Immunohistochemical distribution of secretagogin in the mouse brain.

Introduction: Calcium is essential for the correct functioning of the central nervous system, and calcium-binding proteins help to finely regulate its concentration. Whereas some calcium-binding proteins such as calmodulin are ubiquitous and are present in many cell types, others such as calbindin, calretinin, and parvalbumin are expressed in specific neuronal populations. Secretagogin belongs to this latter group and its distribution throughout the brain is only partially known. In the present work, the distribution of secretagogin-immunopositive cells was studied in the entire brain of healthy adult mice. Methods: Adult male C57BL/DBA mice aged between 5 and 7 months were used. Their whole brain was sectioned and used for immunohistochemistry. Specific neural populations were observed in different zones and nuclei identified according to Paxinos mouse brain atlas. Results: Labelled cells were found with a Golgi-like staining, allowing an excellent characterization of their dendritic and axonal arborizations. Many secretagogin-positive cells were observed along different encephalic regions, especially in the olfactory bulb, basal ganglia, and hypothalamus. Immunostained populations were very heterogenous in both size and distribution, as some nuclei presented labelling in their entire extension, but in others, only scattered cells were present. Discussion: Secretagogin can provide a more complete vision of calcium-buffering mechanisms in the brain, and can be a useful neuronal marker in different brain areas for specific populations.

Oleoylethanolamide Treatment Modulates Both Neuroinflammation and Microgliosis, and Prevents Massive Leukocyte Infiltration to the Cerebellum in a Mouse Model of Neuronal Degeneration.

Neurodegenerative diseases involve an exacerbated neuroinflammatory response led by microglia that triggers cytokine storm and leukocyte infiltration into the brain. PPARα agonists partially dampen this neuroinflammation in some models of brain insult, but neuronal loss was not the triggering cause in any of them. This study examines the anti-inflammatory and immunomodulatory properties of the PPARα agonist oleoylethanolamide (OEA) in the Purkinje Cell Degeneration (PCD) mouse, which exhibits striking neuroinflammation caused by aggressive loss of cerebellar Purkinje neurons. Using real-time quantitative polymerase chain reaction and immunostaining, we quantified changes in pro- and anti-inflammatory markers, microglial density and marker-based phenotype, and overall leukocyte recruitment at different time points after OEA administration. OEA was found to modulate cerebellar neuroinflammation by increasing the gene expression of proinflammatory mediators at the onset of neurodegeneration and decreasing it over time. OEA also enhanced the expression of anti-inflammatory and neuroprotective factors and the Pparα gene. Regarding microgliosis, OEA reduced microglial density-especially in regions where it is preferentially located in PCD mice-and shifted the microglial phenotype towards an anti-inflammatory state. Finally, OEA prevented massive leukocyte infiltration into the cerebellum. Overall, our findings suggest that OEA may change the environment to protect neurons from degeneration caused by exacerbated inflammation.

Oleoylethanolamide Delays the Dysfunction and Death of Purkinje Cells and Ameliorates Behavioral Defects in a Mouse Model of Cerebellar Neurodegeneration.

Oleoylethanolamide (OEA) is an endocannabinoid that has been proposed to prevent neuronal damage and neuroinflammation. In this study, we evaluated the effects of OEA on the disruption of both cerebellar structure and physiology and on the behavior of Purkinje cell degeneration (PCD) mutant mice. These mice exhibit cerebellar degeneration, displaying microtubule alterations that trigger the selective loss of Purkinje cells and consequent behavioral impairments. The effects of different doses (1, 5, and 10 mg/kg, i.p.) and administration schedules (chronic and acute) of OEA were assessed at the behavioral, histological, cellular, and molecular levels to determine the most effective OEA treatment regimen. Our in vivo results demonstrated that OEA treatment prior to the onset of the preneurodegenerative phase prevented morphological alterations in Purkinje neurons (the somata and dendritic arbors) and decreased Purkinje cell death. This effect followed an inverted U-shaped time-response curve, with acute administration on postnatal day 12 (10 mg/kg, i.p.) being the most effective treatment regimen tested. Indeed, PCD mice that received this specific OEA treatment regimen showed improvements in motor, cognitive and social functions, which were impaired in these mice. Moreover, these in vivo neuroprotective effects of OEA were mediated by the PPARα receptor, as pretreatment with the PPARα antagonist GW6471 (2.5 mg/kg, i.p.) abolished them. Finally, our in vitro results suggested that the molecular effect of OEA was related to microtubule stability and structure since OEA administration normalized some alterations in microtubule features in PCD-like cells. These findings provide strong evidence supporting the use of OEA as a pharmacological agent to limit severe cerebellar neurodegenerative processes.

