Arginine methylation of RNA-binding proteins is impaired in Huntington's disease.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion in the HD gene, coding for huntingtin protein (HTT). Mechanisms of HD cellular pathogenesis remain undefined and likely involve disruptions in many cellular processes and functions presumably mediated by abnormal protein interactions of mutant HTT. We previously found HTT interaction with several protein arginine methyl-transferase (PRMT) enzymes. Protein arginine methylation mediated by PRMT enzymes is an important post-translational modification with an emerging role in neurodegeneration. We found that normal (but not mutant) HTT can facilitate the activity of PRMTs in vitro and the formation of arginine methylation complexes. These interactions appear to be disrupted in HD neurons. This suggests an additional functional role for HTT/PRMT interactions, not limited to substrate/enzyme relationship, which may result in global changes in arginine protein methylation in HD. Our quantitative analysis of striatal precursor neuron proteome indicated that arginine protein methylation is significantly altered in HD. We identified a cluster highly enriched in RNA-binding proteins with reduced arginine methylation, which is essential to their function in RNA processing and splicing. We found that several of these proteins interact with HTT, and their RNA-binding and localization are affected in HD cells likely due to a compromised arginine methylation and/or abnormal interactions with mutant HTT. These studies reveal a potential new mechanism for disruption of RNA processing in HD, involving a direct interaction of HTT with methyl-transferase enzymes and modulation of their activity and highlighting methylation of arginine as potential new therapeutic target for HD.

Interaction of huntingtin with PRMTs and its subsequent arginine methylation affects HTT solubility, phase transition behavior and neuronal toxicity.

Huntington's disease (HD) is an incurable neurodegenerative disorder caused by a CAG expansion in the huntingtin gene (HTT). Post-translational modifications of huntingtin protein (HTT), such as phosphorylation, acetylation and ubiquitination, have been implicated in HD pathogenesis. Arginine methylation/dimethylation is an important modification with an emerging role in neurodegeneration; however, arginine methylation of HTT remains largely unexplored. Here we report nearly two dozen novel arginine methylation/dimethylation sites on the endogenous HTT from human and mouse brain and human cells suggested by mass spectrometry with data-dependent acquisition. Targeted quantitative mass spectrometry identified differential arginine methylation at specific sites in HD patient-derived striatal precursor cell lines compared to normal controls. We found that HTT can interact with several type I protein arginine methyltransferases (PRMTs) via its N-terminal domain. Using a combination of in vitro methylation and cell-based experiments, we identified PRMT4 (CARM1) and PRMT6 as major enzymes methylating HTT at specific arginines. Alterations of these methylation sites had a profound effect on biochemical properties of HTT rendering it less soluble in cells and affected its liquid-liquid phase separation and phase transition patterns in vitro. We found that expanded HTT 1-586 fragment can form liquid-like assemblies, which converted into solid-like assemblies when the R200/205 methylation sites were altered. Methyl-null alterations increased HTT toxicity to neuronal cells, while overexpression of PRMT 4 and 6 was beneficial for neuronal survival. Thus, arginine methylation pathways that involve specific HTT-modifying PRMT enzymes and modulate HTT biochemical and toxic properties could provide targets for HD-modifying therapies.

Immortalized striatal precursor neurons from Huntington's disease patient-derived iPS cells as a platform for target identification and screening for experimental therapeutics.

