Longitudinal axons transmit all signals between the brain and spinal cord. Their axon tracts through the brain stem are established by a simple set of pioneer axons with precise trajectories parallel to the floor plate. To identify longitudinal guidance mechanisms in vivo, the overall role of floor plate tissue and the specific roles of Slit/Robo signals were tested. Ectopic induction or genetic deletion of the floor plate diverted longitudinal axons into abnormal trajectories. The expression patterns of the diffusible cues of the Slit family were altered in the floor plate experiments, suggesting their involvement in longitudinal guidance. Genetic tests of Slit1 and Slit2, and the Slit receptors Robo1 and Robo2 were carried out in mutant mice. Slit1;Slit2 double mutants had severe longitudinal errors, particularly for ventral axons, including midline crossing and wandering longitudinal trajectories. Robo1 and Robo2 were largely genetically redundant, and neither appeared to specify specific tract positions. However, combined Robo1 and Robo2 mutations strongly disrupted each pioneer tract. Thus, pioneer axons depend on long-range floor plate cues, with Slit/Robo signaling required for precise longitudinal trajectories.

The enzyme β-secretase (BACE1) is essentially involved in the production of cerebral amyloidogenic pathology in Alzheimer's disease (AD). The measurement of BACE1 activity in cerebrospinal fluid (CSF) has been reported, which may render CSF measurement of BACE1 a potential biomarker candidate of AD. In order to investigate whether BACE1 protein activity is correlated with regional brain atrophy in AD, we investigated the association between CSF levels of BACE1 and MRI-assessed hippocampus volume in patients with AD (n = 30). An increase in CSF-BACE1 activity was associated with decreased left and right hippocampus volume corrected for global head volume in the AD patients. Boot-strapped regression analysis showed that increased CSF levels of BACE1 activity were associated with increased CSF concentration of total tau but not amyloid-β1-42 in AD. White matter hyperintensities did not influence the results. BACE1 activity and protein levels were significantly increased in AD compared to 19 elderly healthy controls. Thus, the CSF biomarker candidate of BACE1 activity was associated with hippocampus atrophy in AD in a robust manner and may reflect neurotoxic amyloid-β-related processes.

We have characterized a system of early neurons that establish the first two major longitudinal tracts in the embryonic mouse forebrain. Axon tracers and antibody labels were used to map the axon projections in the thalamus from embryonic days 9.0-12, revealing several distinct neuron populations that contributed to the first tracts. Each of the early axon populations first grew independently, pioneering a short segment of new tract. However, each axon population soon merged with other axons to form one of only two shared longitudinal tracts, both descending: the tract of the postoptic commissure (TPOC), and, in parallel, the stria medullaris. Thus, the forebrain longitudinal tracts are pioneered by a relay of axons, with distinct axon populations pioneering successive segments of these pathways. The extensive merging of tracts suggests that axon-axon interactions are a major guidance mechanism for longitudinal axons. Several axon populations express tyrosine hydroxylase, identifying the TPOC as a major pathway for forebrain dopaminergic projections. To start a genetic analysis of pioneer axon guidance, we have identified the transcription factor Pax6 as critical for tract formation. In Pax6 mutants, both longitudinal tracts failed to form due to errors by every population of early longitudinal axons. Taken together, these results have identified potentially important interactions between series of pioneer axons and the Pax6 gene as a general regulator of longitudinal tract formation in the forebrain.

During early vertebrate forebrain development, pioneer axons establish a symmetrical scaffold descending longitudinally through the rostral forebrain, thus forming the tract of the postoptic commissure (TPOC). In mouse embryos, this tract begins to appear at embryonic day 9.5 (E9.5) as a bundle of axons tightly constrained at a specific dorsoventral level. We have characterized the participation of the Slit chemorepellants and their Robo receptors in the control of TPOC axon projection. In E9.5–E11.5 mouse embryos, Robo1 and Robo2 are expressed in the nucleus origin of the TPOC (nTPOC), and Slit expression domains flank the TPOC trajectory. These findings suggested that these proteins are important factors in the dorsoventral positioning of the TPOC axons. Consistently with this role, Slit2 inhibited TPOC axon growth in collagen gel cultures, and interfering with Robo function in cultured embryos induced projection errors in TPOC axons. Moreover, absence of both Slit1 and Slit2 or Robo1 and Robo2 in mutant mouse embryos revealed aberrant TPOC trajectories, resulting in abnormal spreading of the tract and misprojections into both ventral and dorsal tissues. These results reveal that Slit-Robo signaling regulates the dorsoventral position of this pioneer tract in the developing forebrain.

The aspartyl protease BACE1 is the rate limiting enzyme in the synthesis of amyloid beta, which accumulation in the human brain is a hallmark of Alzheimer’s disease (AD). BACE1 has been proposed as a surrogate marker of AD; however, very few BACE1 immunoassays have been reported. In the present study we have screened ten BACE1 antibodies by Western blot and several antibody pairs to develop a new BACE1 sandwich ELISA procedure. We identified one pair that showed little background and good reproducibility. Several dilution buffers and sample denaturation methods were tried to partially unfold BACE1 before capture. We found that dilution in PBS followed by 10 min incubation at 50 °C critically improves the performance of the assay. Finally, we successfully measured BACE1 levels in a few human brain and platelet lysates as well as in plasma and AD CSF. We anticipate that this assay will lay the ground to accurately measure BACE1 levels in human tissues, which could facilitate the molecular diagnosis of AD in the near future.

