Toward accurate cerebral blood flow estimation in mice after accounting for anesthesia.

Purpose: To improve the accuracy of cerebral blood flow (CBF) measurement in mice by accounting for the anesthesia effects. Methods: The dependence of CBF on anesthesia dose and time was investigated by simultaneously measuring respiration rate (RR) and heart rate (HR) under four different anesthetic regimens. Quantitative CBF was measured by a phase-contrast (PC) MRI technique. RR was evaluated with a mouse monitoring system (MouseOX) while HR was determined using an ultrashort-TE MRI sequence. CBF, RR, and HR were recorded dynamically with a temporal resolution of 1 min in a total of 19 mice. Linear regression models were used to investigate the relationships among CBF, anesthesia dose, RR, and HR. Results: CBF, RR, and HR all showed a significant dependence on anesthesia dose (p < 0.0001). However, the dose in itself was insufficient to account for the variations in physiological parameters, in that they showed a time-dependent change even for a constant dose. RR and HR together can explain 52.6% of the variations in CBF measurements, which is greater than the amount of variance explained by anesthesia dose (32.4%). Based on the multi-parametric regression results, a model was proposed to correct the anesthesia effects in mouse CBF measurements, specifically C B F c o r r e c t e d = C B F + 0.58 R R - 0.41 H R - 32.66 D o s e . We also reported awake-state CBF in mice to be 142.0 ± 8.8 mL/100 g/min, which is consistent with the model-predicted value. Conclusion: The accuracy of CBF measurement in mice can be improved by using a correction model that accounts for respiration rate, heart rate, and anesthesia dose.

Non-contrast assessment of blood-brain barrier permeability to water in mice: An arterial spin labeling study at cerebral veins.

Blood-brain barrier (BBB) plays a critical role in protecting the brain from toxins and pathogens. However, in vivo tools to assess BBB permeability are scarce and often require the use of exogenous contrast agents. In this study, we aimed to develop a non-contrast arterial-spin-labeling (ASL) based MRI technique to estimate BBB permeability to water in mice. By determining the relative fraction of labeled water spins that were exchanged into the brain tissue as opposed to those that remained in the cerebral veins, we estimated indices of global BBB permeability to water including water extraction fraction (E) and permeability surface-area product (PS). First, using multiple post-labeling delay ASL experiments, we estimated the bolus arrival time (BAT) of the labeled spins to reach the great vein of Galen (VG) to be 691.2 ± 14.5 ms (N = 5). Next, we investigated the dependence of the VG ASL signal on labeling duration and identified an optimal imaging protocol with a labeling duration of 1200 ms and a PLD of 100 ms. Quantitative E and PS values in wild-type mice were found to be 59.9 ± 3.2% and 260.9 ± 18.9 ml/100 g/min, respectively. In contrast, mice with Huntington's disease (HD) revealed a significantly higher E (69.7 ± 2.4%, P = 0.026) and PS (318.1 ± 17.1 ml/100 g/min, P = 0.040), suggesting BBB breakdown in this mouse model. Reproducibility studies revealed a coefficient-of-variation (CoV) of 4.9 ± 1.7% and 6.1 ± 1.2% for E and PS, respectively. The proposed method may open new avenues for preclinical research on pathophysiological mechanisms of brain diseases and therapeutic trials in animal models.

An RNA-targeting CRISPR-Cas13d system alleviates disease-related phenotypes in Huntington's disease models.

Huntington's disease (HD) is a fatal, dominantly inherited neurodegenerative disorder caused by CAG trinucleotide expansion in exon 1 of the huntingtin (HTT) gene. Since the reduction of pathogenic mutant HTT messenger RNA is therapeutic, we developed a mutant allele-sensitive CAGEX RNA-targeting CRISPR-Cas13d system (Cas13d-CAGEX) that eliminates toxic CAGEX RNA in fibroblasts derived from patients with HD and induced pluripotent stem cell-derived neurons. We show that intrastriatal delivery of Cas13d-CAGEX via an adeno-associated viral vector selectively reduces mutant HTT mRNA and protein levels in the striatum of heterozygous zQ175 mice, a model of HD. This also led to improved motor coordination, attenuated striatal atrophy and reduction of mutant HTT protein aggregates. These phenotypic improvements lasted for at least eight months without adverse effects and with minimal off-target transcriptomic effects. Taken together, we demonstrate proof of principle of an RNA-targeting CRISPR-Cas13d system as a therapeutic approach for HD, a strategy with implications for the treatment of other dominantly inherited disorders.

