The anti-leprosy drug clofazimine reduces polyQ toxicity through activation of PPARγ.

BACKGROUND: PolyQ diseases are autosomal dominant neurodegenerative disorders caused by the expansion of CAG repeats. While of slow progression, these diseases are ultimately fatal and lack effective therapies. METHODS: A high-throughput chemical screen was conducted to identify drugs that lower the toxicity of a protein containing the first exon of Huntington's disease (HD) protein huntingtin (HTT) harbouring 94 glutamines (Htt-Q94). Candidate drugs were tested in a wide range of in vitro and in vivo models of polyQ toxicity. FINDINGS: The chemical screen identified the anti-leprosy drug clofazimine as a hit, which was subsequently validated in several in vitro models. Computational analyses of transcriptional signatures revealed that the effect of clofazimine was due to the stimulation of mitochondrial biogenesis by peroxisome proliferator-activated receptor gamma (PPARγ). In agreement with this, clofazimine rescued mitochondrial dysfunction triggered by Htt-Q94 expression. Importantly, clofazimine also limited polyQ toxicity in developing zebrafish and neuron-specific worm models of polyQ disease. INTERPRETATION: Our results support the potential of repurposing the antimicrobial drug clofazimine for the treatment of polyQ diseases. FUNDING: A full list of funding sources can be found in the acknowledgments section.

A new non-aggregative splicing isoform of human Tau is decreased in Alzheimer's disease.

Tauopathies, including Alzheimer's disease (AD) and frontotemporal lobar degeneration with Tau pathology (FTLD-tau), are a group of neurodegenerative disorders characterized by Tau hyperphosphorylation. Post-translational modifications of Tau such as phosphorylation and truncation have been demonstrated to be an essential step in the molecular pathogenesis of these tauopathies. In this work, we demonstrate the existence of a new, human-specific truncated form of Tau generated by intron 12 retention in human neuroblastoma cells and, to a higher extent, in human RNA brain samples, using qPCR and further confirming the results on a larger database of human RNA-seq samples. Diminished protein levels of this new Tau isoform are found by Westernblotting in Alzheimer's patients' brains (Braak I n = 3; Braak II n = 6, Braak III n = 3, Braak IV n = 1, and Braak V n = 10, Braak VI n = 8) with respect to non-demented control subjects (n = 9), suggesting that the lack of this truncated isoform may play an important role in the pathology. This new Tau isoform exhibits similar post-transcriptional modifications by phosphorylation and affinity for microtubule binding, but more interestingly, is less prone to aggregate than other Tau isoforms. Finally, we present evidence suggesting this new Tau isoform could be linked to the inhibition of GSK3ß, which would mediate intron 12 retention by modulating the serine/arginine rich splicing factor 2 (SRSF2). Our results show the existence of an important new isoform of Tau and suggest that further research on this less aggregation-prone Tau may help to develop future therapies for Alzheimer's disease and other tauopathies.

Huntington's disease-specific mis-splicing unveils key effector genes and altered splicing factors.

Correction of mis-splicing events is a growing therapeutic approach for neurological diseases such as spinal muscular atrophy or neuronal ceroid lipofuscinosis 7, which are caused by splicing-affecting mutations. Mis-spliced effector genes that do not harbour mutations are also good candidate therapeutic targets in diseases with more complex aetiologies such as cancer, autism, muscular dystrophies or neurodegenerative diseases. Next-generation RNA sequencing (RNA-seq) has boosted investigation of global mis-splicing in diseased tissue to identify such key pathogenic mis-spliced genes. Nevertheless, while analysis of tumour or dystrophic muscle biopsies can be informative on early stage pathogenic mis-splicing, for neurodegenerative diseases, these analyses are intrinsically hampered by neuronal loss and neuroinflammation in post-mortem brains. To infer splicing alterations relevant to Huntington's disease pathogenesis, here we performed intersect-RNA-seq analyses of human post-mortem striatal tissue and of an early symptomatic mouse model in which neuronal loss and gliosis are not yet present. Together with a human/mouse parallel motif scan analysis, this approach allowed us to identify the shared mis-splicing signature triggered by the Huntington's disease-causing mutation in both species and to infer upstream deregulated splicing factors. Moreover, we identified a plethora of downstream neurodegeneration-linked mis-spliced effector genes that-together with the deregulated splicing factors-become new possible therapeutic targets. In summary, here we report pathogenic global mis-splicing in Huntington's disease striatum captured by our new intersect-RNA-seq approach that can be readily applied to other neurodegenerative diseases for which bona fide animal models are available.

