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•Reduced cholesterol biosynthesis in the brain is an early dysfunction in Huntington's disease.•Liquid chromatography-mass spectrometry is a powerful technique for cholesterol metabolites quantitation in biological samples.•Validation of analytical methods regarding sample preparation and instrumental analysis is critical for metabolic studies.•Identifying reproducible and reliable changes in cholesterol metabolism in Huntington's disease has important implications for developing new biomarkers and new therapies.

Huntington’s disease is caused by a pathologically long (>35) CAG repeat located in the first exon of the Huntingtin gene (HTT). While pathologically expanded CAG repeats are the focus of extensive investigations, non-pathogenic CAG tracts in protein-coding genes are less well characterized. Here, we investigated the function and evolution of the physiological CAG tract in the HTTgene. We show that the poly-glutamine (polyQ) tract encoded by CAGs in the huntingtin protein (HTT) is under purifying selection and subjected to stronger selective pressures than CAG-encoded polyQ tracts in other proteins. For natural selection to operate, the polyQ must perform a function. By combining genome-edited mouse embryonic stem cells and cell assays, we show that small variations in HTT polyQ lengths significantly correlate with cells’ neurogenic potential and with changes in the gene transcription network governing neuronal function. We conclude that during evolution natural selection promotes the conservation and purity of the CAG-encoded polyQ tract and that small increases in its physiological length influence neural functions of HTT. We propose that these changes in HTT polyQ length contribute to evolutionary fitness including potentially to the development of a more complex nervous system.

Chronic lymphocytic leukemia (CLL) is caused by the progressive accumulation of mature CD5+ B cells in secondary lymphoid organs. In vitro data suggest that CD4+ T lymphocytes also sustain survival and proliferation of CLL clones through CD40L/CD40 interactions. In vivo data in animal models are conflicting. To clarify this clinically relevant biological issue, we generated genetically modified Eμ-TCL1 mice lacking CD4+ T cells (TCL1+/+AB0), CD40 (TCL1+/+CD40−/−), or CD8+ T cells (TCL1+/+TAP−/−), and we monitored the appearance and progression of a disease that mimics aggressive human CLL by flow cytometry and immunohistochemical analyses. Findings were confirmed by adoptive transfer of leukemic cells into mice lacking CD4+ T cells or CD40L or mice treated with antibodies depleting CD4 T cells or blocking CD40L/CD40 interactions. CLL clones did not proliferate in mice lacking or depleted of CD4+ T cells, thus confirming that CD4+ T cells are essential for CLL development. By contrast, CD8+ T cells exerted an antitumor activity, as indicated by the accelerated disease progression in TCL1+/+TAP−/− mice. Antigen specificity of CD4+ T cells was marginal for CLL development, because CLL clones efficiently proliferated in transgenic mice whose CD4 T cells had a T-cell receptor with CLL-unrelated specificities. Leukemic clones also proliferated when transferred into wild-type mice treated with monoclonal antibodies blocking CD40 or into CD40L−/− mice, and TCL1+/+CD40−/− mice developed frank CLL. Our data demonstrate that CD8+ T cells restrain CLL progression, whereas CD4+ T cells support the growth of leukemic clones in TCL1 mice through CD40-independent and apparently noncognate mechanisms.

Chronic lymphocytic leukemia (CLL) is caused by the progressive accumulation of mature CD5+B cells in secondary lymphoid organs. In vitro data suggest that CD4+T lymphocytes also sustain survival and proliferation of CLL clones through CD40L/CD40 interactions. In vivo data in animal models are conflicting. To clarify this clinically relevant biological issue, we generated genetically modified Eμ-TCL1 mice lacking CD4+T cells (TCL1+/+AB0), CD40 (TCL1+/+CD40−/−), or CD8+T cells (TCL1+/+TAP−/−), and we monitored the appearance and progression of a disease that mimics aggressive human CLL by flow cytometry and immunohistochemical analyses. Findings were confirmed by adoptive transfer of leukemic cells into mice lacking CD4+T cells or CD40L or mice treated with antibodies depleting CD4 T cells or blocking CD40L/CD40 interactions. CLL clones did not proliferate in mice lacking or depleted of CD4+T cells, thus confirming that CD4+T cells are essential for CLL development. By contrast, CD8+T cells exerted an antitumor activity, as indicated by the accelerated disease progression in TCL1+/+TAP−/−mice. Antigen specificity of CD4+T cells was marginal for CLL development, because CLL clones efficiently proliferated in transgenic mice whose CD4 T cells had a T-cell receptor with CLL-unrelated specificities. Leukemic clones also proliferated when transferred into wild-type mice treated with monoclonal antibodies blocking CD40 or into CD40L−/−mice, and TCL1+/+CD40−/−mice developed frank CLL. Our data demonstrate that CD8+T cells restrain CLL progression, whereas CD4+T cells support the growth of leukemic clones in TCL1 mice through CD40-independent and apparently noncognate mechanisms.

