Evidence for altered cholesterol metabolism in Huntington's disease post mortem brain tissue.

AIMS: Cholesterol plays an essential role in membrane structure and function, being especially important in the brain. Alteration of brain cholesterol synthesis and metabolism has been demonstrated in several Huntington's disease (HD) mouse and cell models; however, less is known about these alterations in human tissue. This study aimed to identify alterations to cholesterol synthetic and metabolic pathways in human HD brain tissue. METHODS: A broad range of cholesterol synthetic precursors, metabolites and oxidation products were measured by gas chromatography-tandem mass spectrometry in five regions of human post mortem HD brain and compared with age- and sex-matched control tissues. The level of enzymes that regulate cholesterol homeostasis, cholesterol 24-hydroxylase and delta(24)-sterol reductase were investigated by Western blotting and qPCR in putamen. RESULTS: The most significant changes were localized to the putamen, where a 60% decrease in 24(S)-hydroxycholesterol, 30% increase in cholesterol and 100-200% increase in synthetic precursors (lathosterol, zymosterol and desmosterol) was detected. The enzymes cholesterol 24-hydroxylase and delta(24)-sterol reductase were also significantly decreased in HD putamen as compared with control tissues. Free radical-generated cholesterol oxidation products 7-keto cholesterol and 7ß-hydroxycholesterol were also increased by 50-70% in HD putamen. CONCLUSION: Human HD brain has significantly decreased cholesterol metabolism and disrupted cholesterol homeostasis. Our data also indicate that lipid oxidative stress accompanies HD pathology.

Cannabidiol (CBD) treatment improves spatial memory in 14-month-old female TAU58/2 transgenic mice.

Frontotemporal dementia (FTD) and Alzheimer's disease (AD) share the pathological hallmark of intracellular neurofibrillary tangles, which result from the hyperphosphorylation of microtubule associated protein tau. The P301S mutation in human tau carried by TAU58/2 transgenic mice results in brain pathology and behavioural deficits relevant to FTD and AD. The phytocannabinoid cannabidiol (CBD) exhibits properties beneficial for multiple pathological processes evident in dementia. Therefore, 14-month-old female TAU58/2 transgenic and wild type-like (WT) littermates were treated with 100 mg/kg CBD or vehicle i.p. starting three weeks prior to conducting behavioural paradigms relevant to FTD and AD. TAU58/2 females exhibited impaired motor function, reduced bodyweight and less anxiety behaviour compared to WT. Impaired spatial reference memory of vehicle-treated transgenic mice was restored by chronic CBD treatment. Chronic CBD also reduced anxiety-like behaviours and decreased contextual fear-associated freezing in all mice. Chronic remedial CBD treatment ameliorated several disease-relevant phenotypes in 14-month-old TAU58/2 transgenic mice, suggesting potential for the treatment of tauopathy-related behavioural impairments including cognitive deficits.

The behavioural phenotype of 14-month-old female TAU58/2 transgenic mice.

Frontotemporal dementia (FTD) and Alzheimer's disease (AD) exhibit intracellular inclusions [neurofibrillary tangles (NFT's)] of microtubule-associated protein tau that contributes to neuronal dysfunction and death. Mutations in the microtubule-associated protein tau (MAPT) gene leads to tau hyperphosphorylation and promotes NFT formation. The TAU58/2 transgenic mouse model expresses mutant human tau (P301S mutation) and exhibits behavioural abnormalities relevant to dementia in early adulthood. Here we comprehensively determined the behavioural phenotype of TAU58/2 transgenic female mice at 14 months of age using test paradigms relevant to FTD and AD. TAU58/2 females showed a significant motor deficit and lower bodyweight compared to WT littermates. Transgenic females failed to habituate to the test arena in the light-dark test. Interestingly, transgenics did not exhibit an anxiolytic-like phenotype and intermediate-term spatial learning in the cheeseboard test was intact. However, a significant learning deficit was detected in the 1st trial across test days indicating impaired long-term spatial memory. In addition, the preference for a previously rewarded location was absent in transgenic females during probe trial testing. Finally, TAU58/2 mice had a defective acoustic startle response and impaired sensorimotor gating. In conclusion TAU58/2 mice exhibit several behavioural deficits that resemble those observed in human FTD and AD. Additionally, we observed a novel startle response deficit in these mice. At 14 months of age, TAU58/2 females represent a later disease stage and are therefore a potentially useful model to test efficacy of therapeutics to reverse or ameliorate behavioural deficits in post-onset tauopapthy-related neurodegenerative disorders.

