Single nuclei RNA-seq reveals a medium spiny neuron glutamate excitotoxicity signature prior to the onset of neuronal death in an ovine Huntington's disease model.

Huntington's disease (HD) is a neurodegenerative genetic disorder caused by an expansion in the CAG repeat tract of the huntingtin (HTT) gene resulting in behavioural, cognitive, and motor defects. Current knowledge of disease pathogenesis remains incomplete, and no disease course-modifying interventions are in clinical use. We have previously reported the development and characterisation of the OVT73 transgenic sheep model of HD. The 73 polyglutamine repeat is somatically stable and therefore likely captures a prodromal phase of the disease with an absence of motor symptomatology even at 5-years of age and no detectable striatal cell loss. To better understand the disease-initiating events we have undertaken a single nuclei transcriptome study of the striatum of an extensively studied cohort of 5-year-old OVT73 HD sheep and age matched wild-type controls. We have identified transcriptional upregulation of genes encoding N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors in medium spiny neurons, the cell type preferentially lost early in HD. Further, we observed an upregulation of astrocytic glutamate uptake transporters and medium spiny neuron GABAA receptors, which may maintain glutamate homeostasis. Taken together, these observations support the glutamate excitotoxicity hypothesis as an early neurodegeneration cascade-initiating process but the threshold of toxicity may be regulated by several protective mechanisms. Addressing this biochemical defect early may prevent neuronal loss and avoid the more complex secondary consequences precipitated by cell death.

Proteomic Analysis of an Induced Pluripotent Stem Cell Model Reveals Strategies to Treat Juvenile Myelomonocytic Leukemia.

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of early childhood with a poor survival rate, thus there is a requirement for improved treatment strategies. Induced pluripotent stem cells offer the ability to model disease and develop new treatment strategies. JMML is frequently associated with mutations in PTPN11. Children with Noonan syndrome, a development disorder, have an increased incidence of JMML associated with specific germline mutations in PTPN11. We undertook a proteomic assessment of myeloid cells derived from induced pluripotent stem cells obtained from Noonan syndrome patients with PTPN11 mutations, either associated or not associated with an increased incidence of JMML. We report that the proteomic perturbations induced by the leukemia-associated PTPN11 mutations are associated with TP53 and NF-Kκb signaling. We have previously shown that MYC is involved in the differential gene expression observed in Noonan syndrome patients associated with an increased incidence of JMML. Thus, we employed drugs to target these pathways and demonstrate differential effects on clonogenic hematopoietic cells derived from Noonan syndrome patients, who develop JMML and those who do not. Further, we demonstrated these small molecular inhibitors, JQ1 and CBL0137, preferentially extinguish primitive hematopoietic cells from sporadic JMML patients as opposed to cells from healthy individuals.

Cerebral deficiency of vitamin B5 (d-pantothenic acid; pantothenate) as a potentially-reversible cause of neurodegeneration and dementia in sporadic Alzheimer's disease.

Alzheimer's disease (AD) is the most common cause of age-related neurodegeneration and dementia, and there are no available treatments with proven disease-modifying actions. It is therefore appropriate to study hitherto-unknown aspects of brain structure/function in AD to seek alternative disease-related mechanisms that might be targeted by new therapeutic interventions with disease-modifying actions. During hypothesis-generating metabolomic studies of brain, we identified apparent differences in levels of vitamin B5 between AD cases and controls. We therefore developed a method based on gas chromatography-mass spectrometry by which we quantitated vitamin B5 concentrations in seven brain regions from nine AD cases and nine controls. We found that widespread, severe cerebral deficiency of vitamin B5 occurs in AD. This deficiency was worse in those regions known to undergo severe damage, including the hippocampus, entorhinal cortex, and middle temporal gyrus. Vitamin B5 is the obligate precursor of CoA/acetyl-CoA (acetyl-coenzyme A), which plays myriad key roles in the metabolism of all organs, including the brain. In brain, acetyl-CoA is the obligate precursor of the neurotransmitter acetylcholine, and the complex fatty-acyl groups that mediate the essential insulator role of myelin, both processes being defective in AD; moreover, the large cerebral vitamin B5 concentrations co-localize almost entirely to white matter. Vitamin B5 is well tolerated when administered orally to humans and other mammals. We conclude that cerebral vitamin B5 deficiency may well cause neurodegeneration and dementia in AD, which might be preventable or even reversible in its early stages, by treatment with suitable oral doses of vitamin B5.

Vitamin B5 (d-pantothenic acid) localizes in myelinated structures of the rat brain: Potential role for cerebral vitamin B5 stores in local myelin homeostasis.

Vitamin B5 (d-pantothenic acid; pantothenate) is an essential trace nutrient that functions as the obligate precursor of coenzyme A (CoA), through which it plays key roles in myriad biological processes, including many that regulate carbohydrate, lipid, protein, and nucleic acid metabolism. In the brain, acetyl-CoA is necessary for synthesis of the complex fatty-acyl chains of myelin, and of the neurotransmitter acetylcholine. We recently found that cerebral pantothenate is markedly lowered, averaging ∼55% of control values in cases of Huntington's disease (HD) including those who are pre-symptomatic, and that regions where pantothenate is lowered correspond to those which are more severely damaged. Here we sought to determine the previously unknown distribution of pantothenate in the normal-rat brain, and whether the diabetic rat might be useful as a model for altered cerebral pantothenate metabolism. We employed histological staining (Nissl) to identify brain structures; immunohistochemistry with anti-pantothenate antibodies to determine the distribution of pantothenate in caudate putamen and cerebellum; and gas-chromatography/mass-spectrometry to quantitate levels of pantothenate and other metabolites in normal- and