Outer radial glia (oRG) emerge as cortical progenitor cells that support the development of an enlarged outer subventricular zone (oSVZ) and the expansion of the neocortex. The in vitrogeneration of oRG is essential to investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 signaling pathway using LIF, which is not expressed in guided cortical organoids, we define a cortical organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The oSVZ comprises progenitor cells expressing specific oRG markers such as GFAP, LIFR,and HOPX,closely matching human fetal oRG. Finally, incorporating neural crest-derived, LIF-producing cortical pericytes into cortical organoids recapitulates the effects of LIF treatment. These data indicate that increasing the cellular complexity of the organoid microenvironment promotes the emergence of oRG and supports a platform to study oRG in hPSC-derived brain organoids routinely.

Human induced pluripotent stem cells (hiPSCs) were first generated in 2007, but the full translational potential of this valuable tool has yet to be realized. The potential applications of hiPSCs are especially relevant to neurology, as brain cells from patients are rarely available for research. hiPSCs from individuals with neuropsychiatric or neurodegenerative diseases have facilitated biological and multi-omics studies as well as large-scale screening of chemical libraries. However, researchers are struggling to improve the scalability, reproducibility and quality of this descriptive disease modelling. Addressing these limitations will be the first step towards a new era in hiPSC research — that of predictive disease modelling — involving the correlation and integration of in vitro experimental data with longitudinal clinical data. This approach is a key element of the emerging precision medicine paradigm, in which hiPSCs could become a powerful diagnostic and prognostic tool. Here, we consider the steps necessary to achieve predictive modelling of neurodegenerative disease with hiPSCs, using Huntington disease as an example.

Stem cell engineering of striatal medium spiny neurons (MSNs) is a promising strategy to understand diseases affecting the striatum and for cell-replacement therapies in different neurological diseases. Protocols to generate cells from human pluripotent stem cells (PSCs) are scarce and how well they recapitulate the endogenous fetal cells remains poorly understood. We have developed a protocol that modulates cell seeding density and exposure to specific morphogens that generates authentic and functional D1- and D2-MSNs with a high degree of reproducibility in 25 days of differentiation. Single-cell RNA sequencing (scRNA-seq) shows that our cells can mimic the cell-fate acquisition steps observed in vivoin terms of cell type composition, gene expression, and signaling pathways. Finally, by modulating the midkine pathway we show that we can increase the yield of MSNs. We expect that this protocol will help decode pathogenesis factors in striatal diseases and eventually facilitate cell-replacement therapies for Huntington’s disease (HD).

Decreased expression of neuronal genes such as brain‐derived neurotrophic factor (BDNF) is associated with several neurological disorders. One molecular mechanism associated with Huntington disease (HD) is a discrete increase in the nuclear activity of the transcriptional repressor REST/NRSF binding to repressor element‐1 (RE1) sequences. High‐throughput screening of a library of 6,984 compounds with luciferase‐assay measuring REST activity in neural derivatives of human embryonic stem cells led to identify two benzoimidazole‐5‐carboxamide derivatives that inhibited REST silencing in a RE1‐dependent manner. The most potent compound, X5050, targeted REST degradation, but neither REST expression, RNA splicing nor binding to RE1 sequence. Differential transcriptomic analysis revealed the upregulation of neuronal genes targeted by REST in wild‐type neural cells treated with X5050. This activity was confirmed in neural cells produced from human induced pluripotent stem cells derived from a HD patient. Acute intraventricular delivery of X5050 increased the expressions of BDNF and several other REST‐regulated genes in the prefrontal cortex of mice with quinolinate‐induced striatal lesions. This study demonstrates that the use of pluripotent stem cell derivatives can represent a crucial step toward the identification of pharmacological compounds with therapeutic potential in neurological affections involving decreased expression of neuronal genes associated to increased REST activity, such as Huntington disease. StemCells2013;31:1816‐1828

