The mGluR5 positive allosteric modulator, CDPPB, ameliorates pathology and phenotypic signs of a mouse model of Huntington's disease.

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by a polyglutamine expansion in the amino-terminal region of the huntingtin protein (htt), leading to motor dysfunction, cognitive decline, psychiatric alterations, and death. The metabotropic glutamate receptor 5 (mGluR5) has been implicated in HD and we have recently demonstrated that mGluR5 positive allosteric modulators (PAMs) are neuroprotective in vitro. In the present study we demonstrate that the mGluR5 PAM, CDPPB, is a potent neuroprotective drug, in vitro and in vivo, capable of delaying HD-related symptoms. The HD mouse model, BACHD, exhibits many HD features, including neuronal cell loss, htt aggregates, motor incoordination and memory impairment. However, chronic treatment of BACHD mice with CDPPB 1.5 mg/kg s.c. for 18 weeks increased the activation of cell signaling pathways important for neuronal survival, including increased AKT and ERK1/2 phosphorylation and augmented the BDNF mRNA expression. CDPPB chronic treatment was also able to prevent the neuronal cell loss that takes place in the striatum of BACHD mice and decrease htt aggregate formation. Moreover, CDPPB chronic treatment was efficient to partially ameliorate motor incoordination and to rescue the memory deficit exhibited by BACHD mice. Importantly, no toxic effects or stereotypical behavior were observed upon CDPPB chronic treatment. Thus, CDPPB is a potential drug to treat HD, preventing neuronal cell loss and htt aggregate formation and delaying HD symptoms.

Gliotoxicity, reverse transcriptase activity and retroviral RNA in monocyte/macrophage culture supernatants from patients with multiple sclerosis.

In investigating a possible link between a novel retroviral agent (provisionally called MSRV), recently characterised in multiple sclerosis (MS), and the neuropathology of MS, it was found that there was a significant correlation between gliotoxicity and reverse transcriptase activity in monocyte/macrophage culture supernatants (MMCS) unique to MS patients. MMCS from healthy controls and patients with other neurological diseases did not display either gliotoxicity or reverse transcriptase activity. The observed gliotoxic effect was an initial, intermediate filament network disorganization and subsequent cell death which was specific to astrocytes and oligodendrocytes. The reverse transcriptase activity and MSRV-specific RNA were observed during the first 2 weeks of culture in MMCS from patients with active MS. The further elucidation of the molecular form(s) of this gliotoxic factor and its original source may be crucial in elucidating important etiopathogenic mechanisms in MS.

A gliotoxic factor and multiple sclerosis.

The pathogenesis of multiple sclerosis (MS) is unknown. Searching for possible toxic factors, it was found that 3-day exposure to heat-treated cerebrospinal fluid (CSF) from MS patients caused apoptotic death of astrocytes and oligodendrocytes, but not fibroblasts, myoblasts, Schwann cells, endothelial cells and neurons, in vitro. CSFs from other inflammatory or non-inflammatory neurological diseases showed no toxicity. Exposure of these glial cells to partially purified MS CSF produced DNA fragmentation, apoptotic bodies, chromatin condensation, cell shrinkage, and changes in the levels of known cytokines. A cytotoxic factor, called gliotoxin, was characterized chromatographically as a stable 17-kDa glycoprotein. Since this protein is highly cytotoxic for astrocytes and oligodendrocytes, it may represent an initial pathogenic factor, leading to the neuropathological features of MS, such as blood-brain barrier involvement and demyelination.

Metabotropic glutamate receptor 5 positive allosteric modulators are neuroprotective in a mouse model of Huntington's disease.

