Using the Braak staging for neurofibrillary changes as an objective indicator of the progression of Alzheimer's disease, we have performed a systematic search for global gene expression changes in the prefrontal cortex during the course of Alzheimer's disease. In the prefrontal cortex, senile plaques and neurofibrillary changes start to appear around Braak stage III, allowing for the detection of changes in gene expression before, during and after the onset of Alzheimer's disease neuropathology. Two distinct patterns of tightly co-regulated groups of genes were observed: (i) an increase in expression in early Braak stages, followed by a decline in expression in later stages (the UPDOWN clusters; containing 865 genes) and (ii) a decrease in expression in early Braak stages, followed by an increase in expression in later stages (the DOWNUP clusters; containing 983 genes). The most profound changes in gene expression were detected between Braak stages II and III, just before or at the onset of plaque pathology and neurofibrillary changes in the prefrontal cortex. We also observed an increase in intracellular beta amyloid staining from Braak stages I to III and a clear decrease in Braak stages IV to VI. These data suggest a link between specific gene expression clusters and Alzheimer's disease-associated neuropathology in the prefrontal cortex. Gene ontology over-representation and functional gene network analyses indicate an increase in synaptic activity and changes in plasticity during the very early pre-symptomatic stage of the disease. In later Braak stages, the decreased expression of these genes suggests a reduction in synaptic activity that coincides with the appearance of plaque pathology and neurofibrillary changes and the clinical diagnosis of mild cognitive impairment. The interaction of the ApoE genotype with the expression levels of the genes in the UPDOWN and DOWNUP clusters demonstrates that the accelerating role of ApoE-ε4 in the progression of Alzheimer's disease is reflected in the temporal changes in gene expression presented here. Since the UPDOWN cluster contains several genes involved in amyloid precursor protein processing and beta amyloid clearance that increase in expression in parallel with increased intracellular beta amyloid load, just before the onset of plaque pathology in the prefrontal cortex, we hypothesize that the temporally orchestrated increase in genes involved in synaptic activity represents a coping mechanism against increased soluble beta amyloid levels. As these gene expression changes occur before the appearance of Alzheimer's disease-associated neuropathology, they provide an excellent starting point for the identification of new targets for the development of therapeutic strategies aimed at the prevention of Alzheimer's disease.

A series of novel 3,4-diarylpyrazolines was synthesized and evaluated in cannabinoid (hCB(1) and hCB(2)) receptor assays. The 3,4-diarylpyrazolines elicited potent in vitro CB(1) antagonistic activities and in general exhibited high CB(1) vs CB(2) receptor subtype selectivities. Some key representatives showed potent pharmacological in vivo activities after oral dosing in both a CB agonist-induced blood pressure model and a CB agonist-induced hypothermia model. Chiral separation of racemic 67, followed by crystallization and an X-ray diffraction study, elucidated the absolute configuration of the eutomer 80 (SLV319) at its C(4) position as 4S. Bioanalytical studies revealed a high CNS-plasma ratio for the development candidate 80. Molecular modeling studies showed a relatively close three-dimensional structural overlap between 80 and the known CB(1) receptor antagonist rimonabant (SR141716A). Further analysis of the X-ray diffraction data of 80 revealed the presence of an intramolecular hydrogen bond that was confirmed by computational methods. Computational models and X-ray diffraction data indicated a different intramolecular hydrogen bonding pattern in the in vivo inactive compound 6. In addition, X-ray diffraction studies of 6 revealed a tighter intermolecular packing than 80, which also may contribute to its poorer absorption in vivo. Replacement of the amidine -NH(2) moiety with a -NHCH(3) group proved to be the key change for gaining oral biovailability in this series of compounds leading to the identification of 80.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra. We have studied alterations in gene expression in the substantia nigra, the caudate nucleus and putamen of four PD patients and four matched controls using custom designed Agilent microarrays. To gain insight into changes in gene expression during early stages of dopaminergic neurodegeneration, we selectively investigated the relatively spared parts of the PD substantia nigra, and correlated gene expression changes with alterations in neuronal density. We identified changes in the expression of 287 transcripts in the substantia nigra, 16 transcripts in the caudate nucleus and four transcripts in the putamen. For selected transcripts, transcriptional alterations were confirmed with qPCR on a larger set of seven PD cases and seven matched controls. We detected concerted changes in functionally connected groups of genes. In the PD substantia nigra, we observed strong evidence for a reduction in neurotrophic support and alterations in axon guidance cues. As the changes occur in relatively spared parts of the PD substantia nigra, they suggest novel disease mechanisms involving neurotrophic support and axon guidance in early stages of cellular stress events, ultimately leading to dopaminergic cell death in PD.

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The endocannabinoid (eCB) system is widely expressed throughout the central nervous system (CNS) and the functionality of type-1 cannabinoid receptors in neurons is well documented. In contrast, there is little knowledge about type-2 cannabinoid receptors (CB 2 Rs) in the CNS. Here, we show that CB 2 Rs are located intracellularly in layer II/III pyramidal cells of the rodent medial prefrontal cortex (mPFC) and that their activation results in IP 3 Rdependent opening of Ca 2+ -activated Cl − channels. To investigate the functional role of CB 2 R activation, we induced neuronal firing and observed a CB 2 R-mediated reduction in firing frequency. The description of this unique CB 2 R-mediated signaling pathway, controlling neuronal excitability, broadens our knowledge of the influence of the eCB system on brain function.calcium-activated chloride current | firing rate | whole-cell current | voltage clamp | intracellular calcium stores T he endocannabinoid (eCB) system is involved in many functions of the CNS, including executive functions associated with the prefrontal cortex, such as decision-making and working memory (1). The eCB system consists of at least two G proteincoupled receptors (GPCRs), type-1 cannabinoid receptor (CB 1 R) and type-2 cannabinoid receptor (CB 2 R), lipid endogenous ligands (e.g., anandamide and 2-arachidonoylglycerol), and various enzymes responsible for the synthesis and degradation of the endogenous ligands (2-6). CB 1 Rs are among the most abundantly expressed GPCRs in the rat brain and their role, predominantly as presynaptic receptors, in modulating neurotransmission is clearly established (5,7,8). In contrast with CB 1 R, the presence and function of CB 2 R in the brain has long been a matter of debate (9). CB 2 Rs are found primarily in the immune system and were initially regarded as the "peripheral" cannabinoid receptor (10,11). This generally accepted idea is challenged by the description of CNS CB 2 R gene expression in rats and wild-type mice (12-14) and the identification of functional CB 2 Rs on glial cells and neurons (15)(16)(17)(18). In addition to the current view that supports the expression of functional CB 2 Rs in neurons upon brain stress or damage (19), it has been reported that CB 2 Rs could play a role in general CNS physiology (20)(21)(22). These developments emphasize the importance of understanding how CB 2 R activation affects neuronal functioning. To demonstrate the presence of functional CB 2 Rs in the rodent medial prefrontal cortex (mPFC) and to elucidate their functional role, we used Western blotting, a radioactive binding assay, and electrophysiological techniques (whole-cell current and voltage clamp) on layer II/III pyramidal neurons. ResultsFunctional CB 2 Rs in the mPFC. The presence of CB 2 Rs in the rat mPFC was demonstrated by a Western blot performed on homogenated mPFC samples (Fig. 1A). A band of the expected molecular weight for CB 2 R was detected, which was absent when the primary antibody was incubated with immunizing peptide (Fig. 1A)....