KIBRA (KIdney/BRAin protein) regulates learning and memory and stabilizes Protein kinase Mζ.

The WWC1 gene has been genetically associated with human episodic memory performance, and its product KIdney/BRAin protein (KIBRA) has been shown to interact with the atypical protein kinase protein kinase M ζ (PKMζ). Although recently challenged, PKMζ remains a candidate postsynaptic regulator of memory maintenance. Here, we show that PKMζ is subject to rapid proteasomal degradation and that KIBRA is both necessary and sufficient to counteract this process, thus stabilizing the kinase and maintaining its function for a prolonged time. We define the binding sequence on KIBRA, a short amino acid motif near the C-terminus. Both hippocampal knock-down of KIBRA in rats and KIBRA knock-out in mice result in decreased learning and memory performance in spatial memory tasks supporting the notion that KIBRA is a player in episodic memory. Interestingly, decreased memory performance is accompanied by decreased PKMζ protein levels. We speculate that the stabilization of synaptic PKMζ protein levels by KIBRA may be one mechanism by which KIBRA acts in memory maintenance. KIBRA/WWC1 has been genetically associated with human episodic memory. KIBRA has been shown to be post-synaptically localized, but its function remained obscure. Here, we show that KIBRA shields PKMζ, a kinase previously linked to memory maintenance, from proteasomal degradation via direct interaction. KIBRA levels in the rodent hippocampus correlate closely both to spatial memory performance in rodents and to PKMζ levels. Our findings support a role for KIBRA in memory, and unveil a novel function for this protein.

Gene expression changes in spinal motoneurons of the SOD1(G93A) transgenic model for ALS after treatment with G-CSF.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is an incurable fatal motoneuron disease with a lifetime risk of approximately 1:400. It is characterized by progressive weakness, muscle wasting, and death ensuing 3-5 years after diagnosis. Granulocyte-colony stimulating factor (G-CSF) is a drug candidate for ALS, with evidence for efficacy from animal studies and interesting data from pilot clinical trials. To gain insight into the disease mechanisms and mode of action of G-CSF, we performed gene expression profiling on isolated lumbar motoneurons from SOD1(G93A) mice, the most frequently studied animal model for ALS, with and without G-CSF treatment. RESULTS: Motoneurons from SOD1(G93A) mice present a distinct gene expression profile in comparison to controls already at an early disease stage (11 weeks of age), when treatment was initiated. The degree of deregulation increases at a time where motor symptoms are obvious (15 weeks of age). Upon G-CSF treatment, transcriptomic deregulations of SOD1(G93A) motoneurons were notably restored. Discriminant analysis revealed that SOD1 mice treated with G-CSF has a transcriptom close to presymptomatic SOD1 mice or wild type mice. Some interesting genes modulated by G-CSF treatment relate to neuromuscular function such as CCR4-NOT or Prss12. CONCLUSIONS: Our data suggest that G-CSF is able to re-adjust gene expression in symptomatic SOD1(G93A) motoneurons. This provides further arguments for G-CSF as a promising drug candidate for ALS.

A novel flow cytometry-based technique to measure adult neurogenesis in the brain.

The stimulation of neurogenesis is an exciting novel therapeutic option for diseases of the central nervous system, ranging from depression to neurodegeneration. One major bottleneck in screening approaches for neurogenesis-inducing compounds is the very demanding in vivo quantification of newborn neurons based on stereological techniques. To effectively develop compounds in this area, novel fast and reliable techniques for quantification of in vivo neurogenesis are needed. In this study, we introduce a flow cytometry-based method for quantifying newly generated neurons in the brain based on the counting of cell nuclei from dissected brain regions. Important steps involve density sedimentation of the cell nuclei, and staining for the proliferation marker bromodeoxy uridine and nuclear cell type markers such as NeuN. We demonstrate the ability of the technique to detect increased neurogenesis in the hippocampus of animals which underwent physical exercise and received fluoxetine treatment.

KIBRA: A New Gateway to Learning and Memory?

The genetic locus encoding KIBRA, a member of the WWC family of proteins, has recently been shown to be associated with human memory performance through genome-wide single nucleotide polymorphism screening. Gene expression analysis and a variety of functional studies have further indicated that such a role is biologically plausible for KIBRA. Here, we review the existing literature, illustrate connections between the different lines of evidence, and derive models based on KIBRA's function(s) in the brain that can be further tested experimentally.