BACKGROUND AND PURPOSE: Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin protein. We have previously demonstrated that the cell signalling of the metabotropic glutamate receptor 5 (mGluR5) is altered in a mouse model of HD. Although mGluR5-dependent protective pathways are more activated in HD neurons, intracellular Ca²âº release is also more pronounced, which could contribute to excitotoxicity. In the present study, we aim to investigate whether mGluR5 positive allosteric modulators (PAMs) could activate protective pathways without triggering high levels of Ca²âº release and be neuroprotective in HD. EXPERIMENTAL APPROACH: We performed a neuronal cell death assay to determine which drugs are neuroprotective, Western blot and Ca²âº release experiments to investigate the molecular mechanisms involved in this neuroprotection, and object recognition task to determine whether the tested drugs could ameliorate HD memory deficit. KEY RESULTS: We find that mGluR5 PAMs can protect striatal neurons from the excitotoxic neuronal cell death promoted by elevated concentrations of glutamate and NMDA. mGluR5 PAMs are capable of activating Akt without triggering increased intracellular Ca²âº concentration ([Ca²âº]i ); and Akt blockage leads to loss of PAM-mediated neuroprotection. Importantly, PAMs' potential as drugs that may be used to treat neurodegenerative diseases is highlighted by the neuroprotection exerted by mGluR5 PAMs on striatal neurons from a mouse model of HD, BACHD. Moreover, mGluR5 PAMs can activate neuroprotective pathways more robustly in BACHD mice and ameliorate HD memory deficit. CONCLUSIONS AND IMPLICATIONS: mGluR5 PAMs are potential drugs that may be used to treat neurodegenerative diseases, especially HD.

Multiple phosphorylation of stathmin. Identification of four sites phosphorylated in intact cells and in vitro by cyclic AMP-dependent protein kinase and p34cdc2.

Stathmin is a ubiquitous, highly conserved phosphoprotein which most likely acts as a relay integrating various intracellular pathways regulating cell proliferation, differentiation, and functions. At least 14 molecular forms of stathmin have been identified so far, which migrate as 2 unphosphorylated and 12 increasingly phosphorylated spots (M(r) = 19,000-23,000; pI = 6.2-5.6) on two-dimensional electrophoretic gels, and whose pattern may reflect the state of activation of cells. We found that stathmin could be phosphorylated in vitro by at least three different protein kinases: cAMP-dependent protein kinase, p34cdc2, and casein kinase II, cAMP-dependent protein kinase catalyzed the phosphorylation of stathmin on serines 16 (K-R-A-S) and 63 (R-R-K-S), whereas p34cdc2 induced phosphorylation on serines 25 (I-L-S-P-R) and 38 (P-L-S-P-P-K-K-K). Interestingly, phosphorylation by both kinases together yielded all of the phosphoforms of stathmin identified so far. Two-dimensional phosphopeptide analysis allowed us to demonstrate that the same four sites were exclusively found to be phosphorylated in vivo, in brain tissue as well as in control or nerve growth factor-stimulated PC12 cells. In this latter case, the major site phosphorylated in response to nerve growth factor being serine 25, it is likely that a kinase such as a mitogen-activated protein kinase, known to be activated by growth factors, might directly phosphorylate stathmin. The phosphopeptide map analysis allowed further identification of the specific combinations among the four sites whose phosphorylation is responsible for the characteristic two-dimensional polyacrylamide gel electrophoresis migration of the resulting stathmin forms both in vitro and in vivo and revealed the existence of likely structural interactions between the sites phosphorylated. In conclusion, our results show that phosphorylation of serines 16, 25, 38, and 63 accounts for all of the major functional stathmin forms observed in vivo. The present identification of these sites will foster a better understanding of some intracellular mechanisms involved in the diverse physiological regulation of the proliferation, differentiation, and functions of cells, including the role of stathmin in these processes as a relay integrating diverse signaling pathways.

Identification and molecular characterization of Unc-33-like phosphoprotein (Ulip), a putative mammalian homolog of the axonal guidance-associated unc-33 gene product.

The control of neuritic extension and guidance is critical for the development, maturation, and regeneration of functional neuronal circuits. We identified a neuronal 64-85 kDa phosphoprotein, the expression of which in mouse brain is regulated during development, reaching a peak at approximately 5 d postnatal, when maturation of neurons and synaptic connections is highly active. The amino acid sequence of the mouse protein deduced from its cloned cDNA reveals similarities with that of the neuritic outgrowth- and guidance-related product of the unc-33 gene in Caenorhabditis elegans. The regulation of its phosphorylation in response to nerve growth factor, as well as its localization in neurites and growth cones and at the neuromuscular junction, further indicates that Ulip (for Unc-33-like phosphoprotein) is not only a structural but likely is also a functional mammalian homolog of Unc-33, potentially involved in the control of neuritic outgrowth and axonal guidance.