Early detection of Alzheimer's disease using creatine chemical exchange saturation transfer magnetic resonance imaging.

Detecting Alzheimer's disease (AD) at an early stage brings a lot of benefits including disease management and actions to slow the progression of the disease. Here, we demonstrate that reduced creatine chemical exchange saturation transfer (CrCEST) contrast has the potential to serve as a new biomarker for early detection of AD. The results on wild type (WT) mice and two age-matched AD models, namely tauopathy (Tau) and Aß amyloidosis (APP), indicated that CrCEST contrasts of the cortex and corpus callosum in the APP and Tau mice were significantly reduced compared to WT counterpart at an early stage (6-7 months) (p < 0.011). Two main causes of the reduced CrCEST contrast, i.e. cerebral pH and creatine concentration, were investigated. From phantom and hypercapnia experiments, CrCEST showed excellent sensitivity to pH variations. From MRS results, the creatine concentration in WT and AD mouse brain was equivalent, which suggests that the reduced CrCEST contrast was dominated by cerebral pH change involved in the progression of AD. Immunohistochemical analysis revealed that the abnormal cerebral pH in AD mice may relate to neuroinflammation, a known factor that can cause pH reduction.

Nemo-like kinase reduces mutant huntingtin levels and mitigates Huntington's disease.

Nemo-like kinase (NLK), an evolutionarily conserved serine/threonine kinase, is highly expressed in the brain, but its function in the adult brain remains not well understood. In this study, we identify NLK as an interactor of huntingtin protein (HTT). We report that NLK levels are significantly decreased in HD human brain and HD models. Importantly, overexpression of NLK in the striatum attenuates brain atrophy, preserves striatal DARPP32 levels and reduces mutant HTT (mHTT) aggregation in HD mice. In contrast, genetic reduction of NLK exacerbates brain atrophy and loss of DARPP32 in HD mice. Moreover, we demonstrate that NLK lowers mHTT levels in a kinase activity-dependent manner, while having no significant effect on normal HTT protein levels in mouse striatal cells, human cells and HD mouse models. The NLK-mediated lowering of mHTT is associated with enhanced phosphorylation of mHTT. Phosphorylation defective mutation of serine at amino acid 120 (S120) abolishes the mHTT-lowering effect of NLK, suggesting that S120 phosphorylation is an important step in the NLK-mediated lowering of mHTT. A further mechanistic study suggests that NLK promotes mHTT ubiquitination and degradation via the proteasome pathway. Taken together, our results indicate a protective role of NLK in HD and reveal a new molecular target to reduce mHTT levels.

Small molecule modulator of protein disulfide isomerase attenuates mutant huntingtin toxicity and inhibits endoplasmic reticulum stress in a mouse model of Huntington's disease.

Huntington's disease (HD) is caused by a cytosine-adenine-guanine (CAG) trinucleotide repeat expansion in the huntingtin (HTT) gene encoding an elongated polyglutamine tract within the N-terminal of the huntingtin protein (Htt) and leads to Htt misfolding, aberrant protein aggregation, and progressive appearance of disease symptoms. Chronic activation of endoplasmic reticulum (ER) stress by mutant Htt (mHtt) results in cellular dysfunction and ultimately cell death. Protein disulfide isomerase (PDI) is a chaperone protein located in the ER. Our previous studies demonstrated that mHtt caused PDI to accumulate at mitochondria-associated ER membranes and triggered cell death, and that modulating PDI activity using small molecules protected cells again mHtt toxicity in cell and brain slice models of HD. In this study, we demonstrated that PDI is upregulated in the HD human brain, in cell and mouse models. Chronic administration of a reversible, brain penetrable small molecule PDI modulator, LOC14 (20 mg/kg/day), significantly improved motor function, attenuated brain atrophy and extended survival in the N171-82Q HD mice. Moreover, LOC14 preserved medium spiny neuronal marker dopamine- and cyclic-AMP-regulated phosphoprotein of molecular weight 32 000 (DARPP32) levels in the striatum of HD mice. Mechanistic study revealed that LOC14 suppressed mHtt-induced ER stress, indicated by repressing the abnormally upregulated ER stress proteins in HD models. These findings suggest that LOC14 is promising to be further optimized for clinical trials of HD, and modulation of signaling pathways coping with ER stress may constitute an attractive approach to reduce mHtt toxicity and identify new therapeutic targets for treatment of HD.

