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The enormous, 2-3-million-year evolutionary expansion of hominin neocortices to the current enormity enabled humans to take over the planet. However, there appears to have been a glitch, and it occurred without a compensatory expansion of the entorhinal cortical (EC) gateway to the hippocampal memory-encoding system needed to manage the processing of the increasing volume of neocortical data converging on it. The resulting age-dependent connectopathic glitch was unnoticed by the early short-lived populations. It has now surfaced as Alzheimer's disease (AD) in today's long-lived populations. With advancing age, processing of the converging neocortical data by the neurons of the relatively small lateral entorhinal cortex (LEC) inflicts persistent strain and high energy costs on these cells. This may result in their hyper-release of harmless Aβ 1-42 monomers into the interstitial fluid, where they seed the formation of toxic amyloid-β oligomers (AβOs) that initiate AD. At the core of connectopathic AD are the postsynaptic cellular prion protein (PrPC). Electrostatic binding of the negatively charged AβOs to the positively charged N-terminus of PrPC induces hyperphosphorylation of tau that destroys synapses. The spread of these accumulating AβOs from ground zero is supported by Aβ's own production mediated by target cells' Ca2+-sensing receptors (CaSRs). These data suggest that an early administration of a strongly positively charged, AβOs-interacting peptide or protein, plus an inhibitor of CaSR, might be an effective AD-arresting therapeutic combination.

Understanding the pathophysiology of Alzheimer's disease (AD) in the principal human neural cells is necessary for finding therapeutics for this illness. To help do this, we have been using freshly cultured functionally normal cerebral cortical adult human astrocytes (NAHAs) and postnatal neurons. The findings show that amyloid-β oligomers (Aβ-os) binding to calcium-sensing receptors (CaSRs) on NAHAs and neuron surfaces trigger signals capable of driving AD pathogenesis. This Aβ•CaSR signalling shifts the amyloid precursor protein (APP) from its α-secretase shedding producing neurotrophic/neuroprotective soluble (s)APPα to its β-secretase cleaving engendering AD-driving Aβ42/Aβ42-os peptides. Aβ•CaSR signalling in NAHAs also drives the release of toxic hyper-phosphorylated Tau proteins in exosomes, and of nitric oxide, and VEGF-A. These several harmful agents comprise the neuron-killing machinery, driving the very slowly spreading AD neurocontagion. VEGF-A over-secretion from Aβ-exposed blood vessel-attached astrocytes induces a functional magnetic resonance imaging- detectable hippocampal neoangiogenesis which indicates approaching AD in amnestic minor cognitive impairment (aMCI) patients. Most important in AD's regard, selective allosteric CaSR antagonists (calcylitics) added to Aβ42/Aβ42-os-exposed NAHAs (or to human neuron cultures) rescue the extracellular shedding of neurotrophic/ neuroprotective sAPPα and suppress all the neurotoxic effects of Aβ•CaSR signalling even when multiple microglial cytokines are also present. Therefore, since the multipotent calcilytics would be reasonably safe and inexpensive drugs for humans, it is worthwhile testing them as AD therapeutics in clinical trials especially in persons in the earliest detectable stages of AD neuropathology progression such as aMCI., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Dentate-gyral granule cells in the hippocampus plus dentate gyrus memory-recording/retrieving machine, unlike most other neurons in the brain, are continuously being generated in the adult brain with the important task of separating overlapping patterns of data streaming in from the outside world via the entorhinal cortex. This "adult neurogenesis" is driven by tools in the mature granule cell's cilium. Here we report our discovery of leptin's LepRb receptor in this cilium. In addition, we discuss how ciliary LepRb signaling might be involved with ciliary p75NTR and SSTR3 receptors in adult neurogenesis and memory formation as well as attenuation of Alzheimer's neuropathology by reducing the production of its toxic amyloid-β-derived drivers.

Evidence has begun emerging for the "contagious" and destructive Aβ42 (amyloid-beta42) oligomers and phosphorylated Tau oligomers as drivers of sporadic Alzheimer's disease (AD), which advances along a pathway starting from the brainstem or entorhinal cortex and leading to cognition-related upper cerebral cortex regions. Seemingly, Aβ42 oligomers trigger the events generating the neurotoxic Tau oligomers, which may even by themselves spread the characteristic AD neuropathology. It has been assumed that only neurons make and spread these toxic drivers, whereas their associated astrocytes are just janitorial bystanders/scavengers. But this view is likely to radically change since normal human astrocytes freshly isolated from adult cerebral cortex can be induced by exogenous Aβ25-35, an Aβ42 proxy, to make and secrete increased amounts of endogenous Aβ42. Thus, it would seem that the steady slow progression of AD neuropathology along specific cognition-relevant brain networks is driven by both Aβ42 and phosphorylated Tau oligomers that are variously released from increasing numbers of "contagion-stricken" members of tightly coupled neuron-astrocyte teams. Hence, we surmise that stopping the oversecretion and spread of the two kinds of "contagious" oligomers by such team members, perhaps via a specific CaSR (Ca(2+)-sensing receptor) antagonist like NPS 2143, might effectively treat AD., (© The Author(s) 2014.)

The "amyloid cascade hypothesis" posits that an extracellular build-up of amyloid-β oligomers (Aβ-os) and polymers (fibrils) subsequently inducing toxic hyperphosphorylated (p)-Tau oligomers (p-Tau-os) and neurofibrillary tangles starts the sporadic late-onset Alzheimer's disease (LOAD) in the aged lateral entorhinal cortex. Conversely, mutated genes cause a diffuse cerebral Aβs/Aβ-os overproduction promoting early-onset familiar AD (EOFAD). Surplus exogenous Aβ-os exert toxic actions at several levels. They reach the nuclei of human astrocyte-neurons teams (ANTs) to enhance the transcription of Aβ precursor protein (APP) and β-secretase/BACE1 genes. The overexpressed APP and BACE1 proteins act in concert with γ-secretase to overproduce endogenous Aβs/Aβ-os, of which a few enter the nuclei to upkeep Aβs overproduction, while the rest gather in the cytoplasm, damage mitochondria, and are oversecreted. Simultaneously, extracellular Aβ-os bind the ANTs' calcium-sensing receptors (CaSRs) activating signalings that hinder the proteolysis and hence favor the surplus hoarding/secretion of Aβs/Aβ-os. Overreleased Aβ-os spread, reach growing numbers of adjacent ANTs to recruit them to overproduce/oversecrete further Aβ-os amounts via the just mentioned mechanisms. Alongside, Aβ•CaSR signalings elicit a noxious overproduction/overrelease of nitric oxide (NO) and vascular endothelial growth factor (VEGF)-A from ANTs' astrocytes. While astrocytes survive the toxic onslaught, neurons die. Thus, AD progression is driven by ceaselessly self-sustaining neurotoxic cycles, which engender first Aβ-os and later p-Tau-os that cooperatively destroy increasingly wider cognition-related cortical areas. Notably, a highly selective allosteric CaSR antagonist (calcilytic), like NPS 2143, does preserve human cortical postnatal HCN-1A neurons viability notwithstanding the presence of exogenous Aβ-os by suppressing the otherwise elicited oversecretion and spread of newly synthesized Aβ-os. Therefore, if given at minimal cognitive impairment or earlier stages, calcilytics could halt AD progression and preserve the patients' cortical neurons, cognitive abilities, and eventually life.