Pathogenic SREK1 decrease in Huntington's disease lowers TAF1 mimicking X-linked dystonia parkinsonism.

Huntington's disease and X-linked dystonia parkinsonism are two monogenic basal ganglia model diseases. Huntington's disease is caused by a polyglutamine-encoding CAG repeat expansion in the Huntingtin (HTT) gene leading to several toxic interactions of both the expanded CAG-containing mRNA and the polyglutamine-containing protein, while X-linked dystonia parkinsonism is caused by a retrotransposon insertion in the TAF1 gene, which decreases expression of this core scaffold of the basal transcription factor complex TFIID. SRSF6 is an RNA-binding protein of the serine and arginine-rich (SR) protein family that interacts with expanded CAG mRNA and is sequestered into the characteristic polyglutamine-containing inclusion bodies of Huntington's disease brains. Here we report decreased levels of the SRSF6 interactor and regulator SREK1-another SR protein involved in RNA processing-which includes TAF1 as one of its targets. This led us to hypothesize that Huntington's disease and X-linked dystonia parkinsonism pathogeneses converge in TAF1 alteration. We show that diminishing SRSF6 through RNA interference in human neuroblastoma cells leads to a decrease in SREK1 levels, which, in turn, suffices to cause diminished TAF1 levels. We also observed decreased SREK1 and TAF1 levels in striatum of Huntington's disease patients and transgenic model mice. We then generated mice with neuronal transgenic expression of SREK1 (TgSREK1 mice) that, interestingly, showed transcriptomic alterations complementary to those in Huntington's disease mice. Most importantly, by combining Huntington's disease and TgSREK1 mice we verify that SREK1 overexpression corrects TAF1 deficiency and attenuates striatal atrophy and motor phenotype of Huntington's disease mice. Our results therefore demonstrate that altered RNA processing upon SREK1 dysregulation plays a key role in Huntington's disease pathogenesis and pinpoint TAF1 as a likely general determinant of selective vulnerability of the striatum in multiple neurological disorders.

Pharmacokinetic evaluation of marbofloxacin after intravenous administration at different ages in llama crias, and pharmacokinetic/pharmacodynamic analysis by Monte Carlo simulation.

In llama crias (tekes), Escherichia coli and Staphylococcus aureus are major pathogens, and marbofloxacin could be a suitable choice. The objectives of this study were (a) to evaluate the serum pharmacokinetics of marbofloxacin (5 mg/kg) after intravenous administration in tekes and simulate a multidose regimen; (b) to emulate pharmacokinetic profiles after single dose and steady-state conditions by Monte Carlo simulation (c) to determine the MIC of regional strains of Escherichia coli and Staphylococcus aureus; (d) to perform a PK/PD analysis by Monte Carlo simulation. Pharmacokinetics of marbofloxacin was evaluated in six animals at 3, 10, 24, 50, and 80 days after birth. Marbofloxacin were determined by HPLC. A steady-state multi-dose simulation was carried out, and concentration-time profiles were generated by Monte Carlo simulation. MIC of marbofloxacin against regional E. coli and S. aureus strains were also determined. Finally, a PK/PD analysis was conducted by Monte Carlo simulation. After pharmacokinetic analysis, clearance showed a trend to increase (0.14 and 0.18 L kg-1  hr-1 ), and AUC (36.74 and 15.21 µg hr-1  ml-1 ) and Vss (3.06 and 3.37 L/kg) trended to decrease at 3 and 80 days-old, respectively, showing accumulation ~50% in animals with 3 days. All strains tested of E. coli (MIC90  = 0.06 µg/ml) and S. aureus (MIC90  = 0.25 µg/ml) were susceptible to marbofloxacin. PK/PD analysis suggests that the therapeutic regimen of marbofloxacin could be effective for infections caused by E. coli strains in animals between 3 and 80 days, with a CFR for Cmax /MIC > 10 of 100% and for AUC24 /MIC > 125 of 99.99%; and for infections produced by S. aureus in animals between 3 and 24 days old, with a CFR for Cmax /MIC > 10 of 93.08% and for AUC24 /MIC > 60 of 97.01%, but a higher dose should be used in older animals, because PK/PD endpoints were not met.