2,4 DNP improves motor function, preserves medium spiny neuronal identity, and reduces oxidative stress in a mouse model of Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the first exon of the gene huntingtin. There is no treatment to prevent or delay the disease course of HD currently. Oxidative stress and mitochondrial dysfunction have emerged as key determinants of the disease progression in HD. Therefore, counteracting mutant huntingtin (mHtt)-induced oxidative stress and mitochondrial dysfunction appears as a new approach to treat this devastating disease. Interestingly, mild mitochondrial uncoupling improves neuronal resistance to stress and facilitates neuronal survival. Mild mitochondrial uncoupling can be induced by the proper dose of 2,4-dinitrophenol (DNP), a proton ionophore that was previously used for weight loss. In this study, we evaluated the effects of chronic administration of DNP at three doses (0.5, 1, 5mg/kg/day) on mHtt-induced behavioral deficits and cellular abnormalities in the N171-82Q HD mouse model. DNP at a low dose (1mg/kg/day) significantly improved motor function and preserved medium spiny neuronal marker DARPP32 and postsynaptic protein PSD95 in the striatum of HD mice. Further mechanistic study suggests that DNP at this dose reduced oxidative stress in HD mice, which was indicated by reduced levels of F2-isoprostanes in the brain of HD mice treated with DNP. Our data indicated that DNP provided behavioral benefit and neuroprotective effect at a weight neutral dose in HD mice, suggesting that the potential value of repositioning DNP to HD treatment is warranted in well-controlled clinical trials in HD.

Oriented growth of poly(m-phenylenediamine) on Calotropis gigantea fiber for rapid adsorption of ciprofloxacin.

A facile polymerization of m-phenylenediamine (mPD) in methanol/water (50:50, v/v) was performed via conventional chemical oxidative method by using Calotropis gigantea fiber (CGF) as the natural biotemplate. The as-prepared CGF oriented poly(m-phenylenediamine) (PmPD), i.e. CGF-O-PmPD, exhibits a well defined fiber-oriented morphology. The presence of PmPD layer enables CGF-O-PmPD to present roughen surface with N-rich functional groups that can show high performance for the adsorption of ciprofloxacin. The variables affecting the adsorption capacity were systematically investigated, including contact time, initial concentration, initial pH, ion strength, and so forth. The experimental data reveal that with increasing the amount of mPD from 0.2, 0.5 to 2.0 g, the adsorption capacity for ciprofloxacin shows a monotonic decrease, while the adsorption kinetics show a monotonic increase, with the adsorption percentage from >50%, >60% to >70% within 10 s, demonstrating its superfast adsorption kinetics for ciprofloxacin. In addition, an increasing adsorption capacity is observed over the pH range studied, with the adsorption capacity from 0.73 to 6.7 mg g-1 at pH 2.0 to 64.9-77.3 mg g-1 at pH 10.0. After five adsorption-desorption cycles, the adsorption capacity of CGF-O-PmPD for ciprofloxacin shows no significant decrease, indicating its excellent reusability and potential application in treating antibiotic-containing wastewater.

Early white matter abnormalities, progressive brain pathology and motor deficits in a novel knock-in mouse model of Huntington's disease.

White matter abnormalities have been reported in premanifest Huntington's disease (HD) subjects before overt striatal neuronal loss, but whether the white matter changes represent a necessary step towards further pathology and the underlying mechanism of these changes remains unknown. Here, we characterized a novel knock-in mouse model that expresses mouse HD gene homolog (Hdh) with extended CAG repeat- HdhQ250, which was derived from the selective breeding of HdhQ150 mice. HdhQ250 mice manifest an accelerated and robust phenotype compared with its parent line. HdhQ250 mice exhibit progressive motor deficits, reduction in striatal and cortical volume, accumulation of mutant huntingtin aggregation, decreased levels of DARPP32 and BDNF and altered striatal metabolites. The abnormalities detected in this mouse model are reminiscent of several aspects of human HD. In addition, disturbed myelination was evident in postnatal Day 14 HdhQ250 mouse brain, including reduced levels of myelin regulatory factor and myelin basic protein, and decreased numbers of myelinated axons in the corpus callosum. Thinner myelin sheaths, indicated by increased G-ratio of myelin, were also detected in the corpus callosum of adult HdhQ250 mice. Moreover, proliferation of oligodendrocyte precursor cells is altered by mutant huntingtin both in vitro and in vivo. Our data indicate that this model is suitable for understanding comprehensive pathogenesis of HD in white matter and gray matter as well as developing therapeutics for HD.