The excess vascular endothelial growth factor (VEGF) produced in the Alzheimer's disease (AD) brain can harm neurons, blood vessels, and other components of the neurovascular units (NVUs). But could astrocytes partaking in networks of astrocyte-neuron teams and connected to blood vessels of NVUs contribute to VEGF production? We have shown with cultured cerebral cortical normal (i.e., untransformed) adult human astrocytes (NAHAs) that exogenous amyloid-β peptides (Aβs) stimulate the astrocytes to make and secrete large amounts of Aβs and nitric oxide by a mechanism mediated through the calcium-sensing receptor (CaSR). Here, we report that exogenous Aβs stimulate the NAHAs to produce and secrete even VEGF-A through a CaSR-mediated mechanism. This is indicated by the ability of Aβs to specifically bind the CaSR, and the capability of a CaSR activator, the "calcimimetic" NPS R-568, to imitate, and of the CaSR antagonist, "calcilytic" NPS 2143, to inhibit, the Aβs stimulation of VEGF-A production and secretion by the NAHAs. Thus, Aβs that accumulate in the AD brain may make the astrocytes that envelop and functionally collaborate with neurons into multi-agent AD-driving "machines" via a CaSR signaling mechanism(s). These observations suggest the possibility that CaSR allosteric antagonists such as NPS 2143 might impede AD progression.

Primary cilia are dynamic sensory organelles that continuously undergo structural modifications in response to environmental and cellular signals, many of which exhibit rhythmic patterns. Building on our previous findings of rhythmic cilia-related gene expression in diurnal primates (baboon), this study extends the investigation to the nocturnal mouse brain to identify circadian patterns of cilia gene expression across brain regions. We used computational techniques and transcriptomic data from four publicly available databases, to examine the circadian expression of cilia-associated genes within six brain areas: brainstem, cerebellum, hippocampus, hypothalamus, striatum, and suprachiasmatic nucleus. Our analysis reveals that a substantial proportion of cilia transcripts exhibit circadian rhythmicity across the examined regions, with notable overrepresentation in the striatum, hippocampus, and cerebellum. We also demonstrate region-specific variations in the abundance and timing of circadian cilia genes' peaks, indicating an adaptation to the distinct physiological roles of each brain region. Additionally, we show that the rhythmic patterns of cilia transcripts are shifted under various physiological and pathological conditions, including modulation of the dopamine system, high-fat diet, and epileptic conditions, indicating the adaptable nature of cilia transcripts' oscillation. While limited to a few mouse brain regions, our study provides initial insights into the distinct circadian patterns of cilia transcripts and highlights the need for future research to expand the mapping across wider brain areas to fully understand the role of cilia's spatiotemporal dynamics in brain functions. [ABSTRACT FROM AUTHOR]

It is generally assumed that the neuropathology of sporadic (late-onset or nonfamilial) Alzheimer's disease (AD) is driven by the overproduction and spreading of first Amyloid-βx-42 (Aβ42) and later hyperphosphorylated (hp)-Tau oligomeric "infectious seeds". Hitherto, only neurons were held to make and spread both oligomer types; astrocytes would just remove debris. However, we have recently shown that exogenous fibrillar or soluble Aβ peptides specifically bind and activate the Ca(2+)-sensing receptors (CaSRs) of untransformed human cortical adult astrocytes and postnatal neurons cultured in vitro driving them to produce, accrue, and secrete surplus endogenous Aβ42. While the Aβ-exposed neurons start dying, astrocytes survive and keep oversecreting Aβ42, nitric oxide (NO), and vascular endothelial growth factor (VEGF)-A. Thus astrocytes help neurons' demise. Moreover, we have found that a highly selective allosteric CaSR agonist ("calcimimetic"), NPS R-568, mimics the just mentioned neurotoxic actions triggered by Aβ●CaSR signaling. Contrariwise, and most important, NPS 2143, a highly selective allosteric CaSR antagonist ("calcilytic"), fully suppresses all the Aβ●CaSR signaling-driven noxious actions. Altogether our findings suggest that the progression of AD neuropathology is promoted by unceasingly repeating cycles of accruing exogenous Aβ42 oligomers interacting with the CaSRs of swelling numbers of astrocyte-neuron teams thereby recruiting them to overrelease additional Aβ42 oligomers, VEGF-A, and NO. Calcilytics would beneficially break such Aβ/CaSR-driven vicious cycles and hence halt or at least slow the otherwise unstoppable spreading of AD neuropathology.

The "amyloid-β (Aβ) hypothesis" posits that accumulating Aβ peptides (Aβs) produced by neurons cause Alzheimer's disease (AD). However, the Aβs contribution by the more numerous astrocytes remains undetermined. Previously we showed that fibrillar (f)Aβ25-35, an Aβ42 proxy, evokes a surplus endogenous Aβ42 production/accumulation in cortical adult human astrocytes. Here, by using immunocytochemistry, immunoblotting, enzymatic assays, and highly sensitive sandwich ELISA kits, we investigated the effects of fAβ25-35 and soluble (s)Aβ25-35 on Aβ42 and Aβ40 accumulation/secretion by human cortical astrocytes and HCN-1A neurons and, since the calcium-sensing receptor (CaSR) binds Aβs, their modulation by NPS 2143, a CaSR allosteric antagonist (calcilytic). The fAβ25-35-exposed astrocytes and surviving neurons produced, accumulated, and secreted increased amounts of Aβ42, while Aβ40 also accrued but its secretion was unchanged. Accordingly, secreted Aβ42/Aβ40 ratio values rose for astrocytes and neurons. While slightly enhancing Aβ40 secretion by fAβ25-35-treated astrocytes, NPS 2143 specifically suppressed the fAβ25-35-elicited surges of endogenous Aβ42 secretion by astrocytes and neurons. Therefore, NPS 2143 addition always kept Aβ42/Aβ40 values to baseline or lower levels. Mechanistically, NPS 2143 decreased total CaSR protein complement, transiently raised proteasomal chymotrypsin activity, and blocked excess NO production without affecting the ongoing increases in BACE1/β-secretase and γ-secretase activity in fAβ25-35-treated astrocytes. Compared to fAβ25-35, sAβ25-35 also stimulated Aβ42 secretion by astrocytes and neurons and NPS 2143 specifically and wholly suppressed this effect. Therefore, since NPS 2143 thwarts any Aβ/CaSR-induced surplus secretion of endogenous Aβ42 and hence further vicious cycles of Aβ self-induction/secretion/spreading, calcilytics might effectively prevent/stop the progression to full-blown AD., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The pathophysiological mechanisms underlying the onset and inexorable progression of the late‑onset form of Alzheimer's disease (AD) are still the object of controversy. This review takes stock of some most recent advancements of this field concerning the complex roles played by the amyloid‑β (Aβ)‑binding p75 neurotrophin receptor (p75NTR) and calcium‑sensing receptor (CaSR) and by the primary cilia in AD. Apart from their physiological roles, p75NTR is more intensely expressed in the hippocampus of human AD brains and Aβ‑bound p75NTR triggers cell death, whereas Aβ‑bound CaSR signalling induces the de novo synthesis and release of nitric oxide (NO), vascular endothelial growth factor (VEGF)‑A and Aβ peptides (Aβs), particularly on the part of normal adult human astrocytes. The latter effect could significantly increase the pool of Aβ‑ and NO‑producing nerve cells favouring the progressive spread of a self‑sustaining and self‑reinforcing 'infectious' mechanism of neural and vascular (i.e. blood-brain barrier) cell damage. Interestingly, primary cilia concentrate p75NTR receptors in their membranes and are abnormally structured/damaged in transgenic (Tg) AD‑model mice, which could impact on the adult neurogenesis occurring in the dentate gyrus's subgranular zone (SGZ) that is necessary for new memory encoding, thereby favouring typical AD cognitive decline. Altogether, these findings may pave the way to novel therapeutic approaches to AD, particularly in its mild cognitive impairment (MCI) and pre‑MCI stages of development.