Functional characterization of phosphorylation of 69-kDa human choline acetyltransferase at serine 440 by protein kinase C.

Choline acetyltransferase, the enzyme that synthesizes the transmitter acetylcholine in cholinergic neurons, is a substrate for protein kinase C. In the present study, we used mass spectrometry to identify serine 440 in recombinant human 69-kDa choline acetyltransferase as a protein kinase C phosphorylation site, and site-directed mutagenesis to determine that phosphorylation of this residue is involved in regulation of the enzyme's catalytic activity and binding to subcellular membranes. Incubation of HEK293 cells stably expressing wild-type 69-kDa choline acetyltransferase with the protein kinase C activator phorbol 12-myristate 13-acetate showed time- and dose-related increases in specific activity of the enzyme; in control and phorbol ester-treated cells, the enzyme was distributed predominantly in cytoplasm (about 88%) with the remainder (about 12%) bound to cellular membranes. Mutation of serine 440 to alanine resulted in localization of the enzyme entirely in cytoplasm, and this was unchanged by phorbol ester treatment. Furthermore, activation of mutant enzyme in phorbol ester-treated HEK293 cells was about 50% that observed for wild-type enzyme. Incubation of immunoaffinity purified wild-type and mutant choline acetyltransferase with protein kinase C under phosphorylating conditions led to incorporation of [(32)P]phosphate, with radiolabeling of mutant enzyme being about one-half that of wild-type, indicating that another residue is phosphorylated by protein kinase C. Acetylcholine synthesis in HEK293 cells expressing wild-type choline acetyltransferase, but not mutant enzyme, was increased by about 17% by phorbol ester treatment.

Expression of transfected stathmin cDNA reveals novel phosphorylated forms associated with developmental and functional cell regulation.

Stathmin is a ubiquitous, highly conserved phosphoprotein, which most likely acts as an intracellular relay integrating various transduction pathways triggered by extracellular signals. Two post-translational isoforms (alpha and beta) have been previously identified whose increasingly phosphorylated forms migrate as a set of isoelectric variant spots (molecular mass 19 kDa; pI 6.2-5.6) on two-dimensional electrophoretic gels. In parallel with the phosphorylation of these forms of stathmin, two sets of three proteins migrating with slightly higher apparent molecular masses (21 and 23 kDa respectively) also incorporated radioactive phosphate in response to cell regulation through various transduction pathways. These phosphoproteins, previously referred to as proteins '16' and '17', share several biochemical properties with stathmin and are recognized by antibodies directed to stathmin or to stathmin peptides. Furthermore, when rat stathmin cDNA was transfected into mouse myogenic C2 cells, it directed the expression of protein sets 16 and 17 together with the 19 kDa forms of stathmin, as detected with a species-specific anti-stathmin antiserum. Proteins 16 and 17 are thus novel phosphorylated derivatives of stathmin, encoded by the same cDNA as its previously identified 19 kDa forms. These results increase the known complexity and diversity of stathmin patterns, which may yield the molecular support for its proposed role as a relay integrating various signals which regulate the proliferation, differentiation and functions of cells during development and adult life.

Expression, purification and characterization of recombinant human choline acetyltransferase: phosphorylation of the enzyme regulates catalytic activity.

