The recent demonstration of K+ channel dysfunction in fibroblasts from Alzheimer disease (AD) patients and past observations of Ca2+-mediated K+ channel modulation during memory storage suggested that AD, which is characterized by memory loss and other cognitive deficits, might also involve dysfunction of intracellular Ca2+ mobilization. Bombesin-induced Ca2+ release, which is inositol trisphosphate-mediated, is shown here to be greatly enhanced in AD fibroblasts compared with fibroblasts from control groups. Bradykinin, another activator of phospholipase C, elicits similar enhancement of Ca2+ signaling in AD fibroblasts. By contrast, thapsigargin, an agent that releases Ca2+ by direct action on the endoplasmic reticulum, produced no differences in Ca2+ increase between AD and control fibroblasts. Depolarization-induced Ca2+ influx data previously demonstrated the absence of between-group differences of Ca2+ pumping and/or buffering. There was no correlation between the number of passages in tissue culture and the observed Ca2+ responses. Furthermore, cells of all groups were seeded and analyzed at the same densities. Radioligand binding experiments indicated that the number and affinity of bombesin receptors cannot explain the observed differences. These and previous observations suggest that the differences in bombesin and bradykinin responses in fibroblasts and perhaps other cell types are likely to be due to alteration of inositol trisphosphatemediated release of intracellular Ca2+.A number of cellular changes have been observed in fibroblasts from patients with Alzheimer disease (AD). These include abnormality of glucose and energy-related metabolism (1), defective release of a cholinergic factor (2), abnormal f8-amyloid expression and processing (3), changes in Ca2+ metabolism (30-34), and altered p-adrenergic-induced cAMP formation (4). The recent demonstration of K+ channel dysfunction in AD fibroblasts (5, 6) and past observations of Ca2+-mediated K+ channel modulation during memory storage (7) suggested that AD, which is characterized by memory loss and other cognitive deficits (8, 9), might also involve dysfunction of intracellular Ca2+ mobilization. Bombesin (10-12), an agent that activates phospholipase C (PLC) to generate inositol 1,4,5-trisphosphate (1P3) (13)(14)(15) different for AD and control fibroblasts. f-Amyloid protein (23-25) itself, while causing the previously observed inactivation of K+ channels in AD fibroblasts, had no effect on the bombesin-elicited Ca2+ signals. These and other findings, together with measurements of bombesin receptor number, suggest that PLC/G-protein coupling and/or IP3 receptors are responsible for differences in Ca2+ responses between AD and non-AD fibroblasts. METHODSCell Lines. Human skin fibroblasts (Table 1) were purchased from the Coriell Cell Repositories (Camden, NJ). Cells were seeded and maintained as described (5). The number of passages was not significantly different between groups [AD, 10.9 ± 1.3 (mean ± SEM), n = 10; AC, 11.5 + 0.8, n = 8;...

Activation of protein kinase C (PKC) can mimic the biophysical effects of associative learning on neurons. Furthermore, classical conditioning of the rabbit nictitating membrane (a form of associative learning) produces translocation of PKC activity from the cytosolic to the membrane compartments of the CA1 region of the hippocampus. Evidence is provided here for a significant change in the amount and distribution of PKC within the CA1 cell field of the rabbit hippocampus that is specific to learning. This change is seen at 1 day after learning as focal increments of [3H]phorbol-12,13-dibutyrate binding to PKC in computer-generated images produced from coronal autoradiographs of rabbit brain. In addition, 3 days after learning, the autoradiographs suggest a redistribution of PKC within CA1 from the cell soma to the dendrites.

Postmortem, genetic, brain imaging, and peripheral cell studies all support decreased mitochondrial activity as a factor in the manifestation of Bipolar Disorder (BD). Because abnormal mitochondrial morphology is often linked to altered energy metabolism, we investigated whether changes in mitochondrial structure were present in brain and peripheral cells of patients with BD. Mitochondria from patients with BD exhibited size and distributional abnormalities compared with psychiatrically-healthy age-matched controls. Specifically, in brain, individual mitochondria profiles had significantly smaller areas, on average, in BD samples (P = 0.03). In peripheral cells, mitochondria in BD samples were concentrated proportionately more within the perinuclear region than in distal processes (P = 0.0008). These mitochondrial changes did not appear to be correlated with exposure to lithium. Also, these abnormalities in brain and peripheral cells were independent of substantial changes in the actin or tubulin cytoskeleton with which mitochondria interact. The observed changes in mitochondrial size and distribution may be linked to energy deficits and, therefore, may have consequences for cell plasticity, resilience, and survival in patients with BD, especially in brain, which has a high-energy requirement. The findings may have implications for diagnosis, if they are specific to BD, and for treatment, if they provide clues as to the underlying pathophysiology of BD.