Alzheimer's disease (AD) is the most common human neurodegenerative ailment, the most prevalent (>95%) late-onset type of which has a still uncertain etiology. The progressive decline of cognitive functions, dementia, and physical disabilities of AD is caused by synaptic losses that progressively disconnect key neuronal networks in crucial brain areas, like the hippocampus and temporoparietal cortex, and critically impair language, sensory processing, memory, and conscious thought. AD's two main hallmarks are fibrillar amyloid-β (fAβ) plaques in extracellular spaces and intracellular accumulation of fAβ peptides and neurofibrillary tangles (NFTs). It is still undecided whether either or both these AD hallmarks cause or result from the disease. Recently, the dysregulation of calcium homeostasis has been advanced as a novel cause of AD. In this case, a suitable candidate of AD driver would be the Aβ peptides-binding/activated calcium-sensing receptor (CaSR), whose intracellular signalling is triggered by Aβ peptides. In this review, we briefly discuss CaSR's roles in normal adult human astrocytes (NAHAs) and their possible impacts on AD., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Perylenequinones that generate reactive oxygen species (ROS) when illuminated with visible light have been recommended as photodynamic chemotherapeutic agents. One of these is calphostin C (CalC), the action of the photo-activated derivative of which, CalCphiE, has been ascribed to its ability to selectively and irreversibly inhibit protein kinase Cs (PKCs). But recent results of experiments with neoplastic rat fibroblasts and human breast and uterine cervix cancer cells have revealed that the action of CalCphiE involves more than PKC inhibition. Besides suppressing PKC activity, CalCphiE rapidly causes endoplasmic reticulum (ER) stress in breast cancer cells and the selective complete oxidation and proteasomal destruction of the functionally essential nuclear envelope protein lamin B1, in human cervical carcinoma (HCC) cells and neoplastic rat fibroblasts. When these lamin B1-lacking cells are placed in the dark, cytoplasmic membrane-linked PKC activities suddenly rebound and apoptogenesis is initiated as indicated by the immediate release of cytochrome c from mitochondria and later on the activation of caspases. Hence, CalCphiE is a photodynamic cytocidal agent attacking multiple targets in cancer cells and it would be worth determining, even for their best applicative use, whether other perylenequinones also share the so far unexpectedly complex deadly properties of the CalCphiE.

Considerable attention has recently been focused on the postnatal persistence of neurogenesis in the dentate gyrus of the hippocampus and the roles of signals from the primary cilium in the different functions of an increasing number of tissues and their malfunctions. Here we summarize the evidence that ties sonic hedgehog-triggered proliferogenic signaling from the primary cilia on granule cell progenitors in the adult dentate subgranular zone to maintain a pool of new "blank slate" dentate granule cells. These can be recruited to bundle and encode novel inputs flowing from various regions of the brain into the dentate gyrus via the entorhinal cortex without altering and erasing the synaptic patterns from previous inputs inscribed on older granule cells.

Dietary Ca(2+) reduces colon cell proliferation and carcinogenesis, but it becomes ineffective or even tumor-promoting during carcinogenesis. It appears that Ca(2+) and the colon cell CaSR together brake the massive cell production in normal colon crypts. The rapid proliferation of the transit-amplifying (TA) progeny of the colon stem cells at the bases of the crypts is driven by the "Wnt" signaling mechanism that stimulates proliferogenic genes and prevents apoptogenesis. It appears that TA cell cycling stops and terminal differentiation starts when the cells reach a higher level in the crypt where there is enough external Ca(2+) to stimulate the expression of CaSRs, the signals from which stimulate the expression of E-cadherin. At this point the APC (adenomatous polyposis coli) protein appears and some of it enters the nucleus. There it removes the apoptogenesis shield and stops the beta-cateninTcf-4 complex from driving further TA cell proliferation by releasing beta-catenin from the nucleus, and delivering it to cytoplasmic APCaxinGSK-3beta complexes for ultimate proteasomal destruction. Cytoplasmic beta-catenin is prevented from returning to the nucleus by destruction in APCaxinGSK-3beta complexes or locked by the emerging E-cadherin into adherens junctions which link the cell to proliferatively shut-down functioning cells with APC-dependent cytoskeletons moving up and out of the crypt. A common first step in colon carcinogenesis is the loss of functional APC which results in the retention of proliferogenic nuclear beta-cateninTcf-4. This drives the eventual appearance of mutation accumulating, apoptosis-resistant clones the proliferation of which cannot be inhibited by external Ca(2+) because of CaSR-disabling gene mutations.

Although the calcium-sensing receptor (CaSR) is expressed by all types of nerve cells in widespread areas of the human central nervous system (CNS), so far its roles in brain pathophysiology remain largely unknown. Here, we review the available evidence concerning the stages of development of sporadic late-onset Alzheimer's disease (LOAD) and the roles therein played by CaSR signaling. As the brain ages, its ability to dispose of dangerous synapse-targeting soluble amyloid beta-(1-42) (sAbeta42) oligomers released from normal neuronal activity declines. As their levels slowly rise, these oligomers increasingly target and eliminate synapses and prevent synapse formation, thereby eroding the foundations of memory formation and cognitive functions. In this initial stage, neurons, even though synaptically impaired, remain alive. Concurrently, sAbeta42 oligomers by binding to CaSR on human astrocytes induce via mitogen activated protein kinase (MAPK) activity the release of huge amounts nitric oxide (NO), which by itself and after conversion to peroxynitrite (ONOO(-)) damages neighboring neurons. When the sAbeta42 oligomers increasingly aggregate into fibrillar plaques, they attract and activate microglial macrophages that, while trying to clear the plaques, produce via Abeta-activated CaSR signaling several proinflammatory cytokines and reactive oxygen species (ROS). Notably, the microglial cytokines, like sAbeta42 oligomers, induce human astrocytes to make large amounts of NO and hence ONOO(-) via CaSR signal-dependent MAPK activity. The microglial cytokines-activated astrocytes might also produce their own sAbeta42, which would combine with neuron- and microglia-released sAbeta42 to increase the fibrillar burden and promote the further production of reactive oxygen species (ROS), NO/ONOO(-), and proinflammatory cytokines to efficiently kill both normal and functionally impaired (undead) neurons. But, on a somewhat positive note, we speculate that the astrocytes' CaSR-stimulated MAPK activities might also induce vascular endothelial growth factor (VEGF) expression and production. This might in turn enhance neuronal stem cells neurogenesis at least in the subgranular zone (SGZ) of the hippocampal dentate gyrus.

There is much evidence that dietary Ca(2+) loading reduces colon cell proliferation and carcinogenesis in humans and rodents, but during carcinogenesis it becomes ineffective or even tumor-promoting. We are beginning to see how Ca(2+) balances the continuous massive cell production in colon crypts by driving the terminal differentiation and eventually the apoptosis of the cells mainly on the mucosal surface, and how this Ca(2+) control is lost during colon carcinogenesis. The rapid proliferation of the transit-amplifying (TA) progeny of the colon stem cells is driven by the so-called "Wnt" signaling mechanism, which involves the stimulation of proliferogenic genes such as those for c-Myc and cyclin D1 and the silencing of the gene for the cell cycle-stopping p21(Cip1/WAF1) protein by nuclear beta-catenin*Tcf-4 complexes. TA cells avoid mitotic damage and premature apoptosis by expressing the protein survivin. It appears that TA cell cycling stops and terminal differentiation starts when the cells reach a higher level in the crypt where there is enough lumenal Ca(2+) to stimulate the expression and activation of CaSRs (Ca(2+)-sensing receptors), the signals from which stimulate the expression of E-cadherin. Along with this, the APC (adenomatous polyposis coli) protein appears and some of it enters the nucleus. There it makes the TA cells susceptible to the eventual apoptotic balancing by stopping survivin expression and the beta-catenin*Tcf-4 complex from driving further cell cycling by releasing beta-catenin from the nucleus, and delivering it to cytoplasmic APC*axin*GSK-3beta complexes for ultimate proteasomal destruction. Cytoplasmic beta-catenin is then prevented from returning to the nucleus by either being intercepted and destroyed by APC*axin*GSK-3beta complexes or locked by the emerging E-cadherin into membrane adherens junctions which tie the cell into the sheet of proliferatively shut-down cells with APC-dependent cytoskeletons moving to the mouth of the crypt and onto the flat mucosal surface. A common first step in sporadic colon carcinogenesis is the loss of functional APC which disorients upwardly directed migration and causes the retention of nuclear beta-catenin and proliferogenic beta-catenin*Tcf-4 complexes as well as genomic instability. Eventually the balance between cell proliferation and terminal differentiation and death is radically tipped in favour of proliferation by the appearance of apoptosis-resistant, survivin-expressing clones of Ca(2+)-insensitive cells which are locked into the proliferative, mutation-prone mode because of CaSR-disabling gene mutations which prevent the stimulation of E-cadherin expression and terminal differentiation.

