Msh3 and Pms1 Set Neuronal CAG-repeat Migration Rate to Drive Selective Striatal and Cortical Pathogenesis in HD Mice.

Modifiers of Huntington's disease (HD) include mismatch repair (MMR) genes; however, their underlying disease-altering mechanisms remain unresolved. Knockout (KO) alleles for 9 HD GWAS modifiers/MMR genes were crossed to the Q140 Huntingtin (mHtt) knock-in mice to probe such mechanisms. Four KO mice strongly ( Msh3 and Pms1 ) or moderately ( Msh2 and Mlh1 ) rescue a triad of adult-onset, striatal medium-spiny-neuron (MSN)-selective phenotypes: somatic Htt DNA CAG-repeat expansion, transcriptionopathy, and mHtt protein aggregation. Comparatively, Q140 cortex also exhibits an analogous, but later-onset, pathogenic triad that is Msh3 -dependent. Remarkably, Q140/homozygous Msh3-KO lacks visible mHtt aggregates in the brain, even at advanced ages (20-months). Moreover, Msh3 -deficiency prevents striatal synaptic marker loss, astrogliosis, and locomotor impairment in HD mice. Purified Q140 MSN nuclei exhibit highly linear age-dependent mHtt DNA repeat expansion (i.e. repeat migration), with modal-CAG increasing at +8.8 repeats/month (R 2 =0.98). This linear rate is reduced to 2.3 and 0.3 repeats/month in Q140 with Msh3 heterozygous and homozygous alleles, respectively. Our study defines somatic Htt CAG-repeat thresholds below which there are no detectable mHtt nuclear or neuropil aggregates. Mild transcriptionopathy can still occur in Q140 mice with stabilized Htt 140-CAG repeats, but the majority of transcriptomic changes are due to somatic repeat expansion. Our analysis reveals 479 genes with expression levels highly correlated with modal-CAG length in MSNs. Thus, our study mechanistically connects HD GWAS genes to selective neuronal vulnerability in HD, in which Msh3 and Pms1 set the linear rate of neuronal mHtt CAG-repeat migration to drive repeat-length dependent pathogenesis; and provides a preclinical platform for targeting these genes for HD suppression across brain regions. One Sentence Summary: Msh3 and Pms1 are genetic drivers of sequential striatal and cortical pathogenesis in Q140 mice by mediating selective CAG-repeat migration in HD vulnerable neurons.

Mapping brain gene coexpression in daytime transcriptomes unveils diurnal molecular networks and deciphers perturbation gene signatures.

Brain tissue transcriptomes may be organized into gene coexpression networks, but their underlying biological drivers remain incompletely understood. Here, we undertook a large-scale transcriptomic study using 508 wild-type mouse striatal tissue samples dissected exclusively in the afternoons to define 38 highly reproducible gene coexpression modules. We found that 13 and 11 modules are enriched in cell-type and molecular complex markers, respectively. Importantly, 18 modules are highly enriched in daily rhythmically expressed genes that peak or trough with distinct temporal kinetics, revealing the underlying biology of striatal diurnal gene networks. Moreover, the diurnal coexpression networks are a dominant feature of daytime transcriptomes in the mouse cortex. We next employed the striatal coexpression modules to decipher the striatal transcriptomic signatures from Huntington's disease models and heterozygous null mice for 52 genes, uncovering novel functions for Prkcq and Kdm4b in oligodendrocyte differentiation and bipolar disorder-associated Trank1 in regulating anxiety-like behaviors and nocturnal locomotion.

Uninterrupted CAG repeat drives striatum-selective transcriptionopathy and nuclear pathogenesis in human Huntingtin BAC mice.