What is the potential impact of the IsoDAR cyclotron on radioisotope production: a review.

The IsoDAR collaboration is developing a high-current cyclotron for a neutrino search experiment. Designed to deliver 10 mA of 60 MeV protons, the current and power of this cyclotron far exceed those of existing accelerators, opening new possibilities for the production of radiopharmaceutical isotopes, producing very high-activity samples in very short times. The cyclotron can also be easily configured to deliver ions other than protons including 1 mA of alpha particles at 240 MeV: this flexibility gives a broad reach into new areas of isotope production. We explain how IsoDAR overcomes the beam limits of commercial cyclotrons, and how it could represent the next step in isotope production rates.

Missing fingers

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Highly Active Antiretroviral Therapy and Gamma Knife Radiosurgery for the Treatment of AIDS-Related Primary Central Nervous System Lymphoma.

BACKGROUND: The criterion standard for the treatment of newly diagnosed primary central nervous system lymphoma (PCNSL) remains high-dose chemotherapy in conjunction with palliative whole-brain radiotherapy; however, there may be a role for novel combined approaches in immunocompromised patients. CASE DESCRIPTION: A 66-year-old man presented with acute cephalalgia, disorientation, and lethargy. His condition was evaluated in the emergency department, and he was admitted with probable hydrocephalus. Magnetic resonance imaging (MRI) of the brain revealed multiple nonspecific brain lesions, predominantly involving the right temporal lobe, which on biopsy led to a diagnosis of PCNSL. Subsequent laboratory studies demonstrated active human immunodeficiency virus (HIV) infection, with a CD4 count of 21 cells/µL and an HIV viral load (VL) of >400,000 copies/mL. The patient was eventually given highly active antiretroviral therapy (HAART). He declined palliative whole-brain radiotherapy but was amenable to gamma knife radiosurgery (GKRS) for treatment of the right temporal brain lesions. Three months later, the patient's neurologic symptoms had improved; similarly, his CD4 count increased to 176 cells/mL, and his HIV viral load was <90 copies/mL. By the 12-month follow-up visit, the patient was asymptomatic, and at 36 months, MRI of the brain demonstrated total remission without new brain lesions. CONCLUSIONS: The criterion standard for treatment of newly diagnosed PCNSL remains high-dose chemotherapy in conjunction with palliative whole-brain radiotherapy; however, there may be a role for novel combined approaches using chemotherapy, HAART, and GKRS to have a positive impact on survival rates of PCNSL related to AIDS.

Cytoskeleton stability is essential for the integrity of the cerebellum and its motor- and affective-related behaviors.

The cerebellum plays a key role in motor tasks, but its involvement in cognition is still being considered. Although there is an association of different psychiatric and cognitive disorders with cerebellar impairments, the lack of time-course studies has hindered the understanding of the involvement of cerebellum in cognitive and non-motor functions. Such association was here studied using the Purkinje Cell Degeneration mutant mouse, a model of selective and progressive cerebellar degeneration that lacks the cytosolic carboxypeptidase 1 (CCP1). The effects of the absence of this enzyme on the cerebellum of mutant mice were analyzed both in vitro and in vivo. These analyses were carried out longitudinally (throughout both the pre-neurodegenerative and neurodegenerative stages) and different motor and non-motor tests were performed. We demonstrate that the lack of CCP1 affects microtubule dynamics and flexibility, defects that contribute to the morphological alterations of the Purkinje cells (PCs), and to progressive cerebellar breakdown. Moreover, this degeneration led not only to motor defects but also to gradual cognitive impairments, directly related to the progression of cellular damage. Our findings confirm the cerebellar implication in non-motor tasks, where the formation of the healthy, typical PCs structure is necessary for normal cognitive and affective behavior.

Pax6 is essential for the maintenance and multi-lineage differentiation of neural stem cells, and for neuronal incorporation into the adult olfactory bulb.