Lamin B1, a major component of the nuclear lamina, anchors the nucleus to the cytoskeletal cage, and controls nuclear orientation, chromosome positioning and, alongside several enzymes, fundamental nuclear functions. Exposing polyomavirus-transformed rat pyF111 fibroblasts and human cervical carcinoma (HCC) C4-I cells for 30 min to photoexcited perylenequinone calphostin C, i.e. Cal C(phiE), an established reactive oxygen species (ROS)-generator and protein kinase C (PKC) inhibitor, caused the cells to selectively oxidize and then totally destroy their nuclear lamin B1 by only 60 min after starting the treatment, i.e. when apoptotic caspases' activities had not yet increased. However, while the oxidized lamin B1 was being destroyed, lamins A/C, the lamin A-associated nuclear envelope protein emerin, and the nucleoplasmic protein cyclin E were neither oxidized nor destroyed. The oxidized lamin B was ubiquitinated and demolished in the proteasome probably by an enhanced peptidyl-glutaminase-like activity. Hence, the Cal C(phiE)-induced rapid and selective lamin B1 oxidation and proteasomal destruction ahead of the activation of apoptotic caspases was by itself a most severe molecular lesion impairing vital nuclear functions. Conversely, Cal C directly added to the cells kept in the dark damaged neither nuclear lamin B1 nor cell viability. Thus, our findings reveal a novel cell-damaging mechanism of a photodynamic tumor therapeutic agent.

Osteocytes and articular chondrocytes sense and respond to the strains imposed on bones and joints by various activities such as breathing and walking. This mechanoresponsiveness is needed to maintain bone and cartilage microstructure and strength. In bone the large number of osteocytes form a vast osteointernet in which the gap junctionally interconnected members are lodged in an extensive lacunocanalicular network. The much smaller number of articular chondrocytes are not interconnected in a chondrointernet. Instead, they are separately lodged in capsules called chondrons. While there are many possible strain-sensing devices, it now appears that the non-motile solitary (primary) cilia protruding like aerials from osteocytes (as well as osteoblasts) and chondrocytes are switches that when toggled by cyclical pulses of lacunocanalicular fluid or cartilage compression send signals such as Ca(2+) surges into the cell to trigger a cascade of events that include appropriate gene activations to maintain and strengthen bone and cartilage. Moreover, the chondrocyte cilium with its Ihh(Indian hedgehog)-activated Smo receptor is a key player along with PTHrP in endochondral bone formation.

Three steps lead to the development of full-blown sporadic or late-onset Alzheimer's disease or dementia (AD). In the young brain, amyloid β-(1-42) (Aβ 42) is a normal aggregation-prone protein product of neuronal activity that is kept at a safe low level by proteolysis in neurons and glial cells, and by expulsion across the blood-brain barrier. But clearance declines with advancing age. Step 1: Because of the normal decline with age of the Aβ 42-clearing mechanisms, toxic amyloid-derived diffusible ligands (ADDLs) made of dodecamers of the aggregation-prone Aβ 42 start accumulating. These Aβ 42 dodecamers selectively target the initially huge numbers of excitatory synapses of neurons and cause them to start slowly dropping, which increasingly impairs plasticity and sooner or later starts noticeably affecting memory formation. At a certain point, this increasing loss of synapses induces the neurons to redirect their still-expressed cell cycle proteins from post-mitotic jobs, such as maintaining synapses, to starting a cell cycle and partially or completely replicating DNA without entering mitosis. The resulting aneuploid or tetraploid neurons survive for as long as 6-12 months as quasi-functional 'undead zombies', with developing tangles of hyperphosphorylated τ protein disrupting the vital anterograde axonal transport of mitochondria and other synapse-vital components. Step 2: The hallmark AD plaques appear as Aβ 42 clearance continues to decline and the formation of Aβ 42 non-diffusible fibrils begins in the aging brain. Step 3: A terminal cytokine-driven maëlstrom begins in the aging brain when microglia, the brain's professional macrophages, are activated in and around the plaques. They produce pro-inflammatory cytokines, such as IFN-γ, IL-1β and TNF-α. One of these, IFN-γ, causes the astrocytes enwrapping the neuronal synapses to express their β-secretase (BACE1) genes and produce and release Aβ 42, which can kill the closely apposed neurons by binding to their p75NTR receptors, which generate apoptogenic signals. Astrocytes are also stimulated by the same cytokines to turn on their nitric oxide synthase (NOS)-2 genes and start pouring large amounts of nitric oxide (NO) and its cytocidal derivative peroxynitrite (ONOO-) directly out onto the closely apposed neurons.

Here, we test whether early visual and OCT rod energy-linked biomarkers indicating pathophysiology in nicotinamide nucleotide transhydrogenase (Nnt)-null 5xFAD mice also occur in Nnt-intact 5xFAD mice and whether these biomarkers can be pharmacologically treated. Four-month-old wild-type or 5xFAD C57BL/6 substrains with either a null (B6J) Nnt or intact Nnt gene (B6NTac) and 5xFAD B6J mice treated for one month with either R-carvedilol + vehicle or only vehicle (0.01% DMSO) were studied. The contrast sensitivity (CS), external limiting membrane-retinal pigment epithelium (ELM-RPE) thickness (a proxy for low pH-triggered water removal), profile shape of the hyperreflective band just posterior to the ELM (i.e., the mitochondrial configuration within photoreceptors per aspect ratio [MCP/AR]), and retinal laminar thickness were measured. Both wild-type substrains showed similar visual performance indices and dark-evoked ELM-RPE contraction. The lack of a light–dark change in B6NTac MCP/AR, unlike in B6J mice, is consistent with relatively greater mitochondrial efficiency. 5xFAD B6J mice, but not 5xFAD B6NTac mice, showed lower-than-WT CS. Light-adapted 5xFAD substrains both showed abnormal ELM-RPE contraction and greater-than-WT MCP/AR contraction. The inner retina and superior outer retina were thinner. Treating 5xFAD B6J mice with R-carvedilol + DMSO or DMSO alone corrected CS and ELM-RPE contraction but not supernormal MCP/AR contraction or laminar thinning. These results provide biomarker evidence for prodromal photoreceptor mitochondrial dysfunction/oxidative stress/oxidative damage, which is unrelated to visual performance, as well as the presence of the Nnt gene. This pathophysiology is druggable in 5xFAD mice. [ABSTRACT FROM AUTHOR]