Choline acetyltransferase synthesizes acetylcholine in cholinergic neurons and, in humans, may be produced in 82- and 69-kDa forms. In this study, recombinant choline acetyltransferase from baculovirus and bacterial expression systems was used to identify protein isoforms by two-dimensional SDS/PAGE and as substrate for protein kinases. Whereas hexa-histidine-tagged 82- and 69-kDa enzymes did not resolve as individual isoforms on two-dimensional gels, separation of wild-type choline acetyltransferase expressed in insect cells revealed at least nine isoforms for the 69-kDa enzyme and at least six isoforms for the 82-kDa enzyme. Non-phosphorylated wild-type choline acetyltransferase expressed in Escherichia coli yielded six (69 kDa) and four isoforms (82 kDa) respectively. Immunofluorescent labelling of insect cells expressing enzyme showed differential subcellular localization with the 69-kDa enzyme localized adjacent to plasma membrane and the 82-kDa enzyme being cytoplasmic at 24 h. By 64 h, the 69-kDa form was in cytoplasm and the 82-kDa form was only present in nucleus. Studies in vitro showed that recombinant 69-kDa enzyme was a substrate for protein kinase C (PKC), casein kinase II (CK2) and alpha-calcium/calmodulin-dependent protein kinase II (alpha-CaM kinase), but not for cAMP-dependent protein kinase (PKA); phosphorylation by PKC and CK2 enhanced enzyme activity. The 82-kDa enzyme was a substrate for PKC and CK2 but not for PKA or alpha-CaM kinase, with only PKC yielding increased enzyme activity. Dephosphorylation of both forms of enzyme by alkaline phosphatase decreased enzymic activity. These studies are of functional significance as they report for the first time that phosphorylation enhances choline acetyltransferase catalytic activity.

Nuclear localization of the 82-kDa form of human choline acetyltransferase.

Choline acetyltransferase is the enzyme catalyzing synthesis of the neurotransmitter acetylcholine in cholinergic neurons. In human, transcripts encoding two forms of the enzyme with apparent molecular masses of 69 and 82 kDa are found in brain and spinal cord; the 82-kDa form differs from the 69-kDa enzyme only in terms of a 118-amino acid extension on its amino terminus. Using green fluorescent protein-tagged choline acetyltransferase, we show that the 82-kDa enzyme is targeted to nuclei of cells, whereas the 69-kDa protein is found in cytoplasm. Expression of site-directed and deletion mutants of the 82-kDa isoform reveals that the extended amino terminus contains a nuclear localization signal in the first nine amino acids which targets the protein to nucleus. This represents the first report of a neurotransmitter-synthesizing enzyme that is localized to the cell nucleus.

[Gliotoxic factor and multiple sclerosis]. / Un facteur gliotoxique et la sclérose en plaques.

Multiple sclerosis in a disease of the central nervous system characterized by perivascular and periventricular lesions of the myelin and immune cell infiltrates and increased permeability of the blood-brain barrier. We have found a cytotoxic factor of the cerebrospinal fluid (CSF) specific for multiple sclerosis patients which has 2 main characteristic effects in vitro on primary or immortalized astrocyte cultures: (1) disruption of the gliofilament network of the cells; and (2) apoptotic cell death induction. Moreover, in vivo, intraventricular injections of minute amounts of partially purified gliotoxic factor in adult rats have striking effects on both the morphology and general organization of astrocytes in the entire brain and the permeability characteristics of the blood brain barrier, which becomes leaky to immunoglobulins. These pathological effects are strongly similar to some of the neuropathological findings reported during the course of MS--They suggest an entirely new hypothesis to explain the active stage of the disease: the presence of a new factor of unknown extrinsic (viral) or intrinsic (cellular) origin, able to disorganize the glial cytoskeleton and glial cell differentiation. This factor is then able to provoke glial cell death. Such glial cell death may result in both demyelination and increased blood brain barrier permeability. Both in vitro and in vivo studies strongly support the idea that this gliotoxic factor plays a central role in the pathogenesis of MS, making its full identification a critical theme for MS research.

Regulation of tyrosine kinase activation and granule release through beta-arrestin by CXCRI.

Chemoattractant-stimulated granule release from neutrophils, basophils and eosinophils is critical for the innate immune response against infectious bacteria. Interleukin 8 (IL-8) activation of the chemokine receptor CXCRI was found to stimulate rapid formation of beta-arrestin complexes with Hck or c-Fgr. Formation of beta-arrestin-Hck complexes led to Hck activation and trafficking of the complexes to granule-rich regions. Granulocytes expressing a dominant-negative beta-arrestin-mutant did not release granules or activate tyrosine kinases after IL-8 stimulation. Thus, beta-arrestins regulate chemokine-induced granule exocytosis, indicating a broader role for beta-arrestins in the regulation of cellular functions than was previously suspected.