The neuroprotective transcription factor ATF5 is decreased and sequestered into polyglutamine inclusions in Huntington's disease.

Activating transcription factor-5 (ATF5) is a stress-response transcription factor induced upon different cell stressors like fasting, amino-acid limitation, cadmium or arsenite. ATF5 is also induced, and promotes transcription of anti-apoptotic target genes like MCL1, during the unfolded protein response (UPR) triggered by endoplasmic reticulum stress. In the brain, high ATF5 levels are found in gliomas and also in neural progenitor cells, which need to decrease their ATF5 levels for differentiation into mature neurons or glia. This initially led to believe that ATF5 is not expressed in adult neurons. More recently, we reported basal neuronal ATF5 expression in adult mouse brain and its neuroprotective induction during UPR in a mouse model of status epilepticus. Here we aimed to explore whether ATF5 is also expressed by neurons in human brain both in basal conditions and in Huntington's disease (HD), where UPR has been described to be partially impaired due to defective ATF6 processing. Apart from confirming that ATF5 is present in human adult neurons, here we report accumulation of ATF5 within the characteristic polyglutamine-containing neuronal nuclear inclusions in brains of HD patients and mice. This correlates with decreased levels of soluble ATF5 and of its antiapoptotic target MCL1. We then confirmed the deleterious effect of ATF5 deficiency in a Caenorhabditis elegans model of polyglutamine-induced toxicity. Finally, ATF5 overexpression attenuated polyglutamine-induced apoptosis in a cell model of HD. These results reflect that decreased ATF5 in HD-probably secondary to sequestration into inclusions-renders neurons more vulnerable to mutant huntingtin-induced apoptosis and that ATF5-increasing interventions might have therapeutic potential for HD.

MAP2 Splicing is Altered in Huntington's Disease.

Dendritic alteration of striatal medium spiny neurons is one of the earliest morphological abnormalities in Huntington's disease (HD). The main microtubule-associated protein in dendrites is MAP2. The low-molecular weight isoforms of MAP2 (LMW-MAP2) are the juvenile forms resulting from exclusion of the sequence encoded by exons E7-E9 and are downregulated after the early stages of neuronal development when E7-E9 exon-including high-molecular weight isoforms (HMW-MAP2) are favored. Splicing alteration has recently been proposed to contribute to HD in view of two pathogenic missplicing events resulting in a highly toxic N-terminal version of mutant huntingtin and in a detrimental imbalance in MAP Tau isoforms with three or four tubulin-binding repeats. Both splicing events are postulated targets of the SR splicing factor SRSF6 which has recently been reported to be dramatically altered in HD. SR proteins often regulate functionally related sets of genes and SRSF6 targets are enriched in genes involved in brain organogenesis including several actin-and tubulin-binding proteins. Here we hypothesized that MAP2 might be target of SRSF6 and altered in HD. By SRSF6 knockdown in neuroblastoma cells, we demonstrate that splicing of MAP2 E7-E9 exons is affected by SRSF6. We then show a disbalance in LMW and HMW MAP2 mRNA isoforms in HD striatum in favor of the juvenile LMW forms together with a decrease in total MAP2 mRNA. This is accompanied by a global decrease in total MAP2 protein due to almost total disappearance of HMW-MAP2 isoforms with preservation of LMW-MAP2 isoforms. Accordingly, the predominant dendritic MAP2 staining in striatal neuropil of control subjects is absent in HD cases. In these, MAP2-immunoreactivity is faint and restricted to neuronal cell bodies often showing a sharp boundary at the base of dendrites. Together, our results highlight the importance of splicing alteration in HD and suggest that MAP2 alteration contributes to dendritic atrophy.