The paired type homeobox 6 (Pax6) transcription factor (TF) regulates multiple aspects of neural stem cell (NSC) and neuron development in the embryonic central nervous system. However, less is known about the role of Pax6 in the maintenance and differentiation of adult NSCs and in adult neurogenesis. Using the +/Sey(Dey) mouse, we have analyzed how Pax6 heterozygosis influences the self-renewal and proliferation of adult olfactory bulb stem cells (aOBSCs). In addition, we assessed its influence on neural differentiation, neuronal incorporation, and cell death in the adult OB, both in vivo and in vitro. Our results indicate that the Pax6 mutation alters Nestin(+)-cell proliferation in vivo, as well as self-renewal, proliferation, and survival of aOBSCs in vitro although a subpopulation of +/Sey(Dey) progenitors is able to expand partially similar to wild-type progenitors. This mutation also impairs aOBSC differentiation into neurons and oligodendrocytes, whereas it increases cell death while preserving astrocyte survival and differentiation. Furthermore, Pax6 heterozygosis causes a reduction in the variety of neurochemical interneuron subtypes generated from aOBSCs in vitro and in the incorporation of newly generated neurons into the OB in vivo. Our findings support an important role of Pax6 in the maintenance of aOBSCs by regulating cell death, self-renewal, and cell fate, as well as in neuronal incorporation into the adult OB. They also suggest that deregulation of the cell cycle machinery and TF expression in aOBSCs which are deficient in Pax6 may be at the origin of the phenotypes observed in this adult NSC population.

Ion source issues for the DAEδALUS neutrino experiment.

The DAEδALUS experiment calls for 10 mA of protons at 800 MeV on a neutrino-producing target. To achieve this record-setting current from a cyclotron system, H2 (+) ions will be accelerated. Loosely bound vibrationally excited H2 (+) ions inevitably produced in conventional ion sources will be Lorentz stripped at the highest energies. Presence of these states was confirmed at the Oak Ridge National Laboratory and strategies were investigated to quench them, leading to a proposed R&D effort towards a suitable ion source for these high-power cyclotrons.

Characterization of the Catania VIS for H2(+).

The Catania VIS 2.46 GHz source has been installed on a test stand at the Best Cyclotron Systems, in Vancouver, Canada, as part of the DAEδALUS and IsoDAR R&D program. Studies to date include optimization for H2 (+)/p ratio and emittance measurements. Inflection, capture, and acceleration tests will be conducted when a small test cyclotron is completed.

Sex-influence of nicotine and nitric oxide on motor coordination and anxiety-related neurophysiological responses.

RATIONALE: Nitric oxide (NO) is a messenger synthesized in both the neuronal and glial populations by nitric oxide synthase type 1 (NOS1). Nicotine regulates NO production in a sex-dependent manner, both molecules being involved in motor function. OBJECTIVE: The present study evaluates sex differences in motor coordination, general movement, and anxiety-related responses resulting from both constant and continuous nicotine treatment and the genetic depletion of NOS1 activity. METHODS: Male and female mice were analyzed with the open-field and the rotarod tests. To understand the role of NO, knockout mice for NOS1 (NOS1-/-) were analyzed. Nicotine was administered continuously at a dose of 24 mg/kg/day via osmotic mini-pumps over 14 days because the behavioral effects elicited are similar to those observed with discontinuous administration. RESULTS: Data analyses revealed noteworthy sex differences derived from NOS1 depletion. Control NOS1-/- males exhibited an exacerbated anxiety-related response in relation to control NOS1-/- females and control wild-type (WT) males; these differences disappeared in the nicotine-administered NOS1-/- males. Additionally, nicotine administration differentially affected the horizontal movements of NOS1-/- females with respect to WT animals. NO depletion affected male but not female motor coordination improvement along the test days. However, the drug affected female motor coordination only at the end of the administration period. CONCLUSIONS: We show for the first time that NO affects motor and anxiety behaviors in a sex-dependent manner. Moreover, the behavioral effects of constant nicotine administration are dimorphic and dependent on NO production.

Differential glial activation during the degeneration of Purkinje cells and mitral cells in the PCD mutant mice.

Purkinje Cell Degeneration (PCD) mice harbor a nna1 gene mutation which leads to an early and rapid degeneration of Purkinje cells (PC) between the third and fourth week of age. This mutation also underlies the death of mitral cells (MC) in the olfactory bulb (OB), but this process is slower and longer than in PC. No clear interpretations supporting the marked differences in these neurodegenerative processes exist. Growing evidence suggests that either beneficial or detrimental effects of gliosis in damaged regions would underlie these divergences. Here, we examined the gliosis occurring during PC and MC death in the PCD mouse. Our results demonstrated different glial reactions in both affected regions. PC disappearance stimulated a severe gliosis characterized by strong morphological changes, enhanced glial proliferation, as well as the release of pro-inflammatory mediators. By contrast, MC degeneration seems to promote a more attenuated glial response in the PCD OB compared with that of the cerebellum. Strikingly, cerebellar oligodendrocytes died by apoptosis in the PCD, whereas bulbar ones were not affected. Interestingly, the level of nna1 mRNA under normal conditions was higher in the cerebellum than in the OB, probably related to a faster neurodegeneration and stronger glial reaction in its absence. The glial responses may thus influence the neurodegenerative course in the cerebellum and OB of the mutant mouse brain, providing harmful and beneficial microenvironments, respectively.