Background: Some promising treatments for Huntington's disease (HD) may require pre-clinical testing in large animals. Minipig is a suitable species because of its large gyrencephalic brain and long lifespan. Objective: To generate HD transgenic (TgHD) minipigs encoding huntingtin (HTT)1–548 under the control of human HTT promoter. Methods: Transgenesis was achieved by lentiviral infection of porcine embryos. PCR assessment of gene transfer, observations of behavior, and postmortem biochemical and immunohistochemical studies were conducted. Results: One copy of the human HTT transgene encoding 124 glutamines integrated into chromosome 1 q24-q25 and successful germ line transmission occurred through successive generations (F0, F1, F2 and F3 generations). No developmental or gross motor deficits were noted up to 40 months of age. Mutant HTT mRNA and protein fragment were detected in brain and peripheral tissues. No aggregate formation in brain up to 16 months was seen by AGERA and filter retardation or by immunostaining. DARPP32 labeling in WT and TgHD minipig neostriatum was patchy. Analysis of 16 month old sibling pairs showed reduced intensity of DARPP32 immunoreactivity in neostriatal TgHD neurons compared to those of WT. Compared to WT, TgHD boars by one year had reduced fertility and fewer spermatozoa per ejaculate. In vitro analysis revealed a significant decline in the number of WT minipig oocytes penetrated by TgHD spermatozoa. Conclusions: The findings demonstrate successful establishment of a transgenic model of HD in minipig that should be valuable for testing long term safety of HD therapeutics. The emergence of HD-like phenotypes in the TgHD minipigs will require more study.

REST/NRSF is a transcription factor that represses transcription of several neuronal genes by binding to a DNA regulatory motif known as Repressor Element 1/Neuron-restrictive silencer element (RE1/NRSE). In Huntingtons Disease, an inherited degenerative disease affecting the brain, REST/NRSF enters pathologically into the nucleus of affected cells, leading to the activation of the RE1/NRSE sites and causing decreased transcription of several important neuronal genes. Following this discovery, an effort has begun by some of the authors aimed at identifying compounds capable of antagonizing REST/NRSF silencing activity. Here we will review the underlying basis for focusing pharmaceutical efforts on REST/NRSF-RE1/NRSE system as well as some of the strategies for a rational drug design approach. We will highlight approaches aimed at identifying or designing small molecules able to impact REST/NRSF nuclear translocation, its DNA binding or, more generally, the formation of the REST/NRSF transcriptional complex, in the attempt to restore neuronal gene transcription in pathological conditions of the brain.

REST/NRSF is a transcription factor that represses transcription of several neuronal genes by binding to a DNA regulatory motif known as Repressor Element 1/Neuron-restrictive silencer element (RE1/NRSE). In Huntington's Disease, an inherited degenerative disease affecting the brain, REST/NRSF enters pathologically into the nucleus of affected cells, leading to the activation of the RE1/NRSE sites and causing decreased transcription of several important neuronal genes. Following this discovery, an effort has begun by some of the authors aimed at identifying compounds capable of antagonizing REST/NRSF silencing activity. Here we will review the underlying basis for focusing pharmaceutical efforts on REST/NRSF-RE1/NRSE system as well as some of the strategies for a rational drug design approach. We will highlight approaches aimed at identifying or designing small molecules able to impact REST/NRSF nuclear translocation, its DNA binding or, more generally, the formation of the REST/NRSF transcriptional complex, in the attempt to restore neuronal gene transcription in pathological conditions of the brain.