In Huntington's disease (HD), the uninterrupted CAG repeat length, but not the polyglutamine length, predicts disease onset. However, the underlying pathobiology remains unclear. Here, we developed bacterial artificial chromosome (BAC) transgenic mice expressing human mutant huntingtin (mHTT) with uninterrupted, and somatically unstable, CAG repeats that exhibit progressive disease-related phenotypes. Unlike prior mHTT transgenic models with stable, CAA-interrupted, polyglutamine-encoding repeats, BAC-CAG mice show robust striatum-selective nuclear inclusions and transcriptional dysregulation resembling those in murine huntingtin knockin models and HD patients. Importantly, the striatal transcriptionopathy in HD models is significantly correlated with their uninterrupted CAG repeat length but not polyglutamine length. Finally, among the pathogenic entities originating from mHTT genomic transgenes and only present or enriched in the uninterrupted CAG repeat model, somatic CAG repeat instability and nuclear mHTT aggregation are best correlated with early-onset striatum-selective molecular pathogenesis and locomotor and sleep deficits, while repeat RNA-associated pathologies and repeat-associated non-AUG (RAN) translation may play less selective or late pathogenic roles, respectively.

Striatal histamine mechanism in the pathogenesis of restless legs syndrome.

STUDY OBJECTIVES: Restless legs syndrome (RLS) has been hypothesized to be generated by abnormal striatal dopamine transmission. Dopaminergic drugs are effective for the treatment of RLS. However, long-term use of dopaminergic drugs causes adverse effects. We used iron-deficient (ID) and iron-replacement (IR) rats to address the neuropathology of RLS and to determine if a histamine H3 receptor (H3R) antagonist might be a useful treatment. Histamine H3R antagonists have been shown to decrease motor activity. METHODS: Control and ID rats were surgically implanted with electrodes for polysomnographic recording. After 3 days of baseline polysomnographic recordings, rats were systemically injected with the H3R agonist, α-methylhistamine, and antagonist, thioperamide. Recordings were continued after drug injection. Striatal H3R levels from control, ID, and IR rats were determined by western blots. Blood from control, ID, and IR rats was collected for the measurement of hematocrit levels. RESULTS: α-Methylhistamine and thioperamide increased and decreased motor activity, respectively, in control rats. In ID rats, α-methylhistamine had no effect on motor activity, whereas thioperamide decreased periodic leg movement (PLM) in sleep. Sleep-wake states were not significantly altered under any conditions. Striatal H3R levels were highest in ID rats, moderate to low in IR rats, and lowest in control rats. Striatal H3R levels were also found to positively and negatively correlate with PLM in sleep and hematocrit levels, respectively. CONCLUSIONS: A striatal histamine mechanism may be involved in ID anemia-induced RLS. Histamine H3R antagonists may be useful for the treatment of RLS.

Opiates increase the number of hypocretin-producing cells in human and mouse brain and reverse cataplexy in a mouse model of narcolepsy.

The changes in brain function that perpetuate opiate addiction are unclear. In our studies of human narcolepsy, a disease caused by loss of immunohistochemically detected hypocretin (orexin) neurons, we encountered a control brain (from an apparently neurologically normal individual) with 50% more hypocretin neurons than other control human brains that we had studied. We discovered that this individual was a heroin addict. Studying five postmortem brains from heroin addicts, we report that the brain tissue had, on average, 54% more immunohistochemically detected neurons producing hypocretin than did control brains from neurologically normal subjects. Similar increases in hypocretin-producing cells could be induced in wild-type mice by long-term (but not short-term) administration of morphine. The increased number of detected hypocretin neurons was not due to neurogenesis and outlasted morphine administration by several weeks. The number of neurons containing melanin-concentrating hormone, which are in the same hypothalamic region as hypocretin-producing cells, did not change in response to morphine administration. Morphine administration restored the population of detected hypocretin cells to normal numbers in transgenic mice in which these neurons had been partially depleted. Morphine administration also decreased cataplexy in mice made narcoleptic by the depletion of hypocretin neurons. These findings suggest that opiate agonists may have a role in the treatment of narcolepsy, a disorder caused by hypocretin neuron loss, and that increased numbers of hypocretin-producing cells may play a role in maintaining opiate addiction.

Motor hyperactivity of the iron-deficient rat - an animal model of restless legs syndrome.