We have previously established induced pluripotent stem cell (iPSC) models of Huntington's disease (HD), demonstrating CAG-repeat-expansion-dependent cell biological changes and toxicity. However, the current differentiation protocols are cumbersome and time consuming, making preparation of large quantities of cells for biochemical or screening assays difficult. Here, we report the generation of immortalized striatal precursor neurons (ISPNs) with normal (33) and expanded (180) CAG repeats from HD iPSCs, differentiated to a phenotype resembling medium spiny neurons (MSN), as a proof of principle for a more tractable patient-derived cell model. For immortalization, we used co-expression of the enzymatic component of telomerase hTERT and conditional expression of c-Myc. ISPNs can be propagated as stable adherent cell lines, and rapidly differentiated into highly homogeneous MSN-like cultures within 2 weeks, as demonstrated by immunocytochemical criteria. Differentiated ISPNs recapitulate major HD-related phenotypes of the parental iPSC model, including brain-derived neurotrophic factor (BDNF)-withdrawal-induced cell death that can be rescued by small molecules previously validated in the parental iPSC model. Proteome and RNA-seq analyses demonstrate separation of HD versus control samples by principal component analysis. We identified several networks, pathways, and upstream regulators, also found altered in HD iPSCs, other HD models, and HD patient samples. HD ISPN lines may be useful for studying HD-related cellular pathogenesis, and for use as a platform for HD target identification and screening experimental therapeutics. The described approach for generation of ISPNs from differentiated patient-derived iPSCs could be applied to a larger allelic series of HD cell lines, and to comparable modeling of other genetic disorders.

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.

Post-Translational Modifications (PTMs), Identified on Endogenous Huntingtin, Cluster within Proteolytic Domains between HEAT Repeats.

Post-translational modifications (PTMs) of proteins regulate various cellular processes. PTMs of polyglutamine-expanded huntingtin (Htt) protein, which causes Huntington's disease (HD), are likely modulators of HD pathogenesis. Previous studies have identified and characterized several PTMs on exogenously expressed Htt fragments, but none of them were designed to systematically characterize PTMs on the endogenous full-length Htt protein. We found that full-length endogenous Htt, which was immunoprecipitated from HD knock-in mouse and human post-mortem brain, is suitable for detection of PTMs by mass spectrometry. Using label-free and mass tag labeling-based approaches, we identified near 40 PTMs, of which half are novel (data are available via ProteomeXchange with identifier PXD005753). Most PTMs were located in clusters within predicted unstructured domains rather than within the predicted α-helical structured HEAT repeats. Using quantitative mass spectrometry, we detected significant differences in the stoichiometry of several PTMs between HD and WT mouse brain. The mass-spectrometry identification and quantitation were verified using phospho-specific antibodies for selected PTMs. To further validate our findings, we introduced individual PTM alterations within full-length Htt and identified several PTMs that can modulate its subcellular localization in striatal cells. These findings will be instrumental in further assembling the Htt PTM framework and highlight several PTMs as potential therapeutic targets for 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.

Metformin Protects Cells from Mutant Huntingtin Toxicity Through Activation of AMPK and Modulation of Mitochondrial Dynamics.

Huntington's disease (HD) is a devastating neurodegenerative disease caused by the pathological elongation of the CAG repeats in the huntingtin gene. Caloric restriction (CR) has been the most reproducible environmental intervention to improve health and prolong life span. We have demonstrated that CR delayed onset and slowed disease progression in a mouse model of HD. Metformin, an antidiabetic drug, mimics CR by acting on cell metabolism at multiple levels. Long-term administration of metformin improved health and life span in mice. In this study, we showed that metformin rescued cells from mutant huntingtin (HTT)-induced toxicity, as indicated by reduced lactate dehydrogenase (LDH) release from cells and preserved ATP levels in cells expressing mutant HTT. Further mechanistic study indicated that metformin activated AMP-activated protein kinase (AMPK) and that inhibition of AMPK activation reduced its protective effects on mutant HTT toxicity, suggesting that AMPK mediates the protection of metformin in HD cells. Furthermore, metformin treatment prevented mitochondrial membrane depolarization and excess fission and modulated the disturbed mitochondrial dynamics in HD cells. We confirmed that metformin crossed the blood-brain barrier after oral administration and activated AMPK in the mouse brain. Our results urge further evaluation of the clinical potential for use of metformin in HD treatment.

Characterization of Behavioral, Neuropathological, Brain Metabolic and Key Molecular Changes in zQ175 Knock-In Mouse Model of Huntington's Disease.