Changes in the levels and activities of neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), have been described in a number of neurodegenerative disorders, including Huntington disease, Alzheimer disease and Parkinson disease. It is only in Huntington disease, however, that gain-of-function and loss-of-function experiments have linked BDNF mechanistically with the underlying genetic defect. Altogether, these studies have led to the development of experimental strategies aimed at increasing BDNF levels in the brains of animals that have been genetically altered to mimic the aforementioned human diseases, with a view to ultimately influencing the clinical treatment of these conditions. In this article, we will review the current knowledge about the involvement of BDNF in a number of neurodegenerative diseases, with particular emphasis on Huntington disease, and will provide the rationale for and discuss the problems in proposing BDNF treatment as a beneficial and feasible therapeutic approach in the clinic.

Neural stem cells are cells that can self-renew ad infinitumand have the potential to generate immature precursors and mature cells of both neuronal and glial lineages. During recent years, neural stem cells gained attention as major candidates for regenerative and cell replacement therapies in various pathological brain conditions; however, they have recently revealed unforeseen valuable characters. Here the authors review on the state of the field, with a particular focus on the most recent results on the optimisation of neural stem cells isolation/derivation and homogeneous long-term expansion, highlighting advantages of this resource and outlining their potential applications. Bearing in mind that stem-cell based therapies for the diseased and injured brain are still far from being a reality, these cells have been recently opened up to valuable exploitation for disease modelling, drug discovery and toxicology tests as short-term applications.

Abstract: Since their identification Neural Stem Cells (NSCs) have been considered an attractive subject for therapeutic applications to the damaged brain. However, toward such a goal and in spite of the excitement, a great deal of basic research is still needed since thein vitro properties of these cells remain poorly understood and theirin vivo engraftment capacity is of limited efficacy. NSCs derived from fetal and adult neural tissue are actually expanded as cell aggregates, named neurospheres, that grow in suspension. Neurospheres have been shown capable to differentiatein vitro to astrocytes and neurons, even after several passages but are, however, composed by a heterogeneous population of cells at different maturation stages. At the state of the art, an important requirement is for procedures that allow consistent and more uniform neuronal differentiation of the neurospheres cells after intracerebral transplantation. Outside the CNS, another potential source of NSC is the blastocyst, from which rodent and human ES cells have been derived. NSCs have been efficiently derived from ES cells and their potential has been tested bothin vitro andin vivo. Beside the promises offered by the ES cell-derived and adult NSCs, the data available demonstrate that NSCs implanted into the adult or developing brain generate mostly glial cells, suggesting that the control on the differentiative fate remains the main open question. In this context it will be important to understand the real impact of adult mammalian neurogenesis and to investigate more deeply how the environment may be exploited to favour the integration and the survival of cells in the diseased brain. The future development of strategies and the exploitation of target molecules able to improve the rate of neuronal, over glial, differentiation and survival by genetically engineering NSC represents the major challenge in the field. This approach coupled to a range of pharmacological approaches and therapeutic strategies to efficiently recruit endogenous or exogenous NSCs clearly represent one of the most important future challenges for basic and clinical research.

The histopathologic classification of breast cancer stratifies tumors based on tumor grade, stage, and type. Despite an overall correlation with survival, this classification is poorly predictive and tumors with identical grade and stage can have markedly contrasting outcomes. Recently, breast carcinomas have been classified by their gene expression profiles on frozen material. The validation of such a classification on formalin-fixed paraffin-embedded tumor archives linked to clinical information in a high-throughput fashion would have a major impact on clinical practice. The authors tested the ability of tumor tissue microarrays (TMAs) to sub-classify breast cancers using a TMA containing 107 breast cancers. The pattern of expression of 13 different protein biomarkers was assessed by immunohistochemistry and the multidimensional data was analyzed using an unsupervised two-dimensional clustering algorithm. This revealed distinct tumor clusters which divided into two main groups correlating with tumor grade (P<0.001) and nodal status (P0.04). None of the protein biomarkers tested could individually identify these groups. The biological significance of this classification is supported by its similarity with one derived from gene expression microarray analysis. Thus, molecular profiling of breast cancer using a limited number of protein biomarkers in TMAs can sub-classify tumors into clinically and biologically relevant subgroups.

Huntingtin is the protein involved in Huntington disease (HD), an inherited neurodegenerative disease. Research activities have focused on the abnormal functions of mutant huntingtin. However, recent results indicate that wild-type huntingtin has important activities in brain neurons, suggesting that loss of these activities may play a role in HD.