Statins inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which initiates the syntheses of cholesterol and isoprenoid lipids that are needed to provide amyloid peptides for the amyloid cascade. This cascade is believed to induce sporadic or late-onset Alzheimer's disease, which accounts for 90 - 95% of Alzheimer's disease sufferers. Cholesterol is also the prime driver of cerebrovascular disease that (along with amyloid peptides) increasingly appears to be linked to the cognitive deterioration of Alzheimer's disease. Cholesterol is needed to make the lipid rafts that are the platforms for isoprenoid-dependent assembly and activation of raftophilic beta- and gamma-secretases that work in tandem to excise dangerous 40 and 42 amino acid amyloid-beta (Abeta) fragments from amyloid precursor protein, the transmembrane amyloid precursor glycoprotein. When they are excessively produced and can no longer be effectively destroyed or otherwise cleared from the hypoperfused ageing brain, the Abeta42 fragments released from the active synaptic terminals of normally busy neurons (and from stressed neurons unsuccessfully trying to proliferate and producing disruptive tangles of hyperphosphorylated tau-proteins) aggregate into neuritic plaques, which activate glial cells. The pro-inflammatory cytokines and growth factors from the glial cells further damage and kill neurons. As statins strike at several parts of the Alzheimer's disease mechanism (such as the infliction of cholesterol-dependent cerebrovascular damage) by inhibiting HMG-CoA reductase, their long-term use (starting as early as possible during Alzheimer's disease development) should slow or even prevent the progression of Alzheimer's disease. Indeed, there is some evidence of a significantly reduced incidence of Alzheimer's disease among people who have been using statins to reduce hypercholesterolaemia and its cardiovascular effects. To be certain of this, there must be more multi-year trials to specifically assess the effects of statins on sporadic Alzheimer's disease.

Between 1958 and the late 1970s it was learned that PTH (the parathyroid hormone) could directly stimulate the initiation of DNA replication by murine CFU-S (colony-forming unit-spleen) cells via cyclic AMP, stimulate the proliferation of normal and X-irradiated murine and rat bone marrow cells, control hematopoiesis, and increase the survival of X-irradiated mice and rats when injected any time between 18h before and 3h after X-irradiation. Since then, it has been shown that the hematopoietic stem cell niche consists of PTH receptor-bearing, osteoblastic trabecular bone-lining cells that maintain the stem cells' (HSCs') proliferatively quiescent 'stemness' by various gene up-regulating and down-regulating signals caused by the tight adhesion of the HSCs to the osteoblastic niche-lining cells. Stimulating the osteoblastic lining cells with recombinant human PTH-(1-34) (Forteo) causes a cyclic AMP-mediated enlargement of the HSC pool and promotes bone marrow transplant engraftment and growth and the survival of lethally irradiated mice. But this is only the beginning of the exploitation of the PTHs for marrow engraftment. It must now be determined whether the marrow engraftment-enhancing action of this potent bone growth-stimulating PTH can be extended from mice to rats and monkeys. It must be determined whether two other PTH peptides, rhPTH-(1-84) [Preos]and [Leu(27)]cyclo(Glu(22)-Lys(26))hPTH-(1-31)NH(2) [Ostabolin-C]) are as effective as or better than rhPTH-(1-34)(Forteo). Since, all three peptides are on the market, or nearing the market, for safely and strongly stimulating bone growth and treating osteoporosis one or all of them may become valuable tools for safely promoting the engraftment of peripherally harvested HSCs in cancer patients whose bone marrows have been 'emptied' by chemotherapeutic drugs or ionizing radiation.

Reportedly, beta-amyloid peptides (Abeta40 and Abeta42) induce the neurodegenerative changes of Alzheimer's disease (AD) both directly by interacting with components of the cell surface to trigger apoptogenic signaling and indirectly by activating astrocytes and microglia to produce excess amounts of inflammatory cytokines. A possible cell surface target for Abetas is the p75 neurotrophin receptor (p75(NTR)). By using SK-N-BE neuroblastoma cells without neurotrophin receptors or engineered to express the full-length p75(NTR) or various parts of it, we have proven that p75(NTR) does mediate the Abeta-induced cell killing via its intracellular death domain (DD). This signaling via the DD activates caspase-8, which then activates caspase-3 and apoptogenesis. We also found a strong cytocidal interaction of direct p75(NTR)-mediated and indirect pro-inflammatory cytokine-mediated neuronal damage induced by Abeta. In fact, pro-inflammatory cytokines such as TNF-alpha and IL-1beta from Abeta-activated microglia potentiated the neurotoxic action of Aalpha mediated by p75(NTR) signaling. The pro-inflammatory cytokines probably amplify neuronal damage and killing by causing astrocytes to flood their associated neurons with NO and its lethal oxidizing ONOO- derivative. Indeed, we have found that a combination of three major pro-inflammatory cytokines, IL-1beta+IFN-gamma+TNF-alpha, causes normal adult human astrocytes (NAHA) to express nitric oxide synthase-2 (NOS-2) and make dangerously large amounts of NO via mitogen-activated protein kinases (MAPKs). Soluble Abeta40, the major amyloid precursor protein cleavage product, by itself stimulates astrocytes to express NOS-2 and make NO, possibly by activating p75(NTR) receptors, which they share with neurons, and can considerably amplify NOS-2 expression by the pro-inflammatory cytokine trio. These observations have uncovered a deadly synergistic interaction of Abeta peptides with pro-inflammatory cytokines in the neuron-astrocyte functional units of the AD brain. Finally, we have found that p75(NTR) and its DD also mediate the killing of SK-N-BE human neuroblastoma cells by the prion protein fragment PrP106-126. Thus, neurons expressing p75(NTR) as well as pro-inflammatory cytokine receptors are likely the preferential targets of Abetas and prions and the neurodegenerative diseases they cause.

The first experiments demonstrating parathyroid hormone's (PTH's) dramatic bone-building activity in rat pups, using a bovine parathyroid extract called parathormone were reported 74 years ago. Over the next decades, the native parathyroid hormone (human (h)PTH(1-84)) was purified and two of its fragments (hPTH(1-34) and (Leu27)cycloGlu22-Lys26hPTH(1-31)NH2) have been developed for the treatment of osteoporosis. One of these, recombinant (r)hPTH(1-34), is now on the market under the trade name of Forteo. The native hormone has also completed clinical trials and (Leu27)cycloGlu22-Lys26hPTH(1-31)NH2 is in phase II clinical trials under the trade name Ostabolin-C. All three of these peptides potently stimulate bone growth, reinforce bone microstructure weakened by estrogen deprivation and reduce further fracturing. Furthermore, future studies may demonstrate that these peptides also promote the repair of existing fractures and implant anchorage in both healthy and osteoporotic humans. PTHs have the potential to become more successful by using cost-cutting, but still effective, cyclical treatment regimens and by formulating them for non-injectable delivery. This review will discuss the identification of PTH peptides, how they function and their future role in the treatment of osteoporosis.

Protein kinase C (PKC) isoforms regulate cell proliferation and apoptosis. Since the PKC isoenzyme complement varies considerably from cell type to cell type, a PKC's responsiveness to an apoptogenic challenge must be defined for both the type of apoptogen and the type of cell. We have already reported that the changes in the distribution and activity of PKC-delta in apoptosing polyomavirus-infected/transformed Fischer rat embryo pyF111 fibroblasts depend on the type of apoptogen. Here, we show that this is also true for PKC-betaI in pyF111 cells treated with the slow DNA-damaging VP-16 (etoposide) or the fast-acting (in the cytoplasm) calphostin C. These apoptogens caused quite different shifts of the PKC-betaI level and activity in the nuclear membrane (NM) and nucleoplasm (NP), but corresponding changes in the cytosol (CS) and cytoplasmic particulate (CP) fractions. The hefty translocation of PKC-betaI onto the CP fraction and its increased activity there suggest the possible triggering of a cytochrome c/caspase-mediated apoptosis-inducing mechanism common to both agents. The present results are a necessary lead-up to functional proteomic analyses aimed at identifying the molecules forming the local PKC-betaI signalling modules under different conditions.