Impaired PLP-dependent metabolism in brain samples from Huntington disease patients and transgenic R6/1 mice.

Oxidative stress has been described as important to Huntington disease (HD) progression. In a previous HD study, we identified several carbonylated proteins, including pyridoxal kinase and antiquitin, both of which are involved in the metabolism of pyridoxal 5´-phosphate (PLP), the active form of vitamin B6. In the present study, pyridoxal kinase levels were quantified and showed to be decreased both in HD patients and a R6/1 mouse model, compared to control samples. A metabolomic analysis was used to analyze metabolites in brain samples of HD patients and R6/1 mice, compared to control samples using mass spectrometry. This technique allowed detection of increased concentrations of pyridoxal, the substrate of pyridoxal kinase. In addition, PLP, the product of the reaction, was decreased in striatum from R6/1 mice. Furthermore, glutamate and cystathionine, both substrates of PLP-dependent enzymes were increased in HD. This reinforces the hypothesis that PLP synthesis is impaired, and could explain some alterations observed in the disease. Together, these results identify PLP as a potential therapeutic agent.

Mice Lacking Functional Fas Death Receptors Are Protected from Kainic Acid-Induced Apoptosis in the Hippocampus.

The Fas receptor (FasR)/Fas ligand (FasL) system plays a significant role in the process of neuronal loss in neurological disorders. Thus, in the present study, we used a real-time PCR array focused apoptosis (Mouse Apoptosis RT(2) PCR Array) to study the role of the Fas pathway in the apoptotic process that occurs in a kainic acid (KA) mice experimental model. In fact, significant changes in the transcriptional activity of a total of 23 genes were found in the hippocampus of wild-type C57BL/6 mice after 12 h of KA treatment compared to untreated mice. Among the up-regulated genes, we found key factors involved in the extrinsic apoptotic pathway, such as tnf, fas and fasL, and also in caspase genes (caspase -4, caspase-8 and caspase-3). To discern the importance of the FasR/FasL pathway, mice lacking the functional Fas death receptor (lpr) were also treated with KA. After 24 h of neurotoxin treatment, lpr mice exhibited a reduced number of apoptotic positive cells, determined by the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) method in different regions of the hippocampus, when compared to wild-type mice. In addition, treatment of lpr mice with KA did not produce significant changes in the transcriptional activity of genes related to apoptosis in the hippocampus, either in the fas and fas ligand genes or in caspase-4 and caspase-8 and the executioner caspase-3 genes, as occurred in wild-type C57BL/6 mice. Thus, these data provide direct evidence that Fas signalling plays a key role in the induction of apoptosis in the hippocampus following KA treatment, making the inhibition of the death receptor pathway a potentially suitable target for excitotoxicity neuroprotection in neurological conditions such as epilepsy.

Reduced striatal dopamine DA D2 receptor function in dominant-negative GSK-3 transgenic mice.

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase with constitutive activity involved in cellular architecture, gene expression, cell proliferation, fate decision and apoptosis, among others. GSK-3 expression is particularly high in brain where it may be involved in neurological and psychiatric disorders such as Alzheimer׳s disease, bipolar disorder and major depression. A link with schizophrenia is suggested by the antipsychotic drug-induced GSK-3 regulation and by the involvement of the Akt/GSK-3 pathway in dopaminergic neurotransmission. Taking advantage of the previous development of dominant negative GSK-3 transgenic mice (Tg) showing a selective reduction of GSK-3 activity in forebrain neurons but not in dopaminergic neurons, we explored the relationship between GSK-3 and dopaminergic neurotransmission in vivo. In microdialysis experiments, local quinpirole (DA D2-R agonist) in dorsal striatum reduced dopamine (DA) release significantly less in Tg mice than in wild-type (WT) mice. However, local SKF-81297 (selective DA D1-R agonist) in dorsal striatum reduced DA release equally in both control and Tg mice indicating a comparable function of DA D1-R in the direct striato-nigral pathway. Likewise, systemic quinpirole administration - acting preferentially on presynaptic DA D2- autoreceptors to modulate DA release-reduced striatal DA release similarly in both control and Tg mice. Quinpirole reduced locomotor activity and induced c-fos expression in globus pallidus (both striatal DA D2-R-mediated effects) significantly more in WT than in Tg mice. Taking together, the present results show that dominant negative GSK-3 transgenic mice show reduced DA D2-R-mediated function in striatum and further support a link between dopaminergic neurotransmission and GSK-3 activity.