BACKGROUND: Abnormal striatal dopamine transmission has been hypothesized to cause restless legs syndrome. Dopaminergic drugs are commonly used to treat restless legs syndrome. However, they cause adverse effects with long-term use. An animal model would allow the systematic testing of potential therapeutic drugs. A high prevalence of restless legs syndrome has been reported in iron-deficient anemic patients. We hypothesized that the iron-deficient animal would exhibit signs similar to those in restless legs syndrome patients. METHODS: After baseline polysomnographic recordings, iron-deficient rats received pramipexole injection. Then, iron-deficient rats were fed a standard rodent diet, and polysomnographic recording were performed for 2 days each week for 4 weeks. RESULTS: Iron-deficient rats have low hematocrit levels and show signs of restless legs syndrome: sleep fragmentation and periodic leg movements in wake and in slow-wave sleep. Iron-deficient rats had a positive response to pramipexole treatment. After the iron-deficient rats were fed the standard rodent diet, hematocrit returned to normal levels, and sleep quality improved, with increased average duration of wake and slow-wave sleep episodes. Periodic leg movements decreased during both waking and sleep. Hematocrit levels positively correlated with the average duration of episodes in wake and in slow-wave sleep and negatively correlated with periodic leg movements in wake and in sleep. Western blot analysis showed that striatal dopamine transporter levels were higher in iron-deficient rats. CONCLUSIONS: The iron-deficient rat is a useful animal model of iron-deficient anemic restless legs syndrome. © 2017 International Parkinson and Movement Disorder Society.

Gender differences between hypocretin/orexin knockout and wild type mice: age, body weight, body composition, metabolic markers, leptin and insulin resistance.

Female hypocretin knockout (Hcrt KO) mice have increased body weight despite decreased food intake compared to wild type (WT) mice. In order to understand the nature of the increased body weight, we carried out a detailed study of Hcrt KO and WT, male, and female mice. Female KO mice showed consistently higher body weight than WT mice, from 4 to 20 months (20-60%). Fat, muscle, and free fluid levels were all significantly higher in adult (7-9 months) as well as old (18-20 months) female KO mice compared to age-matched WT mice. Old male KO mice showed significantly higher fat content (150%) compared to age-matched WT mice, but no significant change in body weight. Respiratory quotient (-19%) and metabolic rates (-14%) were significantly lower in KO mice compared to WT mice, regardless of gender or age. Female KO mice had significantly higher serum leptin levels (191%) than WT mice at 18-20 months, but no difference between male mice were observed. Conversely, insulin resistance was significantly higher in both male (73%) and female (93%) KO mice compared to age- and sex-matched WT mice. We conclude that absence of the Hcrt peptide has gender-specific effects. In contrast, Hcrt-ataxin mice and human narcoleptics, with loss of the whole Hcrt cell, show weight gain in both sexes.

Greatly increased numbers of histamine cells in human narcolepsy with cataplexy.

OBJECTIVE: To determine whether histamine cells are altered in human narcolepsy with cataplexy and in animal models of this disease. METHODS: Immunohistochemistry for histidine decarboxylase (HDC) and quantitative microscopy were used to detect histamine cells in human narcoleptics, hypocretin (Hcrt) receptor-2 mutant dogs, and 3 mouse narcolepsy models: Hcrt (orexin) knockouts, ataxin-3-orexin, and doxycycline-controlled-diphtheria-toxin-A-orexin. RESULTS: We found an average 64% increase in the number of histamine neurons in human narcolepsy with cataplexy, with no overlap between narcoleptics and controls. However, we did not see altered numbers of HDC cells in any of the animal models of narcolepsy. INTERPRETATION: Changes in histamine cell numbers are not required for the major symptoms of narcolepsy, because all animal models have these symptoms. The histamine cell changes we saw in humans did not occur in the 4 animal models of Hcrt dysfunction we examined. Therefore, the loss of Hcrt receptor-2, of the Hcrt peptide, or of Hcrt cells is not sufficient to produce these changes. We speculate that the increased histamine cell numbers we see in human narcolepsy may instead be related to the process causing the human disorder. Although research has focused on possible antigens within the Hcrt cells that might trigger their autoimmune destruction, the present findings suggest that the triggering events of human narcolepsy may involve a proliferation of histamine-containing cells. We discuss this and other explanations of the difference between human narcoleptics and animal models of narcolepsy, including therapeutic drug use and species differences.