Accelerators for America's Future Workshop: Medicine and Biology.

"Medicine and Biology" was one of five working groups of the "Accelerators for America's Future" Workshop held October 2009. The recently-released workshop report stresses that the leadership position of the United States in fields where accelerators play an important part is being seriously eroded because of lack of coordinated agency support for accelerator research and development. This is particularly true for biology and medicine. Radiation therapy with beams of protons and light ions was pioneered in the United States and has proven successful in the treatment of several different tumor sites in the body. Proton therapy is available in the United States in a number of centers; however, all but one contain accelerator and beam-delivery components manufactured abroad. Light-ion therapy is only available overseas. Why has the United States lost its lead in this field? The Working Group noted that in other countries, central governments are subsidizing construction and technology development by their industries, whereas in the United States funding for purchasing and building clinical facilities must be raised from private sources. As a result, most proton facilities in the United States, by virtue of having to recover investment costs, favor reimbursable treatments, detracting from the development of research protocols. The financial hurdle for starting a light-ion facility in the United States has been totally prohibitive for the private-equity market. While technological advances are being made that will provide some reduction in capital costs, the field will not flourish in the United States until effective funding means are developed that do not put the full burden on the private sector.

Mild cerebellar neurodegeneration of aged heterozygous PCD mice increases cell fusion of Purkinje and bone marrow-derived cells.

Bone marrow-derived cells have different plastic properties, especially regarding cell fusion, which increases with time and is prompted by tissue injury. Several recessive mutations, including Purkinje Cell Degeneration, affect the number of Purkinje cells in homozygosis; heterozygous young animals have an apparently normal phenotype but they undergo Purkinje cell loss as they age. Our findings demonstrate that heterozygous pcd mice undergo Purkinje cell loss at postnatal day 300, this slow but steadily progressing cell death starting sooner than has been reported previously and without massive reactive gliosis or inflammation. Here, transplantation of bone marrow stem cells was performed to assess the arrival of bone marrow-derived cells in the cerebellum in these heterozygous mice. Our results reveal that a higher number of cell fusion events occurs in heterozygous animals than in the controls, on days 150 and 300 postnatally. In sum, this study indicates that mild cell death promotes the fusion of bone marrow-derived cells with surviving Purkinje neurons. This phenomenon suggests new therapies for long-lasting neurodegenerative disorders.

Purkinje cell degeneration in pcd mice reveals large scale chromatin reorganization and gene silencing linked to defective DNA repair.

DNA repair protects neurons against spontaneous or disease-associated DNA damage. Dysfunctions of this mechanism underlie a growing list of neurodegenerative disorders. The Purkinje cell (PC) degeneration mutation causes the loss of nna1 expression and is associated with the postnatal degeneration of PCs. This PC degeneration dramatically affects nuclear architecture and provides an excellent model to elucidate the nuclear mechanisms involved in a whole array of neurodegenerative disorders. We used immunocytochemistry for histone variants and components of the DNA damage response, an in situ transcription assay, and in situ hybridization for telomeres to analyze changes in chromatin architecture and function. We demonstrate that the phosphorylation of H2AX, a DNA damage signal, and the trimethylation of the histone H4K20, a repressive mark, in extensive domains of genome are epigenetic hallmarks of chromatin in degenerating PCs. These histone modifications are associated with a large scale reorganization of chromatin, telomere clustering, and heterochromatin-induced gene silencing, all of them key factors in PC degeneration. Furthermore, ataxia telangiectasia mutated and 53BP1, two components of the DNA repair pathway, fail to be concentrated in the damaged chromatin compartments, even though the expression levels of their coding genes were slightly up-regulated. Although the mechanism by which Nna1 loss of function leads to PC neurodegeneration is undefined, the progressive accumulation of DNA damage in chromosome territories irreversibly compromises global gene transcription and seems to trigger PC degeneration and death.

Bone marrow contributes simultaneously to different neural types in the central nervous system through different mechanisms of plasticity.