The accumulation of amyloid beta (Abeta)-peptides and their collection in fibrillar plaques in the human brain are believed to be responsible for Alzheimer's disease. The major neuron killers in the Alzheimer brain include proinflammatory cytokines and NO made by NOS-2 (inducible nitric oxide synthase-2). We have determined the effect of a soluble Abeta peptide, Abeta(1-40), on the expression of NOS-2 in astrocytes using a novel model system consisting of pure cultures of cells from adult human brains that, after the first three passages in vitro, become stably locked into the normal astrocytic phenotype like their counterparts in the adult human brain. Abeta(1-40) alone stimulated quiescent astrocytes to start expressing functional NOS-2 and dumping NO into the culture medium during the next 4 days. But adding three of the proinflammatory cytokines commonly produced in the Alzheimer brain--IFN-gamma, IL-1beta, and TNF-alpha--along with Abeta(1-40) more than trebled NOS-2 expression and doubled NO production. In view of the possibility of myelin breakdown in the Alzheimer brain, we also tested the capability of myelin basic protein (MBP) to stimulate NO production using human astrocytes. We found that MBP mimicked the ability of Abeta(1-40) to induce cells to release NO and adding the cytokine triad along with MBP more than doubled NO production and release. Thus, it appears that Abeta peptides and MBP can join forces with proinflammatory cytokines to enhance the NO-mediated killing of neurons in the Alzheimer brain.

Since NO production by NOS-2 made by astrocytes activated by proinflammatory cytokines contributes to the killing of neurons in variously damaged human brains, knowing the mechanisms responsible for NOS-2 expression should contribute to developing effective therapeutics. The expression and activation of NOS-2 in normal adult human cerebral cortical astrocytes treated with three proinflammatory cytokines, IL-1beta, TNF-alpha, and IFN-gamma, are driven by two separable mechanisms. NOS-2 expression requires a burst of p38 MAPK activity, while the activation of the resulting enzyme protein requires MEK/ERK-dependent BH4 (tetrahydrobiopterin) synthesis between 24 and 24.5 h after adding the cytokines to the culture medium. Here we show that NOS-2 expression in the activated astrocytes requires that the culture medium contain 1.8 mM Ca2+, but it is unaffected by inhibiting calcium-sensing receptors (CASRs) with NPS 89636. However, NOS-2 activation is inhibited by NPS 89626 during the MEK/ERK-dependent stage between 24 and 24.5 h after adding the cytokines, and this inhibition can be overridden by exogenous BH4. Therefore, NOS-2 expression and the subsequent BH4-dependent NOS-2-activation in human astrocytes need 1.8 mM Ca2+ to be in the culture medium, while NOS-2 activation also needs functional CASRs between 24 and 24.5 h after cytokine addition. These findings raise the possibility that calcilytic drugs prevent NO-induced damage and death of human neurons., (Copyright (c) 2005 Wiley-Liss, Inc.)

Forty-seven years ago, the parathyroid hormone (PTH) in one injection of Lilly's old bovine parathyroid extract, PTE, was found to greatly increase the 30-day survival of heavily X-irradiated rats when given from 18 h before to as long as 3 h after irradiation but no later. This was the first indication that PTH might stimulate hematopoiesis. Recent studies have confirmed the relation between PTH and hematopoiesis by showing that hPTH-(1-34)OH increases the size of the hematopoietic stem cell pool in mice. The peptide operates through a cyclic AMP-mediated burst of Jagged 1 production in osteoblastic cells lining the stem cells' niches on trabecular bone surfaces. The osteoblastic cells' Jagged 1 increases the hematopoietic stem cell pool by activating Notch receptors on attached stem cells. PTH-triggered cyclic AMP signals also directly stimulate the proliferation of the hematopoietic stem cells. However, the single PTH injection in the early experiments using PTE probably increased the survival of irradiated rats mainly by preventing the damaged hematopoietic progenitors from irreversibly initiating self-destructive apoptogenesis during the first 5 h after irradiation. It has also been shown that several daily injections of hPTH-(1-34)OH enable lethally irradiated mice to survive by stimulating the growth of transplanted normal bone marrow cells. If the osteogenic PTHs currently entering or on the verge of entering the market for treating osteoporosis can also drive hematopoiesis in humans as well as rodents, they could be potent tools for reducing the damage inflicted on bone marrow by cytotoxic cancer chemotherapeutic drugs and ionizing radiation., (Copyright (c) 2005 Wiley-Liss, Inc.)

We have used backbone N-methylations of parathyroid hormone (PTH) to study the role of these NH groups in the C-terminal amphiphilic alpha-helix of PTH (1-31) in binding to and activating the PTH receptor (P1R). The circular dichroism (CD) spectra indicated the structure of the C-terminal alpha-helix was locally disrupted around the methylation site. The CD spectra differences were explained by assuming a helix disruption for four residues on each side of the site of methylation and taking into account the known dependence of CD on the length of an alpha-helix. Binding and adenylyl cyclase-stimulating data showed that outside of the alpha-helix, methylation of residues Asp30 and Val31 had little effect on structure or activities. Within the alpha-helix, disruption of the structure was associated with increased loss of activity, but for specific residues Val21, Leu24, Arg25, and Leu28 there was a dramatic loss of activities, thus suggesting a more direct role of these NH groups in correct P1R binding and activation. Activity analyses with P1R-delNT, a mutant with its long N-terminal region deleted, gave a different pattern of effects and implicated Ser17, Trp23, and Lys26 as important for its PTH activation. These two groups of residues are located on opposite sides of the helix. These results are compatible with the C-terminal helix binding to both the N-terminal segment and also to the looped-out extracellular region. These data thus provide direct evidence for important roles of the C-terminal domain of PTH in determining high affinity binding and activation of the P1R receptor.

Inflammation in vascular (mostly arterial) walls and heart valves triggered by the trans-endothelial influx of LDL particles and the action of subsequently modified (e.g., by oxidation) LDL particles can trigger true bone formation by valvar fibroblasts, by a subpopulation of re-differentiation-competent VSMCs (vascular smooth muscle cells) or by vascular pericytes. Vascular ossification can lead to heart failure and death. Elderly osteoporotic women who need osteogenic drugs to restore their lost skeletal bone are paradoxically prone to vascular ossification-the "calcification paradox." The recent introduction into the clinic of a potently osteogenic parathyroid hormone peptide, Lilly's rhPTH-(1-34)OH (Forteotrade mark), to reverse skeletal bone loss raises the question of whether this and other potently osteogenic PTHs still in clinical trial might also stimulate vascular ossification in such osteoporotic women. Indeed the VSMCs in human and rat atherosclerotic lesions hyperexpress PTHrP and the PTHR1 (or PTH1R) receptor as do maturing osteoblasts. And the evidence indicates that endogenous PTHrP with its NLS (nuclear/nucleolar localization sequence) does stimulate VSMC proliferation (a prime prerequisite for atheroma formation and ossification) via intranuclear targets that inactivate pRb, the inhibitory G1/S checkpoint regulator, by stimulating its hyperphosphorylation. But neither externally added full-length PTHrP nor the NLS-lacking PTHrP-(1-34)OH gets into the VSMC nucleus and instead they inhibit proliferation and calcification by only activating the cell's PTHR1 receptors. No PTH has an NLS and, as expected from the observations on the externally added PTHrPs, hPTH-(1-34)OH inhibits calcification by VSMCs and cannot stimulate vascular ossification in a diabetic mouse model. Encouraging though this may be for osteoporotics with their "calcification paradox," more work is needed to be sure that the skeletally osteogenic PTHs do not promote vascular ossification with its cardiovascular consequences., ((c) 2005 Wiley-Liss, Inc.)