Huntington's disease (HD) is caused by a polyglutamine expansion in the amino-terminal region of huntingtin. Mutant huntingtin is proteolytically cleaved by caspases, generating amino-terminal aggregates that are toxic for cells. The addition of calpains to total brain homogenates also leads to cleavage of wild-type huntingtin, indicating that proteolysis of mutant and wild-type huntingtin may play a role in HD. Here we report that endogenous wild-type huntingtin is promptly cleaved by calpains in primary neurons. Exposure of primary neurons to glutamate or 3-nitropropionic acid increases intracellular calcium concentration, leading to loss of intact full-length wild-type huntingtin. This cleavage could be prevented by calcium chelators and calpain inhibitors. Degradation of wild-type huntingtin by calcium-dependent proteases thus occurs in HD neurons, leading to loss of wild-type huntingtin neuroprotective activity.

The p75 neurotrophin receptor (p75NTR) belongs to the tumor necrosis factor receptor/nerve growth factor receptor superfamily. In some cells derived from neuronal tissues it causes cell death through a poorly characterized pathway. We developed a neuronal system using conditionally immortalized striatal neurons, in which the expression of p75NTRis inducibly controlled by the ecdysone receptor. In these cells p75NTRinduces apoptosis through its death domain in a nerve growth factor-independent manner. Caspases 9, 6, and 3 are activated by receptor expression indicating the activation of the common effector pathway of apoptosis. Cell death is blocked by a dominant negative form of caspase 9 and Bcl-XLconsistent with a pathway that involves mitochondria. Significantly, the viral flice inhibitory protein E8 protects from p75NTR-induced cell death indicating that death effector domains are involved. A p75NTRconstruct with a deleted death domain dominantly interferes with p75NTRsignaling, implying that receptor multimerization is required. However, in contrast to the other receptors of the family, p75NTR-mediated apoptosis does not involve the adaptor proteins Fas-associated death domain protein or tumor necrosis factor-associated death domain protein, and the apical caspase 8 is not activated. We conclude that p75NTRsignals apoptosis by similar mechanisms as other death receptors but uses different adaptors and apical caspases.

The ST14A cell line was previously derived from embryonic day 14 rat striatal primordia by retroviral transduction of the temperature-sensitive SV40 large T antigen. We showed that cell division and expression of nestin persists at 33°C, the permissive temperature, whereas cell division ceases, nestin expression decreases, and MAP2 expression increases at the nonpermissive temperature of 39°C. In this study, we further characterized the cells and found that they express other general and subtype-specific neuronal characteristics. ST14A cells express enolase and βIII-tubulin. Furthermore, they express the striatal marker DARPP-32, which is up-regulated upon differentiation of the cells by growth in serum-free medium. Stimulation with dopamine, the D2-dopamine receptor agonist quinpirole, or the D1-dopamine receptor agonist SKF82958 results in phosphorylation of CREB. Treatment of the cells with a mixture of reagents which stimulate the MAPK and adenylyl cyclase pathways radically changes the morphology of the ST14A cells. The cells develop numerous neurite-like appearing processes which stain with βIII-tubulin. Moreover, under these conditions, intracellular injection of rectangular depolarizing current stimuli elicits overshooting action potentials with a relatively fast depolarization rate when starting from a strongly hyperpolarized membrane potential. Taken together, these data imply that the ST14A cell line displays some of the characteristics of a medium-size spiny neuron subtype and provides a new tool to elucidate the pathways and molecules involved in medium-size spiny neuron differentiation and disease.

Ciliary neurotrophic factor (CNTF) acts on immature astrocytes that express its trimeric receptor. In contrast, mature astrocytes do not significantly express the specific CNTFα receptor subunit, yet they respond to CNTF administration in vivo.Here we show that this controversy may be solved by a shift in astroglial sensitivity to CNTF over time, related to a change in the type of receptor bound by the cytokine on mature astrocytes. A convergent set of results supports the hypothesis that the CNTF effect is due to the illegitimate binding on the leukemia inhibitory factor receptor (LIFR): (i) it requires high concentration of recombinant rat CNTF; (ii) it involves the Jak/Stat and Ras-MAPK pathways; (iii) it is preserved in CNTFRα−/− cells; (iv) it is potentiated by soluble CNTFRα added to the medium; and (v) it is significantly decreased by a partial antagonist of LIFR. On these bases, we propose a mechanistic model in which, in the adult brain, a CNTF/LIFR interglial system may be modulated by neurons that synthesize CNTFRα.