Many studies have reported the contribution of bone marrow-derived cells (BMDC) to the CNS, raising the possibility of using them as a new source to repair damaged brain tissue or restore neuronal function. This process has mainly been investigated in the cerebellum, in which a degenerative microenvironment has been suggested to be responsible for its modulation. The present study further analyzes the contribution of BMDC to different neural types in other adult brain areas, under both physiological and neurodegenerative conditions, together with the mechanisms of plasticity involved. We grafted genetically marked green fluorescent protein/Cre bone marrow in irradiated recipients: a) the PCD (Purkinje Cell Degeneration) mutant mice, suffering a degeneration of specific neuronal populations at different ages, and b) their corresponding healthy controls. These mice carried the conditional lacZ reporter gene to allow the identification of cell fusion events. Our results demonstrate that BMDC mainly generate microglial cells, although to a lesser extent a clear formation of neuronal types also exists. This neuronal recruitment was not increased by the neurodegenerative processes occurring in PCD mice, where BMDC did not contribute to rescuing the degenerated neuronal populations either. However, an increase in the number of bone marrow-derived microglia was found along the life span in both experimental groups. Six weeks after transplantation more bone marrow-derived microglial cells were observed in the olfactory bulb of the PCD mice compared to the control animals, where the degeneration of mitral cells was in process. In contrast, this difference was not observed in the cerebellum, where Purkinje cell degeneration had been completed. These findings demonstrated that the degree of neurodegenerative environment can foster the recruitment of neural elements derived from bone marrow, but also provide the first evidence that BMDC can contribute simultaneously to different encephalic areas through different mechanisms of plasticity: cell fusion for Purkinje cells and differentiation for olfactory bulb interneurons.

Nucleolar disruption and cajal body disassembly are nuclear hallmarks of DNA damage-induced neurodegeneration in purkinje cells.

The Purkinje cell (PC) degeneration (pcd) phenotype results from mutation in nna1 gene and is associated with the degeneration and death of PCs during the postnatal life. Although the pcd mutation is a model of the ataxic mouse, it shares clinical and pathological characteristics of inherited human spinocerebellar ataxias. PC degeneration in pcd mice provides a useful neuronal system to study nuclear mechanisms involved in DNA damage-dependent neurodegeneration, particularly the contribution of nucleoli and Cajal bodies (CBs). Both nuclear structures are engaged in housekeeping functions for neuronal survival, the biogenesis of ribosomes and the maturation of snRNPs and snoRNPs required for pre-mRNA and pre-rRNA processing, respectively. In this study, we use ultrastructural analysis, in situ transcription assay and molecular markers for DNA damage, nucleoli and CB components to demonstrate that PC degeneration involves the progressive accumulation of nuclear DNA damage associated with disruption of nucleoli and CBs, disassembly of polyribosomes into monoribosomes, ribophagy and shut down of nucleolar and extranucleolar transcription. Microarray analysis reveals that four genes encoding repressors of nucleolar rRNA synthesis (p53, Rb, PTEN and SNF2) are upregulated in the cerebellum of pcd mice. Collectively, these data support that nucleolar and CB alterations are hallmarks of DNA damage-induced neurodegeneration.

Types of cholecystokinin-containing periglomerular cells in the mouse olfactory bulb.

The periglomerular cells (PG) of the olfactory bulb (OB) are involved in the primary processing and the refinement of sensory information from the olfactory epithelium. The neurochemical composition of these neurons has been studied in depth in many species, and over the last decades such studies have focused mainly on the rat. The increasing use of genetic models for research into olfactory function demands a profound characterization of the mouse olfactory bulb, including the chemical composition of bulbar interneurons. Regarding both their connectivity with the olfactory nerve and their neurochemical fate, recently, two different types of PG have been identified in the mouse. In the present report, we analyze both the synaptology and the chemical composition of specific PG populations in the murine olfactory bulb, in particular, those containing the neuropeptide cholecystokinin. Our results demonstrate the existence in the mouse of non-GABAergic PG and that these establish synaptic contacts with the olfactory nerve within the glomeruli. Based on previous classifications, we propose that this population would constitute a new subtype of type 1 mouse PG. In addition, we demonstrate the partial coexistence of cholecystokinin with the calcium-binding proteins neurocalcin and parvalbumin. All these findings add further data to our knowledge of the synaptology and neurochemistry of mouse PG. The differences observed from other rodents reflect the neurochemical heterogeneity of PG in the mammalian OB.