeneration occurring in PNS (sensory) but not CNS (retinal) axons in adult rat. In PNS axons, axotomy activates Neu3 sialidase, increasing the ratio of GM1/GD1a and GM1/GT1b gangliosides immediately after injury in vitro and in vivo. No change in the GM1/GD1a ratio after axotomy was observed in retinal axons (in vitro and in vivo), despite the presence of Neu3 sialidase. Externally applied sialidase converted GD1a ganglioside to GM1 and rescued axon regeneration in CNS axons and in PNS axons after Neu3 sialidase blockade. Neu3 sialidase activation in DRGs is initiated by an influx of extracellular calcium, activating P38MAPK and then Neu3 sialidase. Ganglioside conversion by Neu3 sialidase further activates the ERK pathway. In CNS axons, P38MAPK and Neu3 sialidase were not activated by axotomy.