Metabolism in HD: still a relevant mechanism?

The polyglutamine expansion within huntingtin is the causative factor in the pathogenesis of Huntington's disease (HD). Although the underlying mechanisms by which mutant huntingtin causes neuronal dysfunction and degeneration have not been fully elucidated, compelling evidence suggests that mitochondrial dysfunction and compromised energy metabolism are key players in HD pathogenesis. Longitudinal studies of HD subjects have shown reductions in glucose utilization before the disease clinical onset. Preferential striatal neurodegeneration, a hallmark of HD pathogenesis, also has been associated with interrupted energy metabolism. Data from genetic HD models indicate that mutant huntingtin disrupts mitochondrial bioenergetics and prevents adenosine triphosphate (ATP) generation, implying altered energy metabolism as an important component of HD pathogenesis. Here we revisit the evidence of abnormal energy metabolism in the central nervous system of HD patients, review our current understanding of the molecular mechanisms underlying abnormal metabolism induced by mutant huntingtin, and discuss the promising therapeutic development by halting abnormal metabolism in HD.

Neuroprotective effects of PPAR-γ agonist rosiglitazone in N171-82Q mouse model of Huntington's disease.

Huntington's disease (HD) is a devastating genetic neurodegenerative disease caused by CAG trinucleotide expansion in the exon-1 region of the huntingtin gene. Currently, no cure is available. It is becoming increasingly apparent that mutant Huntingtin (HTT) impairs metabolic homeostasis and causes transcriptional dysregulation. The peroxisome proliferator-activated receptor gamma (PPAR-γ) is a transcriptional factor that plays a key role in regulating genes involved in energy metabolism; recent studies demonstrated that PPAR-γ activation prevented mitochondrial depolarization in cells expressing mutant HTT and attenuated neurodegeneration in various models of neurodegenerative diseases. PPAR-γ-coactivator 1α (PGC-1 α) transcription activity is also impaired by mutant HTT. We now report that the PPAR-γ agonist, rosiglitazone (RSG), significantly attenuated mutant HTT-induced toxicity in striatal cells and that the protective effect of RSG is mediated by activation of PPAR-γ. Moreover, chronic administration of RSG (10 mg/kg/day, i.p) significantly improved motor function and attenuated hyperglycemia in N171-82Q HD mice. RSG administration rescued brain derived neurotrophic factor(BDNF) deficiency in the cerebral cortex, and prevented loss of orexin-A-immunopositive neurons in the hypothalamus of N171-82Q HD mice. RSG also prevented PGC-1α reduction and increased Sirt6 protein levels in HD mouse brain. Our results suggest that modifying the PPAR-γ pathway plays a beneficial role in rescuing motor function as well as glucose metabolic abnormalities in HD.

Striatal neuronal loss correlates with clinical motor impairment in Huntington's disease.

Huntington's disease (HD) is characterized clinically by chorea, motor impairment, psychiatric manifestations, and dementia. Atrophy of the striatum is the neuropathological hallmark of HD, and previous studies have suggested that striatal atrophy correlates more closely with motor impairment than with chorea. Motor impairment, as measured by motor impairment score, correlates with functional disability in HD patients, but chorea does not. In this study, we investigated the relation between neuronal loss and these motor features. We conducted neuropathological and stereologic assessments of neurons in putamen and subthalamic nuclei in HD patients and age-matched controls. In putamen, we estimated the total number and volume of medium spiny neurons labeled with dopamine- and cAMP-regulated phosphoprotein 32 kDa (DARPP-32). In subthalamic nuclei, we estimated the total number of neurons on hematoxylin & eosin/luxol fast blue stains. In putamen of HD, immunohistochemistry showed DARPP-32 neuronal atrophy with extensive disruption of neurites and neuropil; stereologic studies found significant decreases in both the number and size of DARPP-32 neurons; we also detected a significant reduction of overall putamen volume in HD patients, compared to controls. In subthalamic nuclei, there was a mild, but significant, neuronal loss in the HD group. The loss of neurons in putamen and subthalamic nuclei as well as putaminal atrophy were significantly correlated with severity of motor impairment, but not with chorea. Our findings suggest that neuronal loss and atrophy in striatum and neuronal loss in subthalamic nuclei contribute specifically to the motor impairment of HD, but not to chorea.