In vivo inhibition of the mitochondrial H+-ATP synthase in neurons promotes metabolic preconditioning.

A key transducer in energy conservation and signaling cell death is the mitochondrial H(+)-ATP synthase. The expression of the ATPase inhibitory factor 1 (IF1) is a strategy used by cancer cells to inhibit the activity of the H(+)-ATP synthase to generate a ROS signal that switches on cellular programs of survival. We have generated a mouse model expressing a mutant of human IF1 in brain neurons to assess the role of the H(+)-ATP synthase in cell death in vivo. The expression of hIF1 inhibits the activity of oxidative phosphorylation and mediates the shift of neurons to an enhanced aerobic glycolysis. Metabolic reprogramming induces brain preconditioning affording protection against quinolinic acid-induced excitotoxicity. Mechanistically, preconditioning involves the activation of the Akt/p70S6K and PARP repair pathways and Bcl-xL protection from cell death. Overall, our findings provide the first in vivo evidence highlighting the H(+)-ATP synthase as a target to prevent neuronal cell death.

Neuronal apoptosis and motor deficits in mice with genetic inhibition of GSK-3 are Fas-dependent.

Glycogen synthase kinase-3 (GSK-3) inhibitors have been postulated as useful therapeutic tools for the treatment of chronic neurodegenerative and neuropsychiatric diseases. Nevertheless the clinical use of these inhibitors has been limited by their common side effects. Lithium, a non-selective GSK-3 inhibitor has been classically administered to treat bipolar patients but its prescription is decreasing due to its frequent side effects such as hand tremor. This toxicity seems to be higher in the elderly and a clinical trial with lithium for Alzheimer's disease was stopped due to high rate of discontinuation. We have previously described a mechanism for the adverse effects of chronic lithium that involves neuronal apoptosis via Fas signaling. As lithium inhibits many other enzymatic activities such as inositol monophosphatase and histone deacetylase, here we aim to genetically test whether GSK-3 inhibition induces those adverse effects through Fas receptor. For this purpose we took advantage of a transgenic mouse line with decreased GSK-3 activity (Tet/DN-GSK-3 mice) that shows increased rate of neuronal apoptosis as well as motor deficits and brought it to a Fas deficient background (lpr mice). We found that apoptosis induced by GSK-3 inhibition was absent in Fas deficient background. Interestingly, motor deficits were also absent in Fas deficient Tet/DN-GSK-3 mice. These results demonstrate that Fas signaling contributes to the neurological toxicity of GSK-3 inhibition and suggest that a combination of GSK-3 inhibitors with blockers of Fas signaling could help to improve the application of GSK-3 inhibitors to clinics.

Protective neuronal induction of ATF5 in endoplasmic reticulum stress induced by status epilepticus.