Huntington's disease (HD) is caused by an expansion of the trinucleotide poly (CAG) tract located in exon 1 of the huntingtin (Htt) gene leading to progressive neurodegeneration in selected brain regions, and associated functional impairments in motor, cognitive, and psychiatric domains. Since the discovery of the gene mutation that causes the disease, mouse models have been developed by different strategies. Recently, a new model, the zQ175 knock-in (KI) line, was developed in an attempt to have the Htt gene in a context and causing a phenotype that more closely mimics HD in humans. The behavioral phenotype was characterized across the independent laboratories and important features reminiscent of human HD are observed in zQ175 mice. In the current study, we characterized the zQ175 model housed in an academic laboratory under reversed dark-light cycle, including motor function, in vivo longitudinal structural MRI imaging for brain volume, MRS for striatal metabolites, neuropathology, as well as a panel of key disease marker proteins in the striatum at different ages. Our results suggest that homozygous zQ175 mice exhibited significant brain atrophy before the motor deficits and brain metabolite changes. Altered striatal medium spiny neuronal marker, postsynaptic marker protein and complement component C1qC also characterized zQ175 mice. Our results confirmed that the zQ175 KI model is valuable in understanding of HD-like pathophysiology and evaluation of potential therapeutics. Our data also provide suggestions to select appropriate outcome measurements in preclinical studies using the zQ175 mice.

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.

Sirtuin 1 activator SRT2104 protects Huntington's disease mice.

Sirtuin 1 is a nicotinamide adenine dinucleotide-dependent protein deacetylase which regulates longevity and improves metabolism. Activation of Sirtuin 1 confers beneficial effects in models of neurodegenerative diseases. We and others have provided convincing evidence that overexpression of Sirtuin 1 plays a neuroprotective role in mouse models of Huntington's disease. In this study, we report that SRT2104, a small molecule Sirtuin 1 activator, penetrated the blood-brain barrier, attenuated brain atrophy, improved motor function, and extended survival in a mouse model of Huntington's disease. These findings imply a novel therapeutic strategy for Huntington's disease by targeting Sirtuin 1.

Small-molecule TrkB receptor agonists improve motor function and extend survival in a mouse model of Huntington's disease.

Huntington's disease (HD) is a fatal neurodegenerative disease characterized by abnormal motor coordination, cognitive decline and psychiatric disorders. This disease is caused by an expanded CAG trinucleotide repeat in the gene encoding the protein huntingtin. Reduced levels of brain-derived neurotrophic factor (BDNF) in the brain, which results from transcriptional inhibition and axonal transport deficits mediated by mutant huntingtin, have been suggested as critical factors underlying selective neurodegeneration in both HD patients and HD mouse models. BDNF activates its high-affinity receptor TrkB and promotes neuronal survival; restoring BDNF signaling is thus of particular therapeutic interest. In the present study, we evaluated the ability of a small-molecule TrkB agonist 7,8-dihydroxyflavone (7,8-DHF) and its synthetic derivative 4'-dimethylamino-7,8- dihydroxyflavone (4'-DMA-7,8-DHF) to protect neurons in the well-characterized N171-82Q HD mouse model. We found that chronic administration of 7, 8-DHF (5 mg/kg) or 4'-DMA-7,8-DHF (1 mg/kg) significantly improved motor deficits, ameliorated brain atrophy and extended survival in these N171-82Q HD mice. Moreover, 4'-DMA-7,8-DHF preserved DARPP32 levels in the striatum and rescued mutant huntingtin-induced impairment of neurogenesis in the N171-82Q HD mice. These data highlight consideration of TrkB as a therapeutic target in HD and suggest that small-molecule TrkB agonists that penetrate the brain have high potential to be further tested in clinical trials of HD.

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.

Longitudinal characterization of brain atrophy of a Huntington's disease mouse model by automated morphological analyses of magnetic resonance images.