Interrogation of brain miRNA and mRNA expression profiles reveals a molecular regulatory network that is perturbed by mutant huntingtin.

Emerging evidence indicates that microRNAs (miRNAs) may play an important role in the pathogenesis of Huntington's disease (HD). To identify the individual miRNAs that are altered in HD and may therefore regulate a gene network underlying mutant huntingtin-induced neuronal dysfunction in HD, we performed miRNA array analysis combined with mRNA profiling in the cerebral cortex from N171-82Q HD mice. Expression profiles of miRNAs as well as mRNAs in HD mouse cerebral cortex were analyzed and confirmed at different stages of disease progression; the most significant changes of miRNAs in the cerebral cortex were also detected in the striatum of HD mice. Our results revealed a significant alteration of miR-200 family members, miR-200a, and miR-200c in the cerebral cortex and the striatum, at the early stage of disease progression in N171-82Q HD mice. We used a coordinated approach to integrate miRNA and mRNA profiling, and applied bioinformatics to predict a target gene network potentially regulated by these significantly altered miRNAs that might be involved in HD disease progression. Interestingly, miR-200a and miR-200c are predicted to target genes regulating synaptic function, neurodevelopment, and neuronal survival. Our results suggest that altered expression of miR-200a and miR-200c may interrupt the production of proteins involved in neuronal plasticity and survival, and further investigation of the involvement of perturbed miRNA expression in HD pathogenesis is warranted, and may lead to reveal novel approaches for HD therapy.

trans-(-)-ε-Viniferin increases mitochondrial sirtuin 3 (SIRT3), activates AMP-activated protein kinase (AMPK), and protects cells in models of Huntington Disease.

Huntington disease (HD) is an inherited neurodegenerative disorder caused by an abnormal polyglutamine expansion in the protein Huntingtin (Htt). Currently, no cure is available for HD. The mechanisms by which mutant Htt causes neuronal dysfunction and degeneration remain to be fully elucidated. Nevertheless, mitochondrial dysfunction has been suggested as a key event mediating mutant Htt-induced neurotoxicity because neurons are energy-demanding and particularly susceptible to energy deficits and oxidative stress. SIRT3, a member of sirtuin family, is localized to mitochondria and has been implicated in energy metabolism. Notably, we found that cells expressing mutant Htt displayed reduced SIRT3 levels. trans-(-)-ε-Viniferin (viniferin), a natural product among our 22 collected naturally occurring and semisynthetic stilbenic compounds, significantly attenuated mutant Htt-induced depletion of SIRT3 and protected cells from mutant Htt. We demonstrate that viniferin decreases levels of reactive oxygen species and prevents loss of mitochondrial membrane potential in cells expressing mutant Htt. Expression of mutant Htt results in decreased deacetylase activity of SIRT3 and further leads to reduction in cellular NAD(+) levels and mitochondrial biogenesis in cells. Viniferin activates AMP-activated kinase and enhances mitochondrial biogenesis. Knockdown of SIRT3 significantly inhibited viniferin-mediated AMP-activated kinase activation and diminished the neuroprotective effects of viniferin, suggesting that SIRT3 mediates the neuroprotection of viniferin. In conclusion, we establish a novel role for mitochondrial SIRT3 in HD pathogenesis and discovered a natural product that has potent neuroprotection in HD models. Our results suggest that increasing mitochondrial SIRT3 might be considered as a new therapeutic approach to counteract HD, as well as other neurodegenerative diseases with similar mechanisms.

Transgenic mouse model expressing the caspase 6 fragment of mutant huntingtin.