Highly specific role of hypocretin (orexin) neurons: differential activation as a function of diurnal phase, operant reinforcement versus operant avoidance and light level.

Hypocretin (Hcrt) cell loss is responsible for narcolepsy, but Hcrt's role in normal behavior is unclear. We found that Hcrt knock-out mice were unable to work for food or water reward during the light phase. However, they were unimpaired relative to wild-type (WT) mice when working for reward during the dark phase or when working to avoid shock in the light or dark phase. In WT mice, expression of Fos in Hcrt neurons occurs only in the light phase when working for positive reinforcement. Expression was seen throughout the mediolateral extent of the Hcrt field. Fos was not expressed when expected or unexpected unearned rewards were presented, when working to avoid negative reinforcement, or when given or expecting shock, even though these conditions elicit maximal electroencephalogram (EEG) arousal. Fos was not expressed in the light phase when light was removed. This may explain the lack of light-induced arousal in narcoleptics and its presence in normal individuals. This is the first demonstration of such specificity of arousal system function and has implications for understanding the motivational and circadian consequences of arousal system dysfunction. The current results also indicate that comparable and complementary specificities must exist in other arousal systems.

Sleep deprivation under sustained hypoxia protects against oxidative stress.

We previously showed that total sleep deprivation increased antioxidant responses in several rat brain regions. We also reported that chronic hypoxia enhanced antioxidant responses and increased oxidative stress in rat cerebellum and pons, relative to normoxic conditions. In the current study, we examined the interaction between these two parameters (sleep and hypoxia). We exposed rats to total sleep deprivation under sustained hypoxia (SDSH) and compared changes in antioxidant responses and oxidative stress markers in the neocortex, hippocampus, brainstem, and cerebellum to those in control animals left undisturbed under either sustained hypoxia (UCSH) or normoxia (UCN). We measured changes in total nitrite levels as an indicator of nitric oxide (NO) production, superoxide dismutase (SOD) activity and total glutathione (GSHt) levels as markers of antioxidant responses, and levels of thiobarbituric acid-reactive substances (TBARS) and protein carbonyls as signs of lipid and protein oxidation products, respectively. We found that acute (6h) SDSH increased NO production in the hippocampus and increased GSHt levels in the neocortex, brainstem, and cerebellum while decreasing hippocampal lipid oxidation. Additionally, we observed increased hexokinase activity in the neocortex of SDSH rats compared to UCSH rats, suggesting that elevated glucose metabolism may be one potential source of the enhanced free radicals produced in this brain region. We conclude that short-term insomnia under hypoxia may serve as an adaptive response to prevent oxidative stress.

Short-term total sleep deprivation in the rat increases antioxidant responses in multiple brain regions without impairing spontaneous alternation behavior.

Total sleep deprivation (TSD) induces a broad spectrum of cognitive, behavioral and cellular changes. We previously reported that long term (5-11 days) TSD in the rat, by the disk-over-water method, decreases the activity of the antioxidant enzyme superoxide dismutase (SOD) in the brainstem and hippocampus. To gain insight into the mechanisms causing cognitive impairment, here we explore the early associations between metabolic activity, antioxidant responses and working memory (one form of cognitive impairment). Specifically we investigated the impact of short-term (6h) TSD, by gentle handling, on the levels of the endogenous antioxidant, total glutathione (GSHt), and the activities of the antioxidative enzymes, SOD and glutathione peroxidase (GPx). Short-term TSD had no significant impact on SOD activity, but increased GSHt levels in the rat cortex, brainstem and basal forebrain, and GPx activity in the rat hippocampus and cerebellum. We also observed increased activity of hexokinase, (HK), the rate limiting enzyme of glucose metabolism, in the rat cortex and hypothalamus. We further showed that 6h of TSD leads to increased exploratory behavior to a new environment, without impairing spontaneous alternation behavior (SAB) in the Y maze. We conclude that acute (6h) sleep loss may trigger compensatory mechanisms (like increased antioxidant responses) that prevent initial deterioration in working memory.

Rapid changes in glutamate levels in the posterior hypothalamus across sleep-wake states in freely behaving rats.

