Trafficking of JC virus-like particles across the blood-brain barrier.

Hollow viral vectors, such as John Cunningham virus-like particles (JC VLPs), provide a unique opportunity to deliver drug cargo into targeted cells and tissue. Current understanding of the entry of JC virus in brain cells has remained insufficient. In particular, interaction of JC VLPs with the blood-brain barrier (BBB) has not been analyzed in detail. Thus, JC VLPs were produced in this study for investigating the trafficking across the BBB. We performed a carotid artery injection procedure for mouse brain to qualitatively study JC VLPs' in vivo binding and distribution and used in vitro approaches to analyze their uptake and export kinetics in brain endothelial cells. Our results show that clathrin-dependent mechanisms contributed to the entry of VLPs into brain endothelial cells, and exocytosis or transcytosis of VLPs across the BBB was observed in vitro. VLPs were found to interact with sialic acid glycans in mouse brain endothelia. The ability of JC VLPs to cross the BBB can be useful in developing a delivery system for transport of genes and small molecule cargoes to the brain.

Microglial Drug Targets in AD: Opportunities and Challenges in Drug Discovery and Development.

Alzheimer's disease (AD) is a large and increasing unmet medical need with no disease-modifying treatment currently available. Genetic evidence from genome-wide association studies (GWASs) and gene network analysis has clearly revealed a key role of the innate immune system in the brain, of which microglia are the most important element. Single-nucleotide polymorphisms (SNPs) in genes predominantly expressed in microglia have been associated with altered risk of developing AD. Furthermore, microglia-specific pathways are affected on the messenger RNA (mRNA) expression level in post-mortem AD tissue and in mouse models of AD. Together these findings have increased the interest in microglia biology, and numerous scientific reports have proposed microglial molecules and pathways as drug targets for AD. Target identification and validation are generally the first steps in drug discovery. Both target validation and drug lead identification for central nervous system (CNS) targets and diseases entail additional significant obstacles compared to peripheral targets and diseases. This makes CNS drug discovery, even with well-validated targets, challenging. In this article, we will illustrate the special challenges of AD drug discovery by discussing the viability/practicality of possible microglia drug targets including cluster of differentiation 33 (CD33), KCa3.1, kynurenines, ionotropic P2 receptor 7 (P2X7), programmed death-1 (PD-1), Toll-like receptors (TLRs), and triggering receptor expressed in myeloid cells 2 (TREM2).

SORLA regulates calpain-dependent degradation of synapsin.

INTRODUCTION: Sorting-related receptor with A-type repeats (SORLA) is an intracellular sorting receptor in neurons and a major risk factor for Alzheimer disease. METHODS: Here, we performed global proteome analyses in the brain of SORLA-deficient mice followed by biochemical and histopathologic studies to identify novel neuronal pathways affected by receptor dysfunction. RESULTS: We demonstrate that the lack of SORLA results in accumulation of phosphorylated synapsins in cortex and hippocampus. We propose an underlying molecular mechanism by demonstrating that SORLA interacts with phosphorylated synapsins through 14-3-3 adaptor proteins to deliver synapsins to calpain-mediated proteolytic degradation. DISCUSSION: Our results suggest a novel function for SORLA which is in control of synapsin degradation, potentially impacting on synaptic vesicle endocytosis and/or exocytosis.

SORLA-mediated trafficking of TrkB enhances the response of neurons to BDNF.

Stimulation of neurons with brain-derived neurotrophic factor (BDNF) results in robust induction of SORLA, an intracellular sorting receptor of the VPS10P domain receptor gene family. However, the relevance of SORLA for BDNF-induced neuronal responses has not previously been investigated. We now demonstrate that SORLA is a sorting factor for the tropomyosin-related kinase receptor B (TrkB) that facilitates trafficking of this BDNF receptor between synaptic plasma membranes, post-synaptic densities, and cell soma, a step critical for neuronal signal transduction. Loss of SORLA expression results in impaired neuritic transport of TrkB and in blunted response to BDNF in primary neurons; and it aggravates neuromotoric deficits caused by low BDNF activity in a mouse model of Huntington's disease. Thus, our studies revealed a key role for SORLA in mediating BDNF trophic signaling by regulating the intracellular location of TrkB.

Soluble alpha-APP (sAPPalpha) regulates CDK5 expression and activity in neurons.