Huntington's disease (HD) is caused by a polyglutamine expansion in the Huntingtin (Htt) protein. Proteolytic cleavage of Htt into toxic N-terminal fragments is believed to be a key aspect of pathogenesis. The best characterized putative cleavage event is at amino acid 586, hypothesized to be mediated by caspase 6. A corollary of the caspase 6 cleavage hypothesis is that the caspase 6 fragment should be a toxic fragment. To test this hypothesis, and further characterize the role of this fragment, we have generated transgenic mice expressing the N-terminal 586 aa of Htt with a polyglutamine repeat length of 82 (N586-82Q), under the control of the prion promoter. N586-82Q mice show a clear progressive rotarod deficit by 4 months of age, and are hyperactive starting at 5 months, later changing to hypoactivity before early mortality. MRI studies reveal widespread brain atrophy, and histologic studies demonstrate an abundance of Htt aggregates, mostly cytoplasmic, which are predominantly composed of the N586-82Q polypeptide. Smaller soluble N-terminal fragments appear to accumulate over time, peaking at 4 months, and are predominantly found in the nuclear fraction. This model appears to have a phenotype more severe than current full-length Htt models, but less severe than HD mouse models expressing shorter Htt fragments. These studies suggest that the caspase 6 fragment may be a transient intermediate, that fragment size is a factor contributing to the rate of disease progression, and that short soluble nuclear fragments may be most relevant to pathogenesis.

Neuroprotective role of Sirt1 in mammalian models of Huntington's disease through activation of multiple Sirt1 targets.

Huntington's disease is a fatal neurodegenerative disorder caused by an expanded polyglutamine repeat in huntingtin (HTT) protein. We previously showed that calorie restriction ameliorated Huntington's disease pathogenesis and slowed disease progression in mice that model Huntington's disease (Huntington's disease mice). We now report that overexpression of sirtuin 1 (Sirt1), a mediator of the beneficial metabolic effects of calorie restriction, protects neurons against mutant HTT toxicity, whereas reduction of Sirt1 exacerbates mutant HTT toxicity. Overexpression of Sirt1 improves motor function, reduces brain atrophy and attenuates mutant-HTT-mediated metabolic abnormalities in Huntington's disease mice. Further mechanistic studies suggested that Sirt1 prevents the mutant-HTT-induced decline in brain-derived neurotrophic factor (BDNF) concentrations and the signaling of its receptor, TrkB, and restores dopamine- and cAMP-regulated phosphoprotein, 32 kDa (DARPP32) concentrations in the striatum. Sirt1 deacetylase activity is required for Sirt1-mediated neuroprotection in Huntington's disease cell models. Notably, we show that mutant HTT interacts with Sirt1 and inhibits Sirt1 deacetylase activity, which results in hyperacetylation of Sirt1 substrates such as forkhead box O3A (Foxo3a), thereby inhibiting its pro-survival function. Overexpression of Sirt1 counteracts the mutant-HTT-induced deacetylase deficit, enhances the deacetylation of Foxo3a and facilitates cell survival. These findings show a neuroprotective role for Sirt1 in mammalian Huntington's disease models and open new avenues for the development of neuroprotective strategies in Huntington's disease.

Baicalein reduces E46K alpha-synuclein aggregation in vitro and protects cells against E46K alpha-synuclein toxicity in cell models of familiar Parkinsonism.

The E46K is a point mutation in alpha-synuclein (alpha-syn) that causes familial Parkinsonism with Lewy body dementia. We have now generated a cell model of Parkinsonism/Parkinson's disease (PD) and demonstrated cell toxicity after expression of E46K in the differentiated PC12 cells. E46K alpha-syn inhibited proteasome activity and induced mitochondrial depolarization in the cell model. Baicalein has been reported to inhibit fibrillation of wild type alpha-syn in vitro, and to protect neurons against several chemical-induced models of PD. We now report that baicalein significantly attenuated E46K-induced mitochondrial depolarization and proteasome inhibition, and protected cells against E46K-induced toxicity in a cell model of PD. Baicalein also reduced E46K fibrilization in vitro, with a concentration-dependent decrease in beta sheet conformation, though it increased some oligomeric species, and decreased formation of E46K alpha-syn-induced aggregates and rescued toxicity in N2A cells. Taken together, these data indicate that mitochondrial dysfunction, proteasome inhibition and specific aspects of abnormal E46K aggregation accompany E46K alpha-syn-induced cell toxicity, and baicalein can protect as well as altering aggregation properties. Baicalein has potential as a tool to understand the relation between different aggregation species and toxicity, and might be a candidate compound for further validation by using in vivo alpha-syn genetic PD models.