Detection of retinal and blood Aß oligomers with nanobodies.

INTRODUCTION: Abnormal retinal changes are increasingly recognized as an early pathological change in Alzheimer's disease (AD). Although amyloid beta oligomers (Aßo) have been shown to accumulate in the blood and retina of AD patients and animals, it is not known whether the early Aßo deposition precedes their accumulation in brain. METHODS AND RESULTS: Using nanobodies targeting Aß1-40 and Aß1-42 oligomers we were able to detect Aß oligomers in the retina and blood but not in the brain of 3-month-old APP/PS1 mice. Furthermore, Aß plaques were detected in the brain but not the retina of 3-month-old APP/PS1 mice. CONCLUSION: These results suggest that retinal accumulation of Aßo originates from peripheral blood and precedes cognitive decline and Aßo deposition in the brain. This provides a very strong basis to develop and implement an "eye test" for early detection of AD using nanobodies targeting retinal Aß.

CuATSM improves motor function and extends survival but is not tolerated at a high dose in SOD1G93A mice with a C57BL/6 background.

The synthetic copper-containing compound, CuATSM, has emerged as one of the most promising drug candidates developed for the treatment of amyotrophic lateral sclerosis (ALS). Multiple studies have reported CuATSM treatment provides therapeutic efficacy in various mouse models of ALS without any observable adverse effects. Moreover, recent results from an open label clinical study suggested that daily oral dosing with CuATSM slows disease progression in patients with both sporadic and familial ALS, providing encouraging support for CuATSM in the treatment of ALS. Here, we assessed CuATSM in high copy SOD1G93A mice on the congenic C57BL/6 background, treating at 100 mg/kg/day by gavage, starting at 70 days of age. This dose in this specific model has not been assessed previously. Unexpectedly, we report a subset of mice initially administered CuATSM exhibited signs of clinical toxicity, that necessitated euthanasia in extremis after 3-51 days of treatment. Following a 1-week washout period, the remaining mice resumed treatment at the reduced dose of 60 mg/kg/day. At this revised dose, treatment with CuATSM slowed disease progression and increased survival relative to vehicle-treated littermates. This work provides the first evidence that CuATSM produces positive disease-modifying outcomes in high copy SOD1G93A mice on a congenic C57BL/6 background. Furthermore, results from the 100 mg/kg/day phase of the study support dose escalation determination of tolerability as a prudent step when assessing treatments in previously unassessed models or genetic backgrounds.

Medium-Dose Chronic Cannabidiol Treatment Reverses Object Recognition Memory Deficits of APP Swe /PS1ΔE9 Transgenic Female Mice.

Alzheimer's disease (AD) is a neurodegenerative disease that causes behavioral and cognitive impairments. The phytocannabinoid cannabidiol (CBD) has anti-inflammatory, antioxidant, and neuroprotective properties, and in vitro and limited in vivo evidence suggests that CBD possesses therapeutic-like properties for the treatment of AD. Cannabinoids are known to have dose-dependent effects and the therapeutic potential of medium-dose CBD for AD transgenic mice has not been assessed in great detail yet. 12-month-old control and APP Swe /PS1ΔE9 (APPxPS1) transgenic female mice were treated daily via intraperitoneal injection with 5 mg/kg bodyweight CBD (or vehicle) commencing three weeks prior to the assessment of behavioral domains including anxiety, exploration, locomotion, motor functions, cognition, and sensorimotor gating. APPxPS1 mice exhibited a hyperlocomotive and anxiogenic-like phenotype and had wild type-like motor and spatial learning abilities, although AD transgenic mice took generally longer to complete the cheeseboard training (due to a lower locomotion speed). Furthermore spatial learning and reversal learning was delayed by one day in APPxPS1 mice compared to control mice. All mice displayed intact spatial memory and retrieval memory, but APPxPS1 mice showed reduced levels of perseverance in the cheeseboard probe trial. Importantly, vehicle-treated APPxPS1 mice were characterized by object recognition deficits and delayed spatial learning, which were reversed by CBD treatment. Finally, impairments in sensorimotor gating of APPxPS1 mice were not affected by CBD. In conclusion, medium-dose CBD appears to have therapeutic value for the treatment of particular behavioral impairments present in AD patients. Future research should consider the molecular mechanisms behind CBD's beneficial properties for AD transgenic mice.