Huntington's Disease is an inherited neurodegenerative disease that affects the medium spiny neurons in the striatum. The disease is caused by the expansion of a polyglutamine sequence in the N terminus of Huntingtin (Htt), a widely expressed protein. Recently, we have found that Htt is an antiapoptotic protein in striatal cells and acts by preventing caspase-3 activity. Here we report that Htt overexpression in other CNS-derived cells can protect them from more than 20 days exposure to fatal stimuli. In particular, we found that cytochrome ccontinues to be released from mitochondria into the cytosol of cells that overexpress normal Htt. However, procaspase-9 is not processed, indicating that wild-type Htt (wtHtt) acts downstream of cytochrome crelease. These data show that Htt inhibits neuronal cell death by interfering with the activity of the apoptosome complex.

Activation of the Janus kinases (JAK) and signal transducers and activator of transcription (STAT) proteins in response to specific cytokines and growth factors has been investigated primarily in cells of non-neuronal origin. More recently, the JAKs and the STATs have also been found to be active in the developing and mature brain, providing evidence for important roles played by these molecules in the control of neuronal proliferation, survival and differentiation. Nothing, however, is known about their occurrence and role(s) in the aged brain. We, therefore, investigated the presence of Stat3 and Stat1 in aged-rat brain, and have found that the Stat3 protein was markedly down regulated with respect to adult tissue, while Stat1 remained invariant. We also investigated the potential role of some growth factors in the activation of the JAK/STAT in mature neurons, exposing primary neuronal cells to ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Besides CNTF, which is known to recruit Stat3, we found that Stat3 was also tyrosine phosphorylated by bFGF. These data are indicative of an important role of Stat3 and Stat1 in regulating the physiological status of mature neurons.

Huntington's disease is a neurodegenerative disorder caused by CAG expansion that results in expansion of a polyglutamine tract at the extreme N terminus of huntingtin (htt). htt with polyglutamine expansion is proapoptotic in different cell types. Here, we show that caspase inhibitors diminish the toxicity of htt. Additionally, we define htt itself as an important caspase substrate by generating a site-directed htt mutant that is resistant to caspase-3 cleavage at positions 513 and 530 and to caspase-6 cleavage at position 586. In contrast to cleavable htt, caspase-resistant htt with an expanded polyglutamine tract has reduced toxicity in apoptotically stressed neuronal and nonneuronal cells and forms aggregates at a much reduced frequency. These results suggest that inhibiting caspase cleavage of htt may therefore be of potential therapeutic benefit in Huntington's disease.

Transplantation of immature CNS-derived cells into the developing brain is a powerful approach to investigate the factors that regulate neuronal position and phenotype. CNS progenitor cells dissociated from the embryonic striatum and implanted into the brain of embryos of the same species generate cells that reaggregate to form easily recognizable structures that we previously called clusters and cells that disperse and integrate as single cells into the host brain. We sought to determine if the neurons in the clusters differentiate according to their final location or acquire a striatal phenotype in heterotopic positions. We transplanted dissociated cells from the E14 rat medial and lateral ganglionic eminences, either combined or in isolation, into the E16 embryonic rat brain. At all time points, we found clusters of BrdU- and DiI-labelled donor cells located in the forebrain and hindbrain, without any apparent preference for striatum. Immunocytochemical analyses revealed that cells in the clusters expressed DARPP-32 and ARPP-21, two antigens typically co-expressed in striatal medium-sized spiny neurons. In agreement with observations previously noted by several groups, isolated cells integrated into heterologous host areas do not express basal ganglia phenotypes. These data imply that immature striatal neuronal progenitors exert a community effect on each other that is permissive and/or instructive for development of a striatal phenotype in heterotopic locations.