Tiagabine is neuroprotective in the N171-82Q and R6/2 mouse models of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by chorea, incoordination, and shortened life-span, and by huntingtin inclusions and neurodegeneration. We previously screened the 1040 FDA-approved compounds from the NINDS compound library and found that a compound, nipecotic acid, significantly reduced mutant huntingtin aggregations and blocked cell toxicity in an inducible cell model of HD. Because nipecotic acid does not cross the blood-brain barrier (BBB), we studied its analogue, tiagabine, which is able to cross the BBB, in both N171-82Q and R6/2 transgenic mouse models of HD. Tiagabine was administered intraperitoneally at 2 and 5 mg/kg daily in HD mice. We found that tiagabine extended survival, improved motor performance, and attenuated brain atrophy and neurodegeneration in N171-82Q HD mice. These beneficial effects were further confirmed in R6/2 HD mice. The levels of tiagabine at effective doses in mouse serum are comparable to the levels in human patients treated with tiagabine. These results suggest that tiagabine may have beneficial effects in the treatment of HD. Because tiagabine is an FDA-approved drug, it may be a promising candidate for future clinical trials for the treatment of HD.

Single particle characterization of iron-induced pore-forming alpha-synuclein oligomers.

Aggregation of alpha-synuclein is a key event in several neurodegenerative diseases, including Parkinson disease. Recent findings suggest that oligomers represent the principal toxic aggregate species. Using confocal single-molecule fluorescence techniques, such as scanning for intensely fluorescent targets (SIFT) and atomic force microscopy, we monitored alpha-synuclein oligomer formation at the single particle level. Organic solvents were used to trigger aggregation, which resulted in small oligomers ("intermediate I"). Under these conditions, Fe(3+) at low micromolar concentrations dramatically increased aggregation and induced formation of larger oligomers ("intermediate II"). Both oligomer species were on-pathway to amyloid fibrils and could seed amyloid formation. Notably, only Fe(3+)-induced oligomers were SDS-resistant and could form ion-permeable pores in a planar lipid bilayer, which were inhibited by the oligomer-specific A11 antibody. Moreover, baicalein and N'-benzylidene-benzohydrazide derivatives inhibited oligomer formation. Baicalein also inhibited alpha-synuclein-dependent toxicity in neuronal cells. Our results may provide a potential disease mechanism regarding the role of ferric iron and of toxic oligomer species in Parkinson diseases. Moreover, scanning for intensely fluorescent targets allows high throughput screening for aggregation inhibitors and may provide new approaches for drug development and therapy.

Leptin-mediated cell survival signaling in hippocampal neurons mediated by JAK STAT3 and mitochondrial stabilization.

Leptin plays a pivotal role in the regulation of energy homeostasis and metabolism, primarily by acting on neurons in the hypothalamus that control food intake. However, leptin receptors are more widely expressed in the brain suggesting additional, as yet unknown, functions of leptin. Here we show that both embryonic and adult hippocampal neurons express leptin receptors coupled to activation of STAT3 and phosphatidylinositol 3-kinase-Akt signaling pathways. Leptin protects hippocampal neurons against cell death induced by neurotrophic factor withdrawal and excitotoxic and oxidative insults. The neuroprotective effect of leptin is antagonized by the JAK2-STAT3 inhibitor AG-490, STAT3 decoy DNA, and phosphatidylinositol 3-kinase/Akt inhibitors but not by an inhibitor of MAPK. Leptin induces the production of manganese superoxide dismutase and the anti-apoptotic protein Bcl-xL, and stabilizes mitochondrial membrane potential and lessens mitochondrial oxidative stress. Leptin receptor-deficient mice (db/db mice) are more vulnerable to seizure-induced hippocampal damage, and intraventricular administration of leptin protects neurons against seizures. By enhancing mitochondrial resistance to apoptosis and excitotoxicity, our findings suggest that leptin signaling serves a neurotrophic function in the developing and adult hippocampus.