Mouse models of human diseases play crucial roles in understanding disease mechanisms and developing therapeutic measures. Huntington's disease (HD) is characterized by striatal atrophy that begins long before the onset of motor symptoms. In symptomatic HD, striatal volumes decline predictably with disease course. Thus, imaging based volumetric measures have been proposed as outcomes for presymptomatic as well as symptomatic clinical trials of HD. Magnetic resonance imaging of the mouse brain structures is becoming widely available and has been proposed as one of the biomarkers of disease progression and drug efficacy testing. However, three-dimensional and quantitative morphological analyses of the brains are not straightforward. In this paper, we describe a tool for automated segmentation and voxel-based morphological analyses of the mouse brains. This tool was applied to a well-established mouse model of Huntington's disease, the R6/2 transgenic mouse strain. Comparison between the automated and manual segmentation results showed excellent agreement in most brain regions. The automated method was able to sensitively detect atrophy as early as 4 weeks of age and accurately follow disease progression. Comparison between ex vivo and in vivo MRI suggests that the ex vivo end-point measurement of brain morphology is also a valid approach except for the morphology of the ventricles. This is the first report of longitudinal characterization of brain atrophy in a mouse model of Huntington's disease by using automatic morphological analysis.

Long-term potentiation of excitatory synapses on neocortical somatostatin-expressing interneurons.

Synaptic plasticity has been extensively studied in principal neurons of the neocortex, but less work has been done on GABAergic interneurons. Interneurons consist of multiple subtypes and their synaptic properties vary between subtypes. In the present study, we have examined long-term potentiation (LTP) of excitatory synapses on somatostatin (SS)-expressing interneurons in neocortex using transgenic mice that express enhanced green fluorescent protein in these interneurons. We found that a strong theta burst stimulation was required to induce LTP in SS interneurons. LTP was associated with a reduction in paired-pulse facilitation and was not blocked by an N-methyl-d-aspartate receptor (NMDAR) antagonist. LTP was not affected by chelating postsynaptic Ca(2+) with BAPTA, a fast Ca(2+) chelator, and blocking L-type voltage-dependent Ca(2+) channels with nimodipine. Application of forskolin, an activator of adenylate cyclase that increases cyclic adenosine monophosphate (cAMP) concentration, enhanced synaptic transmission and occluded subsequent induction of LTP. Finally, we found that LTP was blocked by protein kinase A (PKA) inhibitors. Our results suggest that excitatory synapses on SS interneurons express a presynaptic form of LTP that is not dependent on NMDARs or postsynaptic Ca(2+) rise but is dependent on the cAMP-PKA signaling pathway.

[Impact of fetal growth restriction on spatial learning and memory abilities of the offspring of rats].

OBJECTIVE: To test the impact of fetal growth restriction (FGR) on the spatial learning and memory abilities of the offspring of rats. METHODS: FGR Model of Sprague-Dawley rats was constructed according to the method of passive smoking. The offspring of the rats were divided into male FGR group, male control group, female FGR group and female control group. Within each group, the rats were randomly divided into three subgroups to be tested at 1, 2, and 4 months of age, respectively (n =10 for each subgroup). Morris water maze task was performed to assess the spatial learning and memory abilities of the rats. RESULTS: The escape latencies to find the platform were shortened with increased training times for all of the rats. At the age of 1 and 2 months, both male and female rats in the FGR group spent more time in finding the platform than their counterparts in the control group (P < 0.05). At the age of 4 months, significant prolonged latency was only found in the female rats. The rats in the FGR group, except the 4 months old male rats, were more likely to use non-effective strategies (random or marginal strategies) to find the platform than the efficient strategies (tendency or straight strategies). The rats in the FGR group stayed in the platform shorter than those in the control groups (P < 0.05). CONCLUSION: FGR can cause gender- and age-specific impairment of spatial learning and memory abilities to the offspring.