TIGHT control of the activity of a therapeutic gene introduced in vivois a major issue in gene therapy research. Appropriate levels of expression may be crucial for gene correction. The tetracycline-sensitive regulatory system is highly effective for transcriptional regulation of foreign genes in mammalian cells. Here we report tight tetracycline-dependent regulation of a luciferase reporter gene transferred into the rat brain in the genetically modified neural precursor cell line ST14A as early as 2 days and until at least 6 days after transplantation. This is the first demonstration of the potential of this regulatory system for the modulation of the expression of therapeutic genes introduced into the central nervous system.

Thein vivoproperties of four different neural stem cell lines, generated from embryonic striatum or hippocampus by immortalization with the temperature-sensitive (s) A58/U19 allele of the SV40 Large T-antigen, have been studied with respect to their ability to survive, differentiate, and integrate after transplantation to the adult rat striatum. The cells were labeled with [3H]thymidine prior to grafting, and combined autoradiography and immunohistochemistry was used to characterize their phenotypic differentiation within the adult brain environment. The results show that all four types of cells survived well, up to at least 1.5–6 months postgrafting, without any signs of tissue perturbation or tumor formation. The cells underwent, on average, 2–3 cell divisions during the first 5 days after implantation and exhibited extensive migration over a distance of 1–1.5 mm from the injection site to become morphologically integrated with the surrounding host striatum. The cell number and tissue distribution attained by 2 weeks remained stable for up to 6 months postgrafting with the exception of one cell line, which showed a 40% loss of cells between 2 and 6 weeks. Twice the number of [3H]thymidine-labeled cells were recovered when the cells were grafted into a 1-week-old excitotoxic striatal lesion, probably due to an increased proliferation of the cells in response to the neuron-depleting depleting lesion. The immortalized cells behaved as multipotent neural progenitors. The vast majority of the cells developed a glial-like morphology, 6–14% being clearly GFAP-positive; however, a small but consistent proportion of them (1–3%) expressed MAP-2 and exhibited neuron-like morphology. In mature transplants about 75–80% of the grafted cells were located in the striatal grey matter, and 10–15% in white matter, some of which are proposed to have differentiated into oligodendrocytes. Remaining 5–10% occurred around small blood vessels (resembling pericytes) and in the subventricular zone underneath the ependyma of the lateral ventricle. It is concluded that thetscell lines are highly suitable for intracerebral transplantation and that they allow the creation of a regionally confined cellular chimeras where the graft-derived glial cells become stably integrated with the resident glial cell matrix.

ABSTRACTThe growth of U-87 or C6 gliomas co-implanted in nude mice with retroviral producer cells (VPC) expressing the herpes simplex virus-thymidine kinase (HSV-tk) gene is only partially impaired by treatment with ganciclovir (GCV). The effect of GCV is even less evident when C6 and VPC are co-implanted into the rat brain. Furthermore, tumors from C6 cells carrying the HSV-tk gene are not eradicated by GCV, although they remain sensitive to GCV when replated in vitro. These limits of the HSV-tk/GCV system in glioma gene therapy may be due to insufficient gene transfer and/or insufficient delivery of GCV to glioma cells. Combination of HSV-tk and one or more cytokines may improve the antitumor efficacy. Among cytokines, interleukin-4 (IL-4) has already been shown to be active against gliomas. In nude mice, GCV treatment inhibited tumor growth more effectively after co-injection of C6 cells with a mixture of VPC transducing IL-4 and HSV-tk genes than after co-injection with either IL-4 or HSV-tk VPC only. In immunocompetent Sprague-Dawley rats, co-injection of IL-4 VPC and C6 cells was also effective in inhibiting the growth of C6 brain tumors, 38% of the animals surviving for at least 2 months. Furthermore, increased and prolonged antitumor efficacy was obtained by transducing both IL-4 and HSV-tk genes.

Ghia: AbbVie: Consultancy, Honoraria, Research Funding; Acerta/AstraZeneca: Consultancy, Honoraria; ArQule: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria; Dynamo: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Juno/Celgene: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Novartis: Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy.