The PARK7 gene encodes a protein, DJ-1, with several functions such as protection of cells from oxidative stress, sperm maturation and fertilization, and chaperone activity. Mutations in the PARK7 gene are associated with autosomal recessive early-onset Parkinson's disease (PD). DJ-1 has been reported to be expressed in multiple cells in the central nerve system. Here, by using both native and denatured Western blots, we examined levels of total DJ-1 and high molecular weight complexes of DJ-1 (HMW) in both the substantia nigra and cortex from rapidly autopsied 18 PD and 9 non-pathological control (NPC) brains. We have discovered that the level of total DJ-1 protein is significantly reduced in the substantia nigra in brains of sporadic PD patients. Moreover, in the PD cortex mitochondria fraction, the HMW DJ-1 complex is significantly lower than in the NPC. These results suggest abnormal DJ-1 expression levels and DJ-1 complex changes may contribute to PD pathogenesis.

Robo1 is a member of the Roundabout (Robo) family of receptors for the Slit axon guidance cues. In mice (and humans), the Robo1 locus has alternative promoters producing two transcript isoforms, Robo1 and Dutt1. These isoforms have unique 5′ termini, predicted to encode distinct N-terminal amino acids, but share the rest of their 3′ exons. To determine the spatial expression of the Robo1 and Dutt1 isoforms, we generated isoform-specific RNA probes, and carried out in situ hybridization on E10.5 mouse embryos, the stage in early neuron differentiation when many major axon pathways are established. The two isoforms had distinct expression patterns that partially overlapped. Dutt1 was the predominant isoform, with widespread expression in regions of post-mitotic neurons and neuroepithelial cells. The Robo1 isoform had a distinct expression pattern restricted to subsets of neurons, many of which were Dutt1-negative. Dutt1 was the main isoform expressed in spinal cord commissural neurons. For both probes, the main hybridization signal was limited to two spots in the nuclei of individual cells. This study shows distinct expression patterns for the Dutt1 and Robo1 alternative promoters in the embryonic nervous system. Keywords axon guidance; neuron; Robo; Slit; longitudinal axon; commissural axon; dorsal root ganglion Results and DiscussionGrowth cones navigate by selectively responding to molecular cues in their environment. Different types of axons differ in their responses to cues, depending on their expression of specific combinations of guidance receptors. In contrast to the great diversity in neuron types and their axon projection patterns, a surprisingly small number of axon guidance receptors have been identified to date. However, single receptor genes could potentially give rise to multiple receptor isoforms through mechanisms such as alternative promoters and differential mRNA splicing.The Slit repellents and their Robo receptors are a major guidance system conserved from invertebrates to vertebrates (Dickson and Gilestro, 2006). The Roundabout (Robo) family of Corresponding author: Grant Mastick, Department of Biology, University of Nevada, Reno NV 89557, Phone: 775-784-6168, FAX: 775-784-1650, gmastick@unr.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. transmembrane receptors was first identified in Drosophila in a mutant screen for genes that control midline crossing by pioneer axons (Seeger et al., 1993). Slit/Robo signaling has a primary function in keeping axons out of the Slit+ midline, both in commissural axons to prevent re-crossing, as well as ...

A series of transition-state analogues of beta-secretases 1 and 2 (BACE1, 2) inhibitors containing fused-ring or biaryl moieties were designed computationally to probe the S2 pocket, synthesized, and tested for BACE1 and BACE2 inhibitory activity. It has been shown that unlike the biaryl analogs, the fused-ring moiety is successfully accommodated in the BACE1 binding site resulting in the ligands with excellent inhibitory activity. Ligand 5b reduced 65% of Aβ40 production in N2a cells stably transfected with Swedish human APP. KeywordsAlzheimer; BACE1; Aspartic protease; computer-aided molecular design Memory loss is the most common characteristic in the clinical manifestation of Alzheimer's disease (AD) which affects the learning of both recent as well as recalled stored information. 1 It has been hypothesized that the therapeutic targeting of Amyloid Precursor Protein (APP) processing may mitigate the formation of toxic fragments and prevents plaque formation. This large transmembrane protein can be cleaved at three distinct sites by proteolytic enzymes collectively referred to as "secretases". 2-5 The key enzyme crucial for the release of amyloidogenic fragments during APP processing is called β-secretase (BACE1). 2 BACE1 enzyme cleaves APP at the N-terminal side of the Aβ sequence to secrete sAPPβ, producing the cell-bound, carboxyl-terminal fragment C99 (also termed C100 or CTF). 3 The C99 fragment is then cleaved by the γ-secretase enzyme in the proteolytic cascade of APP processing. The resulting amyloid-beta peptides, called Aβ40 and Aβ42 because of the number of amino acids they are comprised of, form the extracellular neuritic amyloid plaques -one of the key factors in the pathogenesis of AD. 3It has been shown that BACE1 levels are significantly elevated in vivo in rapidly autopsied brains of sporadic AD patients (< 3 hours) compared with age-matched non-AD patients. 6-*Corresponding author. Tel: +1 312 996 4174; Fax: +1 312 916 7107; email: pap4@uic.edu. a equally contributed to this work b current address is Burnham Institute, San Diego, CA Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptBioorg Med Chem Lett. Author manuscript; available in PMC 2010 January 1. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript8 There has also been reports of a correlation (R 2 = 0.54) between BACE1 activity and amyloid plaques count. 9 It was, therefore, suggested that lowering amyloid plaques in vivo may be achieved by a decrease in production of Aβ40 and Aβ42 through the inhibition of BACE1, thus opening ...