Novel behavioural characteristics of the superoxide dismutase 1 G93A (SOD1G93A ) mouse model of amyotrophic lateral sclerosis include sex-dependent phenotypes.

Amyotrophic lateral sclerosis (ALS) involves the rapid degeneration of upper and lower motor neurons leading to weakening and paralysis of voluntary movements. Mutations in copper-zinc superoxide dismutase 1 (SOD1) are a known genetic cause of ALS, and the SOD1 G93A mouse has been used extensively to investigate molecular mechanisms in ALS. In recent years, evidence suggests that ALS and frontotemporal dementia form a spectrum disorder ranging from motor to cognitive dysfunctions. Thus, we tested male and female SOD1 G93A mice for the first time before the onset of debilitating motor impairments in behavioural domains relevant to both ALS and frontotemporal dementia. SOD1 G93A males displayed reduced locomotion, exploration and increased anxiety-like behaviours compared with control males. Intermediate-term spatial memory was impaired in SOD1 G93A females, whereas long-term spatial memory deficits as well as lower acoustic startle response, and prepulse inhibition were identified in SOD1 G93A mice of both sexes compared with respective controls. Interestingly, SOD1 G93A males exhibited an increased conditioned cue freezing response. Nosing behaviours were also elevated in both male and female SOD1 G93A when assessed in social paradigms. In conclusion, SOD1 G93A mice exhibit a variety of sex-specific behavioural deficits beyond motor impairments supporting the notion of an ALS-frontotemporal spectrum disorder. Thus, SOD1 G93A mice may represent a useful model to test the efficacy of therapeutic interventions on clinical symptoms in addition to declining motor abilities.

Cholesteryl ester levels are elevated in the caudate and putamen of Huntington's disease patients.

Huntington's disease (HD) is an autosomal dominant neurodegenerative illness caused by a mutation in the huntingtin gene (HTT) and subsequent protein (mhtt), to which the brain shows a region-specific vulnerability. Disturbances in neural cholesterol metabolism are established in HD human, murine and cell studies; however, cholesteryl esters (CE), which store and transport cholesterol in the brain, have not been investigated in human studies. This study aimed to identify region-specific alterations in the concentrations of CE in HD. The Victorian Brain Bank provided post-mortem tissue from 13 HD subjects and 13 age and sex-matched controls. Lipids were extracted from the caudate, putamen and cerebellum, and CE were quantified using targeted mass spectrometry. ACAT 1 protein expression was measured by western blot. CE concentrations were elevated in HD caudate and putamen compared to controls, with the elevation more pronounced in the caudate. No differences in the expression of ACAT1 were identified in the striatum. No remarkable differences in CE were detected in HD cerebellum. The striatal region-specific differences in CE profiles indicate functional subareas of lipid disturbance in HD. The increased CE concentration may have been induced as a compensatory mechanism to reduce cholesterol accumulation.

First behavioural assessment of a novel Immp2l knockdown mouse model with relevance for Gilles de la Tourette syndrome and Autism spectrum disorder.

Gilles de la Tourette syndrome (GTS) is a neurodevelopmental disorder, which shares some clinical features with Autism spectrum disorder (ASD). The genetic factors relevant to the development of both disorders are yet to be fully understood, however, some genetic association studies have identified inner mitochondrial membrane peptidase subunit 2 (IMMP2L) as a potential risk gene for both GTS and ASD. The impact of Immp2l deficiency on behavioural domains is currently unknown. A new genetic mouse model for Immp2l was developed. Adult heterozygous (HET) and homozygous (HOMO) Immp2l knockdown (Immp2l KD) mice of both sexes were compared to wild type-like (WT) littermates in the open field (OF), social interaction, novel object recognition, marble burying, and prepulse inhibition (PPI). The effect of acute dexamphetamine (2 mg/kg) on OF behaviour was also determined. OF locomotion was significantly higher in HET compared to HOMO male littermates. Male and female HOMO mice were much more sensitive to the locomotor-stimulating effects of dexamphetamine (DEX), whereas only HOMO males exhibited significant increased DEX-induced OF exploration compared to control groups. HOMO females failed to habituate to an acoustic startle stimulus. Furthermore, compared to HOMO females, HET females showed reduced social interaction, and a similar trend was seen in HET males. The Immp2l KD mouse model possesses moderate face validity for preclinical research into GTS and ASD, in particular as dysfunctional dopaminergic neurotransmission appears to be one mechanism leading to disease presentation. The sex-dependent differences observed in most findings reinforce the strong influence of sex in the pathophysiology of GTS and ASD.