Activating transcription factor 5 (ATF5) is a basic-leucine-zipper transcription factor of the ATF/CREB family. The Atf5 gene generates two transcripts, Atf5α and Atf5ß, of which Atf5α is known to be selectively translated upon endoplasmic reticulum stress response in non-neuronal cells. ATF5 is highly expressed in the developing brain where it modulates proliferation of neural progenitor cells. These cells show a high level of ATF5 that has to decrease to allow them to differentiate into mature neurons or glial cells. This has led to the extended notion that differentiated neural cells do not express ATF5 unless they undergo tumourigenic transformation. However, no systematic analysis of the distribution of ATF5 in adult brain or of its potential role in neuronal endoplasmic reticulum stress response has been reported. By immunostaining here we confirm highest ATF5 levels in neuroprogenitor cells of the embryonic and adult subventricular zone but also found ATF5 in a large variety of neurons in adult mouse brain. By combining Atf5 in situ hybridization and immunohistochemistry for the neuronal marker NeuN we further confirmed Atf5 messenger RNA in adult mouse neurons. Quantitative reverse transcriptase polymerase chain reaction demonstrated that Atf5α is the most abundant transcript in adult mouse encephalon and injection of the endoplasmic reticulum stress inducer tunicamycin into adult mouse brain increased neuronal ATF5 levels. Accordingly, ATF5 levels increased in hippocampal neurons of a mouse model of status epilepticus triggered by intra-amygdala injection of kainic acid, which leads to abnormal hippocampal neuronal activity and endoplasmic reticulum stress. Interestingly, ATF5 upregulation occurred mainly in hippocampal neuronal fields that do not undergo apoptosis in this status epilepticus model such as CA1 and dentate gyrus, thus suggesting a neuroprotective role. This was confirmed in a primary neuronal culture model in which ATF5 overexpression resulted in decreased endoplasmic reticulum stress-induced apoptosis and the opposite result was achieved by Atf5 RNA interference. Furthermore, in vivo administration of the eIF2α phosphatase inhibitor salubrinal resulted in increased ATF5 hippocampal levels and attenuated status epilepticus-induced neuronal death in the vulnerable CA3 subfield. In good agreement with the neuroprotective effect of increased ATF5, we found that apoptosis-resistant epileptogenic foci from patients with temporal lobe epilepsy also showed increased levels of ATF5. Thus, our results demonstrate that adult neurons express ATF5 and that they increase its levels upon endoplasmic reticulum stress as a pro-survival mechanism, thus opening a new field for neuroprotective strategies focused on ATF5 modulation.

CHOP regulates the p53-MDM2 axis and is required for neuronal survival after seizures.

Hippocampal sclerosis is a frequent pathological finding in patients with temporal lobe epilepsy and can be caused by prolonged single or repeated brief seizures. Both DNA damage and endoplasmic reticulum stress have been implicated as underlying molecular mechanisms in seizure-induced brain injury. The CCAAT/enhancer-binding protein homologous protein (CHOP) is a transcriptional regulator induced downstream of DNA damage and endoplasmic reticulum stress, which can promote or inhibit apoptosis according to context. Recent work has proposed inhibition of CHOP as a suitable neuroprotective strategy. Here, we show that transcript and protein levels of CHOP increase in surviving subfields of the hippocampus after prolonged seizures (status epilepticus) in mouse models. CHOP was also elevated in the hippocampus from epileptic mice and patients with pharmacoresistant epilepsy. The hippocampus of CHOP-deficient mice was much more vulnerable to damage in mouse models of status epilepticus. Moreover, compared with wild-type animals, CHOP-deficient mice subject to status epilepticus developed more spontaneous seizures, displayed protracted hippocampal neurodegeneration and a deficit in a hippocampus-dependent object-place recognition task. The absence of CHOP was associated with a supra-maximal induction of p53 after status epilepticus, and inhibition of p53 abolished the cell death-promoting consequences of CHOP deficiency. The protective effect of CHOP could be partly explained by activating transcription of murine double minute 2 that targets p53 for degradation. These data demonstrate that CHOP is required for neuronal survival after seizures and caution against inhibition of CHOP as a neuroprotective strategy where excitotoxicity is an underlying pathomechanism.

GSK-3 Mouse Models to Study Neuronal Apoptosis and Neurodegeneration.