Bipolar disorder (BD) is associated with abnormal circadian rhythms. In treatment responsive BD patients, lithium (Li) stabilizes mood and reduces suicide risk. Li also affects circadian rhythms and expression of ‘clock genes' that control them. However, the extent to which BD, Li and the circadian clock share common biological mechanisms is unknown, and there have been few direct measurements of clock gene function in samples from BD patients. Hence, the role of clock genes in BD and Li treatment remains unclear. Skin fibroblasts from BD patients (N=19) or healthy controls (N=19) were transduced with Per2::luc, a rhythmically expressed, bioluminescent circadian clock reporter gene, and rhythms were measured for 5 consecutive days. Rhythm amplitude and period were compared between BD cases and controls with and without Li. Baseline period was longer in BD cases than in controls. Li 1 mM increased amplitude in controls by 36%, but failed to do so in BD cases. Li 10 mM lengthened period in both BD cases and controls. Analysis of clock gene variants revealed that PER3 and RORA genotype predicted period lengthening by Li, whereas GSK3β genotype predicted rhythm effects of Li, specifically among BD cases. Analysis of BD cases by clinical history revealed that cells from past suicide attempters were more likely to show period lengthening with Li 1 mM. Finally, Li enhanced the resynchronization of damped rhythms, suggesting a mechanism by which Li could act therapeutically in BD. Our work suggests that the circadian clock's response to Li may be relevant to molecular pathology of BD.

The molecular basis for the degeneration of neurons and the deposition of amyloid in plaques and in the cerebrovasculature in Alzheimer's disease (AD) is incompletely understood. We have proposed that one molecule common to these abnormal processes is a fragment of the Alzheimer amyloid precursor protein (APP) comprising the C-terminal 100 amino acids of this molecule (APP-C100). We tested this hypothesis by creating transgenic mice expressing APP-C100 in the brain. We report here that aging (18-28 month) APP-C100 transgenic mice exhibit profound degeneration of neurons and synapses in Ammon's horn and the dentate gyrus of the hippocampal formation. Of the 106 transgenic mice between 8 and 28 months of age that were examined, all of those older than 18 months displayed severe hippocampal degeneration. The numerous degenerating axonal profiles contained increased numbers of neurofilaments, whorls of membrane, and accumulations of debris resembling secondary lysosomes near the cell body. The dendrites of degenerating granule and pyramidal cells contained disorganized, wavy microtubules. Cerebral blood vessels had thickened refractile basal laminae, and microglia laden with debris lay adjacent to larger venous vessels. Mice transgenic for Flag-APP-C100 (in which the hydrophilic Flag tag was fused to the N terminus of APP-C100) showed a similar degree of neurodegeneration in the hippocampal formation as early as 12 months of age. The 45 control mice displayed only occasional necrotic cells and no extensive cell degeneration in the same brain regions. These findings show that APP-C100 is capable of causing some of the neuropathological features of AD.

Body-wide changes in bioenergetics, i.e., energy metabolism, occur in normal aging and disturbed bioenergetics may be an important contributing mechanism underlying late-onset Alzheimer's disease (LOAD). We investigated the bioenergetic profiles of fibroblasts from LOAD patients and healthy controls, as a function of age and disease. LOAD cells exhibited an impaired mitochondrial metabolic potential and an abnormal redox potential, associated with reduced nicotinamide adenine dinucleotide metabolism and altered citric acid cycle activity, but not with disease-specific changes in mitochondrial mass, production of reactive oxygen species, transmembrane instability, or DNA deletions. LOAD fibroblasts demonstrated a shift in energy production to glycolysis, despite an inability to increase glucose uptake in response to IGF-1. The increase of glycolysis and the abnormal mitochondrial metabolic potential in LOAD appeared to be inherent, as they were disease-and not age-specific. Our findings support the hypothesis that impairment in multiple interacting components of bioenergetic metabolism may be a key mechanism contributing to the risk and pathophysiology of LOAD.Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by slow progressive deterioration and death of neurons. A number of interacting factors determine the risk of AD and among the better-studied pathophysiologic pathways, the "amyloid cascade hypothesis" proposes that AD is precipitated by an accumulation of Aβ-containing plaques and tangles of hyperphosphorylated tau (p-tau) 1-3 . This hypothesis is best supported for familial/early-onset forms of AD (EOAD), while less so for the more common sporadic/ late-onset forms (LOAD) 4,5 . In LOAD, accumulation of toxic Aβ and p-tau may not be the initial cause of neural degeneration and may instead be consequences of other causative factors 5,6 . Changes in bioenergetics, i.e., energy metabolism, are part of the normal aging process and disturbed bioenergetics may be a contributing mechanism underlying LOAD 7-10 . These anomalies are body-wide, but affect the brain most substantially because of its exceptionally high-energy requirements. Thus, changes of bioenergetics and metabolism could be at the core of determining the survival capacities of brain cells with age and under stress, with these processes influenced, in turn, by genetic predisposition, epigenetics, environment, and lifestyle.Bioenergetics is the metabolism of various fuel molecules to produce and utilize energy through glycolysis, mitochondrial respiration, that is, oxidative phosphorylation (OxPhos), or the pentose phosphate pathway (PPP). Healthy eukaryotic cells produce ATP about 12% through glycolysis and 88% through OxPhos, on average. In the 1920s, German physician-chemist Otto Warburg discovered that mammalian cancer cells can switch from OxPhos to glycolysis when exposed to low oxygen, called the "Warburg effect" 11 . Unlike proliferating cells, post-mitotic neurons have very little ability to use glycolysis, a...