Mutations in amyloid precursor protein affect its interactions with presenilin/gamma-secretase.

Alzheimer's disease is characterized by accumulation of toxic beta-amyloid (Abeta) in the brain and neuronal death. Several mutations in presenilin (PS1) and beta-amyloid precursor protein (APP) associate with an increased Abeta(42/40) ratio. Abeta(42), a highly fibrillogenic species, is believed to drive Abeta aggregation. Factors shifting gamma-secretase cleavage of APP to produce Abeta(42) are unclear. We investigate the molecular mechanism underlying altered Abeta(42/40) ratios associated with APP mutations at codon 716 and 717. Using FRET-based fluorescence lifetime imaging to monitor APP-PS1 interactions, we show that I716F and V717I APP mutations increase the proportion of interacting molecules earlier in the secretory pathway, resulting in an increase in Abeta generation. A PS1 conformation assay reveals that, in the presence of mutant APP, PS1 adopts a conformation reminiscent of FAD-associated PS1 mutations, thus influencing APP binding to PS1/gamma-secretase. Mutant APP affects both intracellular location and efficiency of APP-PS1 interactions, thereby changing the Abeta(42/40) ratio.

Granulocyte-colony stimulating factor improves outcome in a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that results in progressive loss of motoneurons, motor weakness and death within 1-5 years after disease onset. Therapeutic options remain limited despite a substantial number of approaches that have been tested clinically. In particular, various neurotrophic factors have been investigated. Failure in these trials has been largely ascribed to problems of insufficient dosing or inability to cross the blood-brain barrier (BBB). We have recently uncovered the neurotrophic properties of the haematopoietic protein granulocyte-colony stimulating factor (G-CSF). The protein is clinically well tolerated and crosses the intact BBB. This study examined the potential role of G-CSF in motoneuron diseases. We investigated the expression of the G-CSF receptor in motoneurons and studied effects of G-CSF in a motoneuron cell line and in the SOD1(G93A) transgenic mouse model. The neurotrophic growth factor was applied both by continuous subcutaneous delivery and CNS-targeted transgenic overexpression. This study shows that given at the stage of the disease where muscle denervation is already evident, G-CSF leads to significant improvement in motor performance, delays the onset of severe motor impairment and prolongs overall survival of SOD1(G93A)tg mice. The G-CSF receptor is expressed by motoneurons and G-CSF protects cultured motoneuronal cells from apoptosis. In ALS mice, G-CSF increased survival of motoneurons and decreased muscular denervation atrophy. We conclude that G-CSF is a novel neurotrophic factor for motoneurons that is an attractive and feasible drug candidate for the treatment of ALS.

The LDL receptor-related protein can form homo-dimers in neuronal cells.

The ability of the low density lipoprotein receptor-related protein (LRP) to form homo-dimers was studied in mouse neuroblastoma and human neuroglioma cells as well as in primary cortical cultures from adult mouse brain. Homo-dimerization of LRP light chain (LC) was shown by several methods including co-immunoprecipitation, fluorescence lifetime imaging microscopy, and bimolecular fluorescence complementation assay. The requirement of intact NPXY motifs of LRP LC for homo-dimerization was ruled out by co-immunoprecipitation assay.

Formation of toxic oligomeric alpha-synuclein species in living cells.

BACKGROUND: Misfolding, oligomerization, and fibrillization of alpha-synuclein are thought to be central events in the onset and progression of Parkinson's disease (PD) and related disorders. Although fibrillar alpha-synuclein is a major component of Lewy bodies (LBs), recent data implicate prefibrillar, oligomeric intermediates as the toxic species. However, to date, oligomeric species have not been identified in living cells. METHODOLOGY/PRINCIPAL FINDINGS: Here we used bimolecular fluorescence complementation (BiFC) to directly visualize alpha-synuclein oligomerization in living cells, allowing us to study the initial events leading to alpha-synuclein oligomerization, the precursor to aggregate formation. This novel assay provides us with a tool with which to investigate how manipulations affecting alpha-synuclein aggregation affect the process over time. Stabilization of alpha-synuclein oligomers via BiFC results in increased cytotoxicity, which can be rescued by Hsp70 in a process that reduces the formation of alpha-synuclein oligomers. Introduction of PD-associated mutations in alpha-synuclein did not affect oligomer formation but the biochemical properties of the mutant alpha-synuclein oligomers differ from those of wild type alpha-synuclein. CONCLUSIONS/SIGNIFICANCE: This novel application of the BiFC assay to the study of the molecular basis of neurodegenerative disorders enabled the direct visualization of alpha-synuclein oligomeric species in living cells and its modulation by Hsp70, constituting a novel important tool in the search for therapeutics for synucleinopathies.