Increased GSK-3 activity is believed to contribute to the etiology of chronic disorders like Alzheimer's disease (AD), schizophrenia, diabetes, and some types of cancer, thus supporting therapeutic potential of GSK-3 inhibitors. Numerous mouse models with modified GSK-3 have been generated in order to study the physiology of GSK-3, its implication in diverse pathologies and the potential effect of GSK-3 inhibitors. In this review we have focused on the relevance of these mouse models for the study of the role of GSK-3 in apoptosis. GSK-3 is involved in two apoptotic pathways, intrinsic and extrinsic pathways, and plays opposite roles depending on the apoptotic signaling process that is activated. It promotes cell death when acting through intrinsic pathway and plays an anti-apoptotic role if the extrinsic pathway is occurring. It is important to dissect this duality since, among the diseases in which GSK-3 is involved, excessive cell death is crucial in some illnesses like neurodegenerative diseases, while a deficient apoptosis is occurring in others such as cancer or autoimmune diseases. The clinical application of a classical GSK-3 inhibitor, lithium, is limited by its toxic consequences, including motor side effects. Recently, the mechanism leading to activation of apoptosis following chronic lithium administration has been described. Understanding this mechanism could help to minimize side effects and to improve application of GSK-3 inhibitors to the treatment of AD and to extend the application to other diseases.

Increased neurotransmitter release at the neuromuscular junction in a mouse model of polyglutamine disease.

In Huntington's disease (HD), the expansion of polyglutamine (polyQ) repeats at the N terminus of the ubiquitous protein huntingtin (htt) leads to neurodegeneration in specific brain areas. Neurons degenerating in HD develop synaptic dysfunctions. However, it is unknown whether mutant htt impacts synaptic function in general. To investigate that, we have focused on the nerve terminals of motor neurons that typically do not degenerate in HD. Here, we have studied synaptic transmission at the neuromuscular junction of transgenic mice expressing a mutant form of htt (R6/1 mice). We have found that the size and frequency of miniature endplate potentials are similar in R6/1 and control mice. In contrast, the amplitude of evoked endplate potentials in R6/1 mice is increased compared to controls. Consistent with a presynaptic increase of release probability, synaptic depression under high-frequency stimulation is higher in R6/1 mice. In addition, no changes were detected in the size and dynamics of the recycling synaptic vesicle pool. Moreover, we have found increased amounts of the synaptic vesicle proteins synaptobrevin 1,2/VAMP 1,2 and cysteine string protein-α, and the SNARE protein SNAP-25, concomitant with normal levels of other synaptic vesicle markers. Our results reveal that the transgenic expression of a mutant form of htt leads to an unexpected gain of synaptic function. That phenotype is likely not secondary to neurodegeneration and might be due to a primary deregulation in synaptic protein levels. Our findings could be relevant to understand synaptic toxic effects of proteins with abnormal polyQ repeats.

Impaired ATF6α processing, decreased Rheb and neuronal cell cycle re-entry in Huntington's disease.

The endoplasmic reticulum-stress response is induced in several neurodegenerative diseases and in cellular models of Huntington's disease. However, here we report that the processing of ATF6α to its active nuclear form, one of the three branches of endoplasmic reticulum-stress activation, is impaired in both animal models and Huntington's disease patients. ATF6α has been reported to be essential for the survival of dormant tumour cells that, like neurons, are arrested in the G0-G1 phase of the cell cycle. This effect is mediated by the small GTPase Rheb (Ras-homologue enriched in brain). Our results suggest that the ATF6α/Rheb pathway is altered in Huntington's disease as the decrease in ATF6α processing is accompanied by a decrease in the accumulation of Rheb. These alterations correlate with the aberrant accumulation of cell cycle re-entry markers in post-mitotic neurons which is accompanied by death of a subset of neurons.

NFAT/Fas signaling mediates the neuronal apoptosis and motor side effects of GSK-3 inhibition in a mouse model of lithium therapy.