Therapeutic Effects of Anthocyanins and Environmental Enrichment in R6/1 Huntington's Disease Mice.

BACKGROUND: Huntington's disease (HD) is a progressive neurodegenerative disease with no effective treatment or cure. Environmental enrichment has been used to slow processes leading to ageing and neurodegenerative diseases including HD. Phenolic phytochemicals including anthocyanins have also been shown to improve brain function in ageing and neurodegenerative diseases. OBJECTIVE: This study examined the effects of anthocyanin dietary supplementation and environmental enrichment on behavioural phenotypes and brain cholesterol metabolic alterations in the R6/1 mouse model of HD. METHODS: R6/1 HD mice and their wild-type littermate controls were randomised into the different experimental conditions, involving either environmentally enriched versus standard housing conditions, or anthocyanin versus control diet. Motor dysfunction was assessed from 6 to 26 weeks using the RotaRod and the hind-paw clasping tests. Gas chromatography - tandem mass spectrometry was used to quantify a broad range of sterols in the striatum and cortex of R6/1 HD mice. RESULTS: Anthocyanin dietary supplementation delayed the onset of motor dysfunction in female HD mice. Environmental enrichment improved motor function and the hind paw clasping phenotype in male HD mice only. These mice also had lower levels of cholesterol oxidation products in the cortex compared to standard-housed mice. CONCLUSION: Both anthocyanin supplementation and environmental enrichment are able to improve the motor dysfunction phenotype of R6/1 mice, however the effectiveness of these interventions was different between the two sexes. The interventions examined did not alter brain cholesterol metabolic deficits that have been reported previously in this mouse model of HD.

Brain Cholesterol Synthesis and Metabolism is Progressively Disturbed in the R6/1 Mouse Model of Huntington's Disease: A Targeted GC-MS/MS Sterol Analysis.

BACKGROUND: Cholesterol has essential functions in neurological processes that require tight regulation of synthesis and metabolism. Perturbed cholesterol homeostasis has been demonstrated in Huntington's disease, however the exact role of these changes in disease pathogenesis is not fully understood. OBJECTIVE: This study aimed to comprehensively examine changes in cholesterol biosynthetic precursors, metabolites and oxidation products in the striatum and cortex of the R6/1 transgenic mouse model of Huntington's disease. We also aimed to characterise the progression of the physical phenotype in these mice. METHODS: GC-MS/MS was used to quantify a broad range of sterols in the striatum and cortex of R6/1 and wild type mice at 6, 12, 20, 24 and 28 weeks of age. Motor dysfunction was assessed over 28 weeks using the RotaRod and the hind-paw clasping tests. RESULTS: 24(S)-Hydroxycholesterol and 27-hydroxycholesterol were the major cholesterol metabolites that significantly changed in R6/1 mice. These changes were specifically localised to the striatum and were detected at the end stages of the disease. Cholesterol synthetic precursors (lathosterol and lanosterol) were significantly reduced in the cortex and striatum by 6 weeks of age, prior to the onset of motor dysfunction, as well as the cognitive and affective abnormalities previously reported. Elevated levels of desmosterol, a substrate of delta(24)-sterol reductase (DHCR24), were also detected in R6/1 mice at the end time-point. Female R6/1 mice exhibited a milder weight loss and hind paw clasping phenotype compared to male R6/1 mice, however, no difference in the brain sterol profile was detected between sexes. CONCLUSION: Several steps in cholesterol biosynthetic and metabolic pathways are differentially altered in the R6/1 mouse brain as the disease progresses and this is most severe in the striatum. This provides further insights into early molecular mediators of HD onset and disease progression and identifies candidate molecular targets for novel therapeutic approaches.