Impact of cholesterol level upon APP and BACE proximity and APP cleavage.

Cleavage of APP by BACE is the first proteolytic step in the production of Amyloid beta (Abeta, which accumulates in senile plaques in Alzheimer's disease. BACE-cleavage of APP is thought to happen in endosomes. However, there are controversial data whether APP and BACE can already interact on the cell surface dependent on the cholesterol level. To examine whether APP and BACE come into close proximity on the cell surface in living cells, we employed a novel technique by combining time-resolved Förster resonance energy transfer (FRET) measurements with total internal reflection microscopy (TIRET microscopy). Our data indicate that BACE and APP come into close proximity within the cell, but probably not on the cell surface. To analyze the impact of alterations in cholesterol level upon BACE-cleavage, we measured sAPP secretion. Alteration of APP processing and BACE proximity by cholesterol might be explained by alterations in cell membrane fluidity.

GGA1 acts as a spatial switch altering amyloid precursor protein trafficking and processing.

The beta-amyloid (Abeta) precursor protein (APP) is cleaved sequentially by beta-site of APP-cleaving enzyme (BACE) and gamma-secretase to release the Abeta peptides that accumulate in plaques in Alzheimer's disease (AD). GGA1, a member of the Golgi-localized gamma-ear-containing ARF-binding (GGA) protein family, interacts with BACE and influences its subcellular distribution. We now report that overexpression of GGA1 in cells increased the APP C-terminal fragment resulting from beta-cleavage but surprisingly reduced Abeta. GGA1 confined APP to the Golgi, in which fluorescence resonance energy transfer analyses suggest that the proteins come into close proximity. GGA1 blunted only APP but not notch intracellular domain release. These results suggest that GGA1 prevented APP beta-cleavage products from becoming substrates for gamma-secretase. Direct binding of GGA1 to BACE was not required for these effects, but the integrity of the GAT (GGA1 and TOM) domain of GGA1 was. GGA1 may act as a specific spatial switch influencing APP trafficking and processing, so that APP-GGA1 interactions may have pathophysiological relevance in AD.

Interaction between presenilin 1 and ubiquilin 1 as detected by fluorescence lifetime imaging microscopy and a high-throughput fluorescent plate reader.

Presenilin 1 (PS1) in its active heterodimeric form is the catalytic center of the gamma-secretase complex, an enzymatic activity that cleaves amyloid precursor protein (APP) to produce amyloid beta (Abeta). Ubiquilin 1 is a recently described PS1 interacting protein, the overexpression of which increases PS1 holoprotein levels and leads to reduced levels of functionally active PS1 heterodimer. In addition, it has been suggested that splice variants of the UBQLN1 gene are associated with an increased risk of developing Alzheimer disease (AD). However, it is still unclear whether PS1 and ubiquilin 1 interact when expressed at endogenous levels under normal physiological conditions. Here, we employ three novel fluorescence resonance energy transfer-based techniques to investigate the interaction between PS1 and ubiquilin 1 in intact cells. We consistently find that the ubiquilin 1 N terminus is in close proximity to several epitopes on PS1. We show that ubiquilin 1 interacts both with PS1 holoprotein and heterodimer and that the interaction between PS1 and ubiquilin 1 takes place near the cell surface. Furthermore, we show that the PS1-ubiquilin 1 interaction can be detected between endogenous proteins in primary neurons in vitro as well as in brain tissue of healthy controls and Alzheimer disease patients, providing evidence of its physiological relevance.

Dopamine D1 activation potentiates striatal NMDA receptors by tyrosine phosphorylation-dependent subunit trafficking.