Use of lithium, the mainstay for treatment of bipolar disorder, is limited by its frequent neurological side effects and its risk for overdose-induced toxicity. Recently, lithium has also been proposed as a treatment for Alzheimer disease and other neurodegenerative conditions, but clinical trials have been hampered by its prominent side effects in the elderly. The mechanisms underlying both the positive and negative effects of lithium are not fully known. Lithium inhibits glycogen synthase kinase-3 (GSK-3) in vivo, and we recently reported neuronal apoptosis and motor deficits in dominant-negative GSK-3-transgenic mice. We hypothesized that therapeutic levels of lithium could also induce neuronal loss through GSK-3 inhibition. Here we report induction of neuronal apoptosis in various brain regions and the presence of motor deficits in mice treated chronically with lithium. We found that GSK-3 inhibition increased translocation of nuclear factor of activated T cells c3/4 (NFATc3/4) transcription factors to the nucleus, leading to increased Fas ligand (FasL) levels and Fas activation. Lithium-induced apoptosis and motor deficits were absent when NFAT nuclear translocation was prevented by cyclosporin A administration and in Fas-deficient lpr mice. The results of these studies suggest a mechanism for lithium-induced neuronal and motor toxicity. These findings may enable the development of combined therapies that diminish the toxicities of lithium and possibly other GSK-3 inhibitors and extend their potential to the treatment of Alzheimer disease and other neurodegenerative conditions.

Acute polyglutamine expression in inducible mouse model unravels ubiquitin/proteasome system impairment and permanent recovery attributable to aggregate formation.

The presence of intracellular ubiquitylated inclusions in neurodegenerative disorders and the role of the ubiquitin/proteasome system (UPS) in degrading abnormal hazardous proteins have given rise to the hypothesis that UPS-impairment underlies neurodegenerative processes. However, this remains controversial for polyglutamine disorders such as Huntington disease (HD). Whereas studies in cellular models have provided evidence in favor of UPS-impairment attributable to expression of the N-terminal fragment of mutant huntingtin (N-mutHtt), similar studies on mouse models failed to do so. Furthermore, we have recently shown that the increase in polyubiquitin conjugates reported in the brain of N-mutHtt mice occurs in the absence of a general UPS-impairment. In the present study we aim to clarify the potential of N-mutHtt to impair UPS function in vivo as well as the mechanisms by which neurons may adapt after prolonged exposure to N-mutHtt in genetic models. By combining UPS reporter mice with an inducible mouse model of HD, we demonstrate for the first time polyglutamine-induced global UPS-impairment in vivo. UPS-impairment occurred transiently after acute N-mutHtt expression and restoration correlated with appearance of inclusion bodies (IBs). Consistently, UPS recovery did not take place when IB formation was prevented through administration of N-mutHtt aggregation-inhibitors in both cellular and animal models. Finally, no UPS-impairment was detected in old mice constitutively expressing N-mutHtt despite the age-associated decrease in brain proteasome activity. Therefore, our data reconcile previous contradictory reports by showing that N-mutHtt can indeed impair UPS function in vivo and that N-mutHtt aggregation leads to long lasting restoration of UPS function.

Accumulation of ubiquitin conjugates in a polyglutamine disease model occurs without global ubiquitin/proteasome system impairment.

Aggregation-prone proteins have been suggested to overwhelm and impair the ubiquitin/proteasome system (UPS) in polyglutamine (polyQ) disorders, such as Huntington's disease (HD). Overexpression of an N-terminal fragment of mutant huntingtin (N-mutHtt), an aggregation-prone polyQ protein responsible for HD, obstructs the UPS in cellular models. Furthermore, based on the accumulation of polyubiquitin conjugates in brains of R6/2 mice, which express human N-mutHtt and are one of the most severe polyQ disorder models, it has been proposed that UPS dysfunction is a consistent feature of this pathology, occurring in both in vitro and in vivo models. Here, we have exploited transgenic mice that ubiquitously express a ubiquitin fusion degradation proteasome substrate to directly assess the functionality of the UPS in R6/2 mice or the slower onset R6/1 mice. Although expression of N-mutHtt caused a general inhibition of the UPS in PC12 cells, we did not observe an increase in the levels of proteasome reporter substrate in the brains of R6/2 and R6/1 mice. We show that the increase in ubiquitin conjugates in R6/2 mice can be primarily attributed to an accumulation of large ubiquitin conjugates that are different from the conjugates observed upon UPS inhibition. Together our data show that polyubiquitylated proteins accumulate in R6/2 brain despite a largely operative UPS, and suggest that neurons are able to avoid or compensate for the inhibitory effects of N-mutHtt.