In Chronic Lymphocytic Leukemia (CLL), mature CD5+B cells accumulate in lymphoid organs such as bone marrow and lymph nodes where they proliferate and expand within localized proliferation centers. In vitroand in vivodata suggest that survival and proliferation of CLL cells within proliferation centers may be also dependent on microenvironmental interactions originating from the surrounding cellular elements (e.g. monocyte-derived nurse-like cells, mesenchymal stromal cells, or CD4+T lymphocytes), that deliver both membrane-bound and soluble signals to CLL cells. In particular, the role of CD4+T cells in vivois less defined and data in animal models are conflicting as they appear to sustain CLL clone expansion and survival through CD40L-CD40 interactions, though in approximately 40% of patients with CLL, aggressive leukemic clones appear to be independent of CD40 stimulation.

Values for normative REM sleep without atonia (RSWA) remain unclear. Older age and male sex are associated with greater RSWA, and isolated elevated RSWA has been reported. We aimed to describe normative RSWA and characterize isolated RSWA frequency in adults without REM sleep behavior disorder (RBD).

Huntington's disease (HD) is a neurodegenerative disorder caused by expansion of a CAG repeat in the gene encoding for Huntingtin (Htt), which results in progressive degeneration of the striatal GABAergic/enkephalin neurons. These neurons express both the A2Aand D2receptors, which stimulate and inhibit adenylyl cyclase, respectively. In this study we analyzed the possibility of an involvement of the A2Areceptor and its signaling components in the pathogenesis of HD. We report here that striatal cells expressing mutant Htt exhibit increased binding affinities for the selective A2Areceptor ligand 3H‐SCH‐58261. Furthermore, despite identical basal adenylyl cyclase activity in all cells, forskolin, a direct activator of this enzyme, significantly overstimulated cAMP production in mutant Htt cells with respect to parental or wild‐type Htt‐expressing cells. Michaelis‐Menten analysis of forskolin‐stimulated enzyme activity revealed a specific decrease of Km value in mutant Htt cells, indicating increased sensitivity for the substrate. Remarkably, coupling of the A2Areceptor to adenylyl cyclase was also aberrantly increased. Nevertheless, in all clones, stimulation of cAMP production by 10−7M NECA was fully counteracted by selective A2Areceptor antagonists. Altogether, these data suggest that expression of mutant Htt induces an amplification of adenylyl cyclase‐transduced signals and an aberrant coupling of the A2Areceptor to this transduction system. Given the involvement of adenylyl cyclase in key physiological functions, including cell growth and cell survival, we speculate that these changes may alter the susceptibility of striatal neurons to cell death and may contribute to the development of HD.

While multiple myeloma (MM) is almost invariably preceded by asymptomatic monoclonal gammopathy of undetermined significance (MGUS) and/or smoldering MM (SMM), the alterations of the bone marrow (BM) microenvironment that establish progression to symptomatic disease are circumstantial. Here we show that in Vk*MYC mice harboring oncogene-driven plasma cell proliferative disorder, disease appearance associated with substantial modifications of the BM microenvironment, including a progressive accumulation of both CD8+and CD4+T cells with a dominant T helper type 1 (Th1) response. Progression from asymptomatic to symptomatic MM was characterized by further BM accrual of T cells with reduced Th1 and persistently increased Th2 cytokine production, which associated with accumulation of CD206+Tie2+macrophages, and increased pro-angiogenic cytokines and microvessel density (MVD). Notably, MVD was also increased at diagnosis in the BM of MGUS and SMM patients that subsequently progressed to MM when compared with MGUS and SMM that remained quiescent. These findings suggest a multistep pathogenic process in MM, in which the immune system may contribute to angiogenesis and disease progression. They also suggest initiating a large multicenter study to investigate MVD in asymptomatic patients as prognostic factor for the progression and outcome of this disease.

No relevant conflicts of interest to declare.

While multiple myeloma (MM) is almost invariably preceded by asymptomatic monoclonal gammopathy of undetermined significance (MGUS) and/or smoldering MM (SMM), the alterations of the bone marrow (BM) microenvironment that establish progression to symptomatic disease are circumstantial. Our aim was to identify changes in the BM microenvironment eliciting angiogenesis in MM.

We describe a novel strategy for mRNA normalization in quantitative real-time PCR that is based on expressed Alu repeat amplification as a measure for the mRNA fraction. We show that expressed Alu repeat amplification is a fast, accurate normalization tool that can be successfully used for quantification of selected mRNA in the human transcriptome. This result is particularly important for clinical diagnosis and biomarker validation studies based on mRNA detection in human blood.