Interactions between dopaminergic and glutamatergic afferents in the striatum are essential for motor learning and the regulation of movement. An important mechanism for these interactions is the ability of dopamine, through D1 receptors, to potentiate NMDA glutamate receptor function. Here we show that, in striatal neurons, D1 receptor activation leads to rapid trafficking of NMDA receptor subunits, with increased NR1 and NR2B subunits in dendrites, enhanced coclustering of these subunits with the postsynaptic density scaffolding molecule postsynaptic density-95, and increased surface expression. The dopamine D1 receptor-mediated NMDA receptor trafficking is blocked by an inhibitor of tyrosine kinases. Blockers of tyrosine phosphatases also induce NMDA subunit trafficking, but this effect is nonselective and alters both NR2A- and NR2B-containing receptors. Furthermore, tyrosine phosphatase inhibition leads to the clustering of tyrosine-phosphorylated NR2B subunit along dendritic shafts. Our findings reveal that D1 receptor activation can potentiate striatal NMDA subunit function by directly promoting the surface insertion of the receptor complexes. This effect is regulated by the reciprocal actions of protein tyrosine phosphatases and tyrosine kinases. Modification of these pathways may be a useful therapeutic target for Parkinson's disease and other basal ganglia disorders in which abnormal function of striatal NMDA receptors contributes to the symptoms of the diseases.

Molecular dissection of the interaction between amyloid precursor protein and its neuronal trafficking receptor SorLA/LR11.

SorLA/LR11 is a sorting receptor that regulates the intracellular transport and processing of the amyloid precursor protein (APP) in neurons. SorLA/LR11-mediated binding results in sequestration of APP in the Golgi and in protection from processing into the amyloid-beta peptide (Abeta), the principal component of senile plaques in Alzheimer's disease (AD). To gain insight into the molecular mechanisms governing sorLA and APP interaction, we have dissected the respective protein interacting domains. Using a fluorescence resonance energy transfer (FRET) based assay of protein proximity, we identified binding sites in the extracellular regions of both proteins. Fine mapping by surface plasmon resonance analysis and analytical ultracentrifugation of recombinant APP and sorLA fragments further narrowed down the binding domains to the cluster of complement-type repeats in sorLA that forms a 1:1 stoichiometric complex with the carbohydrate-linked domain of APP. These data shed new light on the molecular determinants of neuronal APP trafficking and processing and on possible targets for intervention with senile plaque formation in patients with AD.

Interaction of the cytosolic domains of sorLA/LR11 with the amyloid precursor protein (APP) and beta-secretase beta-site APP-cleaving enzyme.

sorLA is a recently identified neuronal receptor for amyloid precursor protein (APP) that is known to interact with APP and affect its intracellular transport and processing. Decreased levels of sorLA in the brain of Alzheimer's disease (AD) patients and elevated levels of amyloid-beta peptide (Abeta) in sorLA-deficient mice point to the importance of the receptor in this neurodegenerative disorder. We analyzed APP cleavage in an APP-shedding assay and found that both sorLA and, surprisingly, a sorLA tail construct inhibited APP cleavage in a beta-site APP-cleaving enzyme (BACE)-dependent manner. In line with this finding, sorLA and the sorLA tail significantly reduced secreted Abeta levels when BACE was overexpressed, suggesting that sorLA influences beta-cleavage. To understand the effect of sorLA on APP cleavage by BACE, we analyzed whether sorLA interacts with APP and/or BACE. Because both full-length sorLA and sorLA C-terminal tail constructs were functionally relevant for APP processing, we analyzed sorLA-APP for a potential cytoplasmatic interaction domain. sorLA and C99 coimmunoprecipitated, pointing toward the existence of a new cytoplasmatic interaction site between sorLA and APP. Moreover, sorLA and BACE also coimmunoprecipitate. Thus, sorLA interacts both with BACE and APP and might therefore directly affect BACE-APP complex formation. To test whether sorLA impacts BACE-APP interactions, we used a fluorescence resonance energy transfer assay to evaluate BACE-APP interactions in cells. We discovered that sorLA significantly reduced BACE-APP interactions in Golgi. We postulate that sorLA acts as a trafficking receptor that prevents BACE-APP interactions and hence BACE cleavage of APP.

Role of endocytosis in cellular uptake of sex steroids.