The histamine-containing posterior hypothalamic region (PH-TMN) plays a key role in sleep-wake regulation. We investigated rapid changes in glutamate release in the PH-TMN across the sleep-wake cycle with a glutamate biosensor that allows the measurement of glutamate levels at 1- to 4-s resolution. In the PH-TMN, glutamate levels increased in active waking (AW) and rapid eye movement (REM) sleep compared with quiet waking and nonrapid eye movement (NREM) sleep. There was a rapid (0.6 +/- 1.8 s) and progressive increase in glutamate levels at REM sleep onset. A reduction in glutamate levels consistently preceded the offset of REM sleep by 8 +/- 3 s. Short-duration sleep deprivation resulted in a progressive increase in glutamate levels in the PH-TMN, perifornical-lateral hypothalamus (PF-LH), and cortex. We found that in the PF-LH, glutamate levels took a longer time to return to basal values compared with the time it took for glutamate levels to increase to peak values during AW onset. This is in contrast to other regions we studied in which the return to baseline values after AW was quicker than their rise with waking onset. In summary, we demonstrated an increase in glutamate levels in the PH-TMN with REM/AW onset and a drop in glutamate levels before the offset of REM. High temporal resolution measurement of glutamate levels reveals dynamic changes in release linked to the initiation and termination of REM sleep.

Antioxidant responses to chronic hypoxia in the rat cerebellum and pons.

Obstructive sleep apnea (OSA) is characterized by chronic intermittent hypoxia (CIH) and sleep fragmentation and deprivation. Exposure to CIH results in oxidative stress in the cortex, hippocampus and basal forebrain of rats and mice. We show that sustained and intermittent hypoxia induces antioxidant responses, an indicator of oxidative stress, in the rat cerebellum and pons. Increased glutathione reductase (GR) activity and thiobarbituric acid reactive substance (TBARS) levels were observed in the pons and cerebellum of rats exposed to CIH or chronic sustained hypoxia (CSH) compared with room air (RA) controls. Exposure to CIH or CSH increased GR activity in the pons, while exposure to CSH increased the level of TBARS in the cerebellum. The level of TBARS was increased to a greater extent after exposure to CSH than to CIH in the cerebellum and pons. Increased superoxide dismutase activity (SOD) and decreased total glutathione (GSHt) levels were observed after exposure to CIH compared with CSH only in the pons. We have previously shown that prolonged sleep deprivation decreased SOD activity in the rat hippocampus and brainstem, without affecting the cerebellum, cortex or hypothalamus. We therefore conclude that sleep deprivation and hypoxia differentially affect antioxidant responses in different brain regions.

Sleep deprivation decreases superoxide dismutase activity in rat hippocampus and brainstem.

Sleep deprivation by the disk-over-water technique results in a predictable syndrome of physiological changes in rats. It has been proposed that reactive oxygen species (ROS) may be responsible for some of these effects. A variety of antioxidative enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GPx) help to regulate the level of ROS. In this study we investigated the effects of prolonged (5-11 days) sleep deprivation on the activities of SOD and GPx as well as the metabolic activity of the mitochondria (using alamar blue) in several brain regions (cortex, hippocampus, hypothalamus, brainstem and cerebellum). We show that prolonged sleep deprivation significantly decreased Cu/Zn-SOD activity in the hippocampus and brainstem, suggesting an alteration in the metabolism of ROS resulting in oxidative stress.

Increases in amino-cupric-silver staining of the supraoptic nucleus after sleep deprivation.

Sleep deprived rats undergo a predictable sequence of physiological changes, including changes in skin condition, increased energy expenditure, and altered thermoregulation. Amino-cupric-silver staining was used to identify sleep deprivation related changes in the brain. A significant increase in staining was observed in the supraoptic nucleus (SON) of the hypothalamus of rats with high sleep loss (>45 h) vs. their yoked controls. Follow-up experiments showed that staining was not significantly different in rats sleep deprived for less than 45 h, suggesting that injurious sleep deprivation-related processes occur above a threshold quantity of sleep loss. These anatomical changes suggest that the effects of sleep deprivation may be related to protein metabolism in certain brain regions.