There are three injectable and one oral bone-building (i.e., bone anabolic) parathyroid hormone (PTH) peptides. One of the four, Lilly's injectable teriparatide (Forteo), is currently being used, and the other three are in clinical trials. They are being used or assessed only for treating postmenopausal osteoporosis. However, their potential clinical targets now extend far beyond osteoporosis. They can accelerate the mending of even severe non-union fractures; they will probably be used to strengthen the anchorage of pros-theses to bone; they have been shown to treat psoriasis that has resisted other treatments; they can increase the size of haematopoietic stem cell proliferation and accelerate the endogenous repopulation or repopulation by donor transplants of bone marrow depleted by chemotherapeutic drugs; and they may prevent vascular ossification. Leptin, a member of the cytokine superfamily has a PTH-like osteogenic activity and may even partly mediate PTH action. But leptin has two drawbacks that cloud its therapeutic future. First, apart from directly stimulating osteoblastic cells, it targets cells in the hypothalamic ventromedial nuclei and through them it reduces oestrogenic activity by promoting osteoblast-suppressing adrenergic activity. Second, it stimulates vascular and heart valve ossification, which leads to such events as heart failure and diabetic limb amputations.

Nitric oxide (NO) from astrocytes is one of the signalers used by the brain's extensive glial-neuronal-vascular network, but its excessive production by pro-inflammatory cytokine-stimulated glial cells can be cytodestructive. Here, we show how three pro-inflammatory cytokines (IL-1beta, TNF-alpha, and IFN-gamma) together stimulated the activation, but not the prior expression, of NOS-2 protein via a mechanism involving MEK-ERKs protein kinases in astrocytes from adult human cerebral temporal cortex. The cytokines triggered a transient burst of p38 MAPK activity and the production of NOS-2 mRNA which were followed by bursts of MEK-ERK activities, synthesis of the NOS-2 co-factor tetrahydrobiopterin (BH(4)), a build-up of NOS-2 protein and from it active NOS-2 enzyme. Selectively inhibiting MEK1/MEK2, but not the earlier burst of p38 MAPK activity, with a brief exposure to U0126 between 24 and 24.5 h after adding the cytokine triad affected neither NOS-2 expression nor NOS-2 protein accumulation but stopped BH(4) synthesis and the assembly of the NOS-2 protein into active NOS-2 enzyme. The complete blockage of active NOS-2 production by the brief exposure to U0126 was bypassed by simply adding BH(4) to the culture medium. Therefore, this cytokine triad triggered two completely separable, tandem operating mechanisms in normal human astrocytes, the first being NOS-2 gene expression and accumulation of NOS-2 protein and the second being the synthesis of the BH(4) factor needed to dimerize the NOS-2 protein into active, NO-making NOS-2 enzyme.

Osteogenic peptides are, or have potential to be, therapies for the treatment of osteoporosis, fracture repair, and repair of loosened bone implants. Human parathyroid hormone has been approved for the treatment of post-menopausal osteoporosis. Constrained analogs of PTH and the parathyroid-hormone related peptide (PTHrP) have aided the understanding of how PTH and PTHrP bind to their common receptor and some of these analogs have improved properties that make them possible candidates for clinical trial. Cyclization by lactam formation has shown that a core region of human PTH (hPTH) from residues 16-26 binds as an alpha-helix to the receptor and that the biological effects are remarkably sensitive to ring size. Appropriate cyclization in this region of the molecule not only has yielded analogs with improved receptor activation but also ones less susceptible to protease degradation and thus more active in vivo. Cyclization has been less successful in the N-terminus region, residues 1-12, of hPTH(1-34) with only a cyclization between residues 6 and 10 showing some promise. The growing understanding of how this region binds to the receptor will lead to other productive constraints. This review also covers the potential of a different class of molecule, the osteogenic growth peptide (OPG), as an anabolic bone agent. These molecules have much weaker anabolic effects than PTH and cyclization does not result in improved activity. However, the information gained from these studies may yield analogs with better pharmacological profiles.

The native parathyroid hormone (PTH) and several of its N-terminal adenylyl cyclase-activating fragments and their analogs have become the star stimulators of bone growth for treating osteoporosis, accelerating fracture healing, and strengthening the anchorage of prosthetic bone implants and one of them (Lilly's Forteo--recombinant hPTH-(1-34) has recently arrived in the clinic. But something entirely different has been lurking in the background-the ability of the adenylyl cyclase stimulating hPTH-(1-34) to calm hyperproliferating keratinocytes and reduce psoriatic lesions. By contrast PTH-(7-34) which cannot stimulate adenylyl cyclase actually stimulates keratinocyte proliferation. Normal keratinocytes make PTHrP after they lift off the basal lamina and have stopped cycling. But they have an unconventional PTH/PTHrP receptor which is not coupled to adenylyl cyclase. Psoriatic keratinocytes do not make PTHrP and have only a broken-down, proliferation-limiting terminal differentiation-driving Notch-Notch ligand mechanism. Putting these and other facts together produces a possible picture of an exogenously applied adenylyl cyclase-activating PTH pinch hitting for the missing PTHrP and restoring normal keratinocyte proliferative activity epidermal structure by stimulating dermal fibroblasts which do have the conventional adenylyl cyclase-linked PTHR1 and in response directly or indirectly restore the overlying basal keratinocytes' Notch-Notch ligand terminal differentiation-driving mechanism and consequently a normal epidermal structure., (Copyright 2004 Wiley-Liss, Inc.)

Many, but likely most, neurons in the central nervous system have a nonmotile "primary" cilium extending like an antenna or finger from one of the pair of centrioles in the cell's centrosome into the extracellular space. Since their discovery over 100 years ago, these organelles have been either dismissed as functionless relicts of a bygone era or more often simply ignored. However, it has long been known that the photoreceptor-bearing outer segments of retinal rods and cones are modified primary cilia and it has recently been found that kidney cells' primary cilia are sensitive flowmeters the disabling of which causes polycystic kidney disease. It has also been recently shown that somatostatin sst3 receptors and serotonin 5-HT(6) receptors are selectively sited on neurons in various parts of the rat brain. It seems likely that these selectively receptored neuronal primary cilia will turn out to be the forerunners of a family of cell-signaling devices that help drive various brain functions by sending signals into their own cells and into adjacent cells through gap junctions and via conventional chemical synapses.

The influence of intermittent hPTH(1-34)NH2, hPTH(1-31)NH2, and monocyclic [Leu27]cyclo (Glu22-Lys26)hPTH(1-31)NH2 treatment on callus formation, mechanical strength, and callus tissue mechanical quality of tibial fractures in rats was investigated after 8 and 16 weeks of healing. In the 8 weeks of healing animals, the PTH-peptides were injected subcutaneously during the entire observation period (15 nmol/kg/day [hPTH(1-34)NH2: 15 nmol = 60 microg]), and control animals with fractures were given vehicle. In the 16 weeks of healing animals, the PTH-peptides were injected only during the first 8 weeks of healing (15 nmol/kg/day), after which the animals were left untreated during the rest of the healing period. After the first 8 weeks of healing, increased fracture strength and callus volume were seen in the PTH-treated rats (ultimate load 66%, ultimate stiffness 58%, callus volume 28%), and the three peptides were equally effective. No difference in callus tissue mechanical quality was found between PTH and vehicle animals. After 16 weeks of healing, no differences in fracture strength, callus volume, or callus tissue mechanical quality were seen between PTH and vehicle. When comparing PTH-treated animals at 8 and 16 weeks, fracture strength and callus tissue mechanical quality continued to increase after the withdrawal of PTH (ultimate load 23%, ultimate stress 88%, elastic modulus 87%) and external callus volume declined during this period (27%).

The growing number of patients with osteoporosis in our aging population need "anabolic" drugs to stimulate bone growth, improve bone microarchitecture, and accelerate fracture healing. Potent anabolic agents such as parathyroid hormone (PTH) and some of its adenylyl cyclase-stimulating fragments are either on their way, or have just now reached the clinic. This article discusses how PTHs might stimulate bone growth. The controversial bone anabolic activities of the widely used cholesterol-lowering lipophilic statins and how they might stimulate bone growth are also probed. Also, evidence is presented for leptin, a controller of body fat stores and the ovarian cycle. It has the remarkable property of being an anabolic and antianabolic that uses a hypothalamic factor to restrain osteoblast activity but by itself stimulates osteoblasts and inhibits osteoclasts.