Androgens and estrogens are transported bound to the sex hormone binding globulin (SHBG). SHBG is believed to keep sex steroids inactive and to control the amount of free hormones that enter cells by passive diffusion. Contrary to the free hormone hypothesis, we demonstrate that megalin, an endocytic receptor in reproductive tissues, acts as a pathway for cellular uptake of biologically active androgens and estrogens bound to SHBG. In line with this function, lack of receptor expression in megalin knockout mice results in impaired descent of the testes into the scrotum in males and blockade of vagina opening in females. Both processes are critically dependent on sex-steroid signaling, and similar defects are seen in animals treated with androgen- or estrogen-receptor antagonists. Thus, our findings uncover the existence of endocytic pathways for protein bound androgens and estrogens and their crucial role in development of the reproductive organs.

Neuronal sorting protein-related receptor sorLA/LR11 regulates processing of the amyloid precursor protein.

sorLA (Sorting protein-related receptor) is a type-1 membrane protein of unknown function that is expressed in neurons. Its homology to sorting receptors that shuttle between the plasma membrane, endosomes, and the Golgi suggests a related function in neuronal trafficking processes. Because expression of sorLA is reduced in the brain of patients with Alzheimer's disease (AD), we tested involvement of this receptor in intracellular transport and processing of the amyloid precursor protein (APP) to the amyloid beta-peptide (Abeta), the principal component of senile plaques. We demonstrate that sorLA interacts with APP in vitro and in living cells and that both proteins colocalize in endosomal and Golgi compartments. Overexpression of sorLA in neurons causes redistribution of APP to the Golgi and decreased processing to Abeta, whereas ablation of sorLA expression in knockout mice results in increased levels of Abeta in the brain similar to the situation in AD patients. Thus, sorLA acts as a sorting receptor that protects APP from processing into Abeta and thereby reduces the burden of amyloidogenic peptide formation. Consequently, reduced receptor expression in the human brain may increase Abeta production and plaque formation and promote spontaneous AD.

The low density lipoprotein receptor-related protein (LRP) is a novel beta-secretase (BACE1) substrate.

BACE is a transmembrane protease with beta-secretase activity that cleaves the amyloid precursor protein (APP). After BACE cleavage, APP becomes a substrate for gamma-secretase, leading to release of amyloid-beta peptide (Abeta), which accumulates in senile plaques in Alzheimer disease. APP and BACE are co-internalized from the cell surface to early endosomes. APP is also known to interact at the cell surface and be internalized by the low density lipoprotein receptor-related protein (LRP), a multifunctional endocytic and signaling receptor. Using a new fluorescence resonance energy transfer (FRET)-based assay of protein proximity, fluorescence lifetime imaging (FLIM), and co-immunoprecipitation we demonstrate that the light chain of LRP interacts with BACE on the cell surface in association with lipid rafts. Surprisingly, the BACE-LRP interaction leads to an increase in LRP C-terminal fragment, release of secreted LRP in the media and subsequent release of the LRP intracellular domain from the membrane. Taken together, these data suggest that there is a close interaction between BACE and LRP on the cell surface, and that LRP is a novel BACE substrate.

LRP2/megalin is required for patterning of the ventral telencephalon.

Megalin is a low-density lipoprotein receptor-related protein (LRP2) expressed in the neuroepithelium and the yolk sac of the early embryo. Absence of megalin expression in knockout mice results in holoprosencephaly, indicating an essential yet unidentified function in forebrain development. We used mice with complete or conditional megalin gene inactivation in the embryo to demonstrate that expression of megalin in the neuroepithelium but not in the yolk sac is crucial for brain development. During early forebrain development, megalin deficiency leads to an increase in bone morphogenic protein (Bmp) 4 expression and signaling in the rostral dorsal neuroepithelium, and a subsequent loss of sonic hedgehog (Shh) expression in the ventral forebrain. As a consequence of absent SHH activity, ventrally derived oligodendroglial and interneuronal cell populations are lost in the forebrain of megalin-/- embryos. Similar defects are seen in models with enhanced signaling through BMPs, central regulators of neural tube patterning. Because megalin mediates endocytic uptake and degradation of BMP4, these findings indicate a role for megalin in neural tube specification, possibly by acting as BMP4 clearance receptor in the neuroepithelium.