Psychiatric disorders have clear heritable risk. Several large-scale genome-wide association studies have revealed a strong association between susceptibility for psychiatric disorders, including bipolar disease, schizophrenia, and major depression, and a haplotype located in an intronic region of the L-type voltage gated calcium channel (VGCC) subunit gene CACNA1C (peak associated SNP rs1006737), making it one of the most replicable and consistent associations in psychiatric genetics. In the current study, we used induced human neurons to reveal a functional phenotype associated with this psychiatric risk variant. We generated induced human neurons, or iN cells, from more than 20 individuals harboring homozygous risk genotypes, heterozygous, or homozygous non-risk genotypes at the rs1006737 locus. Using these iNs, we performed electrophysiology and quantitative PCR experiments that demonstrated increased L-type VGCC current density as well as increased mRNA expression of CACNA1C in induced neurons homozygous for the risk genotype, compared to non-risk genotypes. These studies demonstrate that the risk genotype at rs1006737 is associated with significant functional alterations in human induced neurons, and may direct future efforts at developing novel therapeutics for the treatment of psychiatric disease.

APP-BP1 binds to the amyloid precursor protein (APP) carboxyl-terminal domain. Recent work suggests that APP-BP1 participates in a novel ubiquitinylationrelated pathway involving the ubiquitin-like molecule NEDD8. We show here that, in vivo in mammalian cells, APP-BP1 interacts with hUba3, its presumptive partner in the NEDD8 activation pathway, and that the APP-BP1 binding site for hUba3 is within amino acids 443-479. We also provide evidence that the human APP-BP1 molecule can rescue the ts41 mutation in Chinese hamster cells. This mutation previously has been shown to lead to successive S phases of the cell cycle without intervening G 2 , M, and G 1 , suggesting that the product of this gene negatively regulates entry into the S phase and positively regulates entry into mitosis. We show that expression of APP-BP1 in ts41 cells drives the cell cycle through the S-M checkpoint and that this function requires both hUba3 and hUbc12. Overexpression of APP-BP1 in primary neurons causes apoptosis via the same pathway. A specific caspase-6 inhibitor blocks this apoptosis. These findings are discussed in the context of abnormalities in the cell cycle that have been observed in Alzheimer's disease.Amyloid precursor protein (APP), 1 a transmembrane protein, is the source of the ␤-amyloid peptides that accumulate in the brains of patients with Alzheimer's disease (AD). The possibility that APP may act as a signaling receptor was first proposed on the basis of its predicted amino acid sequence, which suggested that APP was a type 1 intrinsic membrane protein consistent with the structure of a cell surface receptor (1). It has now been demonstrated that a percentage of APP is found on the cell surface in neurons (2-4). Cell-surface APP possesses a neurite-promoting activity that is distinct from that of the secreted APP (5), co-localizes with adhesion plaque components (3, 6), and participates in synaptic vesicle recycling (7), suggesting that a percentage of APP may function as a cell surface receptor, transducing signals from the extracellular matrix to the interior of the cell.APP-BP1 was identified by its interaction with the intracellular carboxyl terminus of APP (8), which places this molecule in a position potentially to participate in the transduction of signals from the cell surface into the cell. APP-BP1 initially was found to be homologous to the Arabidopsis auxin resistance gene AXR1, and to the amino terminus of the ubiquitin activating enzyme E1. It was puzzling that APP-BP1 lacked a conserved cysteine required for E1 ubiquitin conjugation activity. However, it was subsequently discovered that eukaryotes express a set of ubiquitin-like proteins that, like ubiquitin, are ligated to other proteins (9, 10). In yeast, one of these ubiquitinlike proteins, Rub1 (related to ubiquitin 1), is activated by a heterodimer consisting of the subunits Ula1 and Uba3. Ula1 and Uba3 are related to the NH 2 -and COOH-terminal domains of the E1 ubiquitin-activating enzyme, respectively, and together fulfill E1-like functions for...