With few exceptions, the non-cycling cells in a vast range of animals including humans have a non-motile primary cilium that extends from the mother centriole of the pair of centrioles in their centrosomes located between their Golgi apparatuses and nuclei. It has very recently been shown that the primary cilium of a dog or a mouse embryonic kidney cell is a fluid flowmeter studded with heterodimeric complexes of mechanoreceptors linked to Ca(2+)-permeable cation channels that when the cilium is bent can send Ca(2+) signals into the cell and beyond to neighboring cells through gap junctions. More than 30 years ago, osteocytes were reported also to have primary cilia, but this was promptly ignored or forgotten. Osteocytes are the bones' strain sensors, which measure skeletal activity from the effects of currents of extracellular fluid caused by their bones being bent and squeezed during various activities such as walking and running. Since bending a kidney cell's primary cilium can send a Ca(2+) wave surging through itself and its neighbors, the bending of an osteocyte's primary cilium by sloshing extracellular fluid is likely to do the same thing and thus be involved in measuring and responding to bone strain., (Copyright 2003 Wiley-Liss, Inc.)

The role of protein kinase C-beta(II) (PKC-beta(II)) in etoposide (VP-16)-induced apoptosis was studied using polyomavirus-transformed pyF111 rat fibroblasts in which PKC-beta(II) specific activity in the nuclear membrane (NM) doubled and the enzyme was cleaved into catalytic fragments. No PKC-beta(II) complexes with lamin B1 and/or active caspases were immunoprecipitable from the NM of proliferating untreated cells, but large complexes of PKC-beta(II) holoprotein and its catalytic fragments with lamin B1, active caspase-3 and -6, and inactive phospho-CDK-1, but not PKC-beta(I) or PKC-delta, could be immunoprecipitated from the NM of VP-16-treated cells, suggesting that PKC-beta(II) is an apoptotic lamin kinase. By 30 min after normal nuclei were mixed with cytoplasms from VP-16-treated, but not untreated, cells, PKC-beta(II) holoprotein had moved from the apoptotic cytoplasm to the normal NM, and lamin B1 was phosphorylated before cleavage by caspase-6. Lamin B1 phosphorylation was partly reduced, but its cleavage was completely prevented, despite the presence of active caspase-6, by adding a selective PKC-betas inhibitor, hispidin, to the apoptotic cytoplasms. Thus, a PKC-beta(II) response to VP-16 seems necessary for lamin B1 cleavage by caspase-6 and nuclear lamina dissolution in apoptosing pyF111 fibroblasts. The possibility of PKC-beta(II) being an apoptotic lamin kinase in these cells was further suggested by lamin B1-bound PKC-delta being inactive or only slightly active and by PKC-alpha not combining with the lamin.

There is a need for anabolic drugs that can stimulate bone growth, improve bone microarchitecture, accelerate fracture healing and thus restore bone strength to oteoporotics. The anabolic agents currently leading the way to the clinic are the parathroid hormone (PTH) and some of its adenylyl cyclase-stimulating fragments. Here we discuss what is known about the genes and their products that are stimulated by PTHR1 receptor signals and in four ways cause a large accumulation of bone-building osteoblasts. We will also discuss the currently controversial anabolic activity of the cholesterol-lowering statins and outline a possible mechanism by which they might stimulate BMP-2 expession and bone growth. Finally, we will present the growing evidence for the body's "fat-o-stat" cytokine-leptin-indirectly restraining bone growth via a hypothalamic factor and at the same serving as a local autocrine/paracrine stimulator of osteoblast activity via IGF-I and an inhibitor of osteoclast generation by stimulating osteoblastic cells' antiosteoclast OPG (osteoprotegerin) expression and reducing their proosteoclast RANKL expression.

In the new millennium, humans will be traveling to Mars and eventually beyond with skeletons that respond to microgravity by self-destructing. Meanwhile in Earth's aging populations growing numbers of men and many more women are suffering from crippling bone loss. During the first decade after menopause all women suffer an accelerating loss of bone, which in some of them is severe enough to result in "spontaneous" crushing of vertebrae and fracturing of hips by ordinary body movements. This is osteoporosis, which all too often requires prolonged and expensive care, the physical and mental stress of which may even kill the patient. Osteoporosis in postmenopausal women is caused by the loss of estrogen. The slower development of osteoporosis in aging men is also due at least in part to a loss of the estrogen made in ever smaller amounts in bone cells from the declining level of circulating testosterone and is needed for bone maintenance as it is in women. The loss of estrogen increases the generation, longevity, and activity of bone-resorbing osteoclasts. The destructive osteoclast surge can be blocked by estrogens and selective estrogen receptor modulators (SERMs) as well as antiosteoclast agents such as bisphosphonates and calcitonin. But these agents stimulate only a limited amount of bone growth as the unaffected osteoblasts fill in the holes that were dug by the now suppressed osteoclasts. They do not stimulate osteoblasts to make bone--they are antiresorptives not bone anabolic agents. (However, certain estrogen analogs and bisphosphates may stimulate bone growth to some extent by lengthening osteoblast working lives.) To grow new bone and restore bone strength lost in space and on Earth we must know what controls bone growth and destruction. Here we discuss the newest bone controllers and how they might operate. These include leptin from adipocytes and osteoblasts and the statins that are widely used to reduce blood cholesterol and cardiovascular damage. But the main focus of this article is necessarily the currently most promising of the anabolic agents, the potent parathyroid hormone (PTH) and certain of its 31- to 38-aminoacid fragments, which are either in or about to be in clinical trial or in the case of Lilly's Forteo [hPTH-(1-34)] tentatively approved by the Food and Drug Administration for treating osteoporosis and mending fractures.

The susceptibility to traumatic fracturing of osteopenic bones, and the spontaneous fracturing of osteoporotic bones by normal body movements caused by the microstructural deterioration and loss of bone, are currently treated with antiresorptive drugs, such as the bisphosphonates, calcitonin, estrogens, and selective estrogen receptor modulators. These antiresorptive agents target osteoclasts and, as their name indicates, reduce or stop bone resorption. They cannot directly stimulate bone formation, increase bone mass above normal values in ovariectomized rat models, or improve microstructure. However, there is a family of agents - the parathyroid hormone (PTH) and some of its fragments and their analogs - which directly stimulate bone growth and improve microstructure independently from impairing osteoclasts. These drugs are about to make their clinical debut in treating patients with osteoporosis and, probably not too far in the future, for accelerating fracture healing. They stimulate osteoblast accumulation and bone formation in three ways via signals from the type 1 PTH/PTH-related protein (PTHR1) receptors on proliferatively inactive preosteoblasts, osteoblasts, osteocytes and bone-lining cells. The receptor signals shut down the proliferative machinery in preosteoblasts and push their maturation to osteoblasts, cause the osteoblastic cells to make and secrete several factors that stimulate the extensive proliferation of osteoprogenitors without PTHRI receptors, stimulate the reversion of bone-lining cells to osteoblasts, and extend osteoblast lifespan and productivity by preventing them from suicidally initiating apoptosis. The first of the PTHs to reach the clinic will be teriparatide [recombinant human (h)PTH-(1-34)], which was recommended for approval in 2001 by the US Food and Drug Administration Endocrinology and Metabolic Drugs Advisory Committee for the treatment of postmenopausal osteoporosis. Teriparatide has been shown to considerably increase cancellous and cortical bone mass, improve bone microstructure, prevent fractures and thus provide benefits that cannot be provided by current antiresorptive drugs, when administered subcutaneously at a daily dose of 20 microg for no longer than 2 years to patients with osteoporosis.