A growing body of evidence suggests a role for soluble alpha-amyloid precursor protein (sAPPalpha) in pathomechanisms of Alzheimer disease (AD). This cleavage product of APP was identified to have neurotrophic properties. However, it remained enigmatic what proteins, targeted by sAPPalpha, might be involved in such neuroprotective actions. Here, we used high-resolution two-dimensional polyacrylamide gel electrophoresis to analyze proteome changes downstream of sAPPalpha in neurons. We present evidence that sAPPalpha regulates expression and activity of CDK5, a kinase that plays an important role in AD pathology. We also identified the cytoprotective chaperone ORP150 to be induced by sAPPalpha as part of this protective response. Finally, we present functional evidence that the sAPPalpha receptor SORLA is essential to mediate such molecular functions of sAPPalpha in neurons.

The pro-neurotrophin receptor sortilin is a major neuronal apolipoprotein E receptor for catabolism of amyloid-ß peptide in the brain.

Apolipoprotein E (APOE) is the major risk factor for sporadic Alzheimer's disease. Among other functions, APOE is proposed to sequester neurotoxic amyloid-ß (Aß) peptides in the brain, delivering them to cellular catabolism via neuronal APOE receptors. Still, the receptors involved in this process remain controversial. Here, we identified the pro-neurotrophin receptor sortilin as major endocytic pathway for clearance of APOE/Aß complexes in neurons. Sortilin binds APOE with high affinity. Lack of receptor expression in mice results in accumulation of APOE and of Aß in the brain and in aggravated plaque burden. Also, primary neurons lacking sortilin exhibit significantly impaired uptake of APOE/Aß complexes despite proper expression of other APOE receptors. Despite higher than normal brain APOE levels, sortilin-deficient animals display anomalies in brain lipid metabolism (e.g., accumulation of sulfatides) seen in APOE-deficient mice, indicating functional deficiency in cellular APOE uptake pathways. Together, our findings identified sortilin as an essential neuronal pathway for APOE-containing lipoproteins in vivo and suggest an intriguing link between Aß catabolism and pro-neurotrophin signaling converging on this receptor.

Production of glycosylated soluble amyloid precursor protein alpha (sAPPalpha) in Leishmania tarentolae.

Soluble amyloid precursor protein alpha (sAPPalpha) is a cleavage product of the amyloid precursor protein (APP), the etiologic agent in Alzheimer's disease (AD). Reduced expression of sAPPalpha was previously found in the brains of AD patients, and it was suggested that sAPPalpha might counteract neurotoxic effects of Abeta, another APP cleavage product with enhanced abundance in Alzheimer's diseased brains. However, little is known about the biological functions of sAPPalpha. Thus, efficient production of this protein is a prerequisite for further studies. The unicellular eukaryotic parasite Leishmania tarentolae has recently emerged as a promising expression system for eukaryotic proteins due to its ability to posttranslationally modify proteins combined with easy cultivation and high protein yield. Interestingly, sAPPalpha produced in L. tarentolae was biologically active and glycosylated. In contrast to nonglycosylated sAPPalpha expressed in Eschericha coli, it also featured higher stability against enzymatic degradation. Detailed analysis of the glycosylation pattern of sAPPalpha produced in L. tarentolae by PGC-LC-ESI-MS/MS N-glycan analysis identified among eukaryotic species the highly conserved core pentasaccharide (Man3GlcNAc2) as being attached to Asn467 of sAPPalpha. Using oxonium ion scanning of CID-MS/MS spectra in combination with ETD fragmentation, we also identified two peptides (peptides 269-288 and 575-587) modified with N-acetyl hexosamine (HexNAc) residues. One of these O-glycosylation sites could be unambiguously assigned to Thr576 of sAPPalpha. This is the first time that O-glycosylation of a recombinant protein expressed in L. tarentolae has been demonstrated. Together, human sAPPalpha produced in L. tarentolae was N- and O-glycosylated on similar sites as described for mammalian-expressed sAPPalpha and showed similar biological activity. This demonstrates that L. tarentolae is a very suitable and simple to handle expression system for mammalian glycoproteins.

Presymptomatic alterations in energy metabolism and oxidative stress in the APP23 mouse model of Alzheimer disease.

Glucose hypometabolism is the earliest symptom observed in the brains of Alzheimer disease (AD) patients. In a former study, we analyzed the cortical proteome of the APP23 mouse model of AD at presymptomatic age (1 month) using a 2-D electrophoresis-based approach. Interestingly, long before amyloidosis can be observed in APP23 mice, proteins associated with energy metabolism were predominantly altered in transgenic as compared to wild-type mice indicating presymptomatic changes in energy metabolism. In the study presented here, we analyzed whether the observed changes were associated with oxidative stress and confirmed our previous findings in primary cortical neurons, which exhibited altered ADP/ATP levels if transgenic APP was expressed. Reactive oxygen species produced during energy metabolism have important roles in cell signaling and homeostasis as they modify proteins. We observed an overall up-regulation of protein oxidation status as shown by increased protein carbonylation in the cortex of presymptomatic APP23 mice. Interestingly, many carbonylated proteins, such as Vilip1 and Syntaxin were associated to synaptic plasticity. This demonstrates an important link between energy metabolism and synaptic function, which is altered in AD. In summary, we demonstrate that changes in cortical energy metabolism and increased protein oxidation precede the amyloidogenic phenotype in a mouse model for AD. These changes might contribute to synaptic failure observed in later disease stages, as synaptic transmission is particularly dependent on energy metabolism.

Kainate promotes alterations in neuronal RNA splicing machinery.

Kainate, a glutamate analogue, activates kainate and AMPA receptors inducing strong synaptic activation. Systemic kainate application to rodents results in seizures, neurodegeneration, and neuronal remodeling in the brain. It is therefore used to investigate molecular mechanisms responsible for these conditions. We analyzed proteome alterations in murine primary cortical neurons after 24 h of kainate treatment. Our 2-D gel based proteomics approach revealed 91 protein alterations, some already associated with kainate-induced pathology. In addition, we found a large number of proteins which have not previously been reported to be associated with kainate-induced pathology. Functional classification of altered proteins revealed that they predominantly participate in mRNA splicing and cytoskeleton remodeling.

SORLA/SORL1, a neuronal sorting receptor implicated in Alzheimer's disease.

The proteolytic breakdown of the amyloid precursor protein (APP) to neurotoxic amyloid-beta peptides in the brain has been recognized as a major pathological pathway in Alzheimer's disease (AD). Yet, the factors that control the processing of APP and their potential contribution to the common sporadic form of AD remain poorly understood. Here, we review recent findings from studies in patients and in animal models that led to the identification of a unique sorting receptor for APP in neurons, designated SORLA/SORL1, that emerges as a key player in amyloidogenic processing and as major genetic risk factor for AD.

Brain-derived neurotrophic factor reduces amyloidogenic processing through control of SORLA gene expression.

Sorting protein-related receptor with A-type repeats (SORLA) is a major risk factor in cellular processes leading to Alzheimer's disease (AD). It acts as sorting receptor for the amyloid precursor protein (APP) that regulates intracellular trafficking and processing into amyloidogenic-beta peptides (A beta). Overexpression of SORLA in neurons reduces while inactivation of gene expression (as in knock-out mouse models) accelerates amyloidogenic processing and senile plaque formation. The current study aimed at identifying molecular pathways that control SORLA gene transcription in vivo and that may contribute to low levels of receptor expression in the brain of patients with AD. Using screening approaches in primary neurons, we identified brain-derived neurotrophic factor (BDNF) as a major inducer of Sorla that activates receptor gene transcription through the ERK (extracellular regulated kinase) pathway. In line with a physiological role as regulator of Sorla, expression of the receptor is significantly impaired in mouse models with genetic (Bdnf(-/-)) or disease-related loss of BDNF activity in the brain (Huntington's disease). Intriguingly, exogenous application of BDNF reduced A beta production in primary neurons and in the brain of wild-type mice in vivo, but not in animals genetically deficient for Sorla. These findings demonstrate that the beneficial effects ascribed to BDNF in APP metabolism act through induction of Sorla that encodes a negative regulator of neuronal APP processing.

A large number of protein expression changes occur early in life and precede phenotype onset in a mouse model for huntington disease.

Huntington disease (HD) is fatal in humans within 15-20 years of symptomatic disease. Although late stage HD has been studied extensively, protein expression changes that occur at the early stages of disease and during disease progression have not been reported. In this study, we used a large two-dimensional gel/mass spectrometry-based proteomics approach to investigate HD-induced protein expression alterations and their kinetics at very early stages and during the course of disease. The murine HD model R6/2 was investigated at 2, 4, 6, 8, and 12 weeks of age, corresponding to absence of disease and early, intermediate, and late stage HD. Unexpectedly the most HD stage-specific protein changes (71-100%) as well as a drastic alteration (almost 6% of the proteome) in protein expression occurred already as early as 2 weeks of age. Early changes included mainly the up-regulation of proteins involved in glycolysis/gluconeogenesis and the down-regulation of the actin cytoskeleton. This suggests a period of highly variable protein expression that precedes the onset of HD phenotypes. Although an up-regulation of glycolysis/gluconeogenesis-related protein alterations remained dominant during HD progression, late stage alterations at 12 weeks showed an up-regulation of proteins involved in proteasomal function. The early changes in HD coincide with a peak in protein alteration during normal mouse development at 2 weeks of age that may be responsible for these massive changes. Protein and mRNA data sets showed a large overlap on the level of affected pathways but not single proteins/mRNAs. Our observations suggest that HD is characterized by a highly dynamic disease pathology not represented by linear protein concentration alterations over the course of disease.

Impairment of adolescent hippocampal plasticity in a mouse model for Alzheimer's disease precedes disease phenotype.

The amyloid precursor protein (APP) was assumed to be an important neuron-morphoregulatory protein and plays a central role in Alzheimer's disease (AD) pathology. In the study presented here, we analyzed the APP-transgenic mouse model APP23 using 2-dimensional gel electrophoresis technology in combination with DIGE and mass spectrometry. We investigated cortex and hippocampus of transgenic and wildtype mice at 1, 2, 7 and 15 months of age. Furthermore, cortices of 16 days old embryos were analyzed. When comparing the protein patterns of APP23 with wildtype mice, we detected a relatively large number of altered protein spots at all age stages and brain regions examined which largely preceded the occurrence of amyloid plaques. Interestingly, in hippocampus of adolescent, two-month old mice, a considerable peak in the number of protein changes was observed. Moreover, when protein patterns were compared longitudinally between age stages, we found that a large number of proteins were altered in wildtype mice. Those alterations were largely absent in hippocampus of APP23 mice at two months of age although not in other stages compared. Apparently, the large difference in the hippocampal protein patterns between two-month old APP23 and wildtype mice was caused by the absence of distinct developmental changes in the hippocampal proteome of APP23 mice. In summary, the absence of developmental proteome alterations as well as a down-regulation of proteins related to plasticity suggest the disturption of a normally occurring peak of hippocampal plasticity during adolescence in APP23 mice. Our findings are in line with the observation that AD is preceded by a clinically silent period of several years to decades. We also demonstrate that it is of utmost importance to analyze different brain regions and different age stages to obtain information about disease-causing mechanisms.

Sortilin-related receptor with A-type repeats (SORLA) affects the amyloid precursor protein-dependent stimulation of ERK signaling and adult neurogenesis.

Sortilin-related receptor with A-type repeats (SORLA) is a sorting receptor that impairs processing of amyloid precursor protein (APP) to soluble (s) APP and to the amyloid beta-peptide in cultured neurons and is poorly expressed in patients with Alzheimer disease (AD). Here, we evaluated the consequences of Sorla gene defects on brain anatomy and function using mouse models of receptor deficiency. In line with a protective role for SORLA in APP metabolism, lack of the receptor results in increased amyloidogenic processing of endogenous APP and in aggravated plaque deposition when introduced into PDAPP mice expressing mutant human APP. Surprisingly, increased levels of sAPP caused by receptor deficiency correlate with pro-found stimulation of neuronal ERK signaling and with enhanced neurogenesis, providing in vivo support for neurotrophic functions of sAPP. Our data document a role for SORLA not only in control of plaque burden but also in APP-dependent neuronal signaling and suggest a molecular explanation for increased neurogenesis observed in some AD patients.

SorLA/LR11 regulates processing of amyloid precursor protein via interaction with adaptors GGA and PACS-1.

SorLA has been recognized as a novel sorting receptor that regulates trafficking and processing of the amyloid precursor protein (APP) and that represents a significant risk factor for sporadic Alzheimer disease. Here, we investigated the cellular mechanisms that control intracellular trafficking of sorLA and their relevance for APP processing. We demonstrate that sorLA acts as a retention factor for APP in trans-Golgi compartments/trans-Golgi network, preventing release of the precursor into regular processing pathways. Proper localization and activity of sorLA are dependent on functional interaction with GGA and PACS-1, adaptor proteins involved in protein transport to and from the trans-Golgi network. Aberrant targeting of sorLA to the recycling compartment or the plasma membrane causes faulty APP trafficking and imbalance in non-amyloidogenic and amyloidogenic processing fates. Thus, our findings identified altered routing of sorLA as a major cellular mechanism contributing to abnormal APP processing and enhanced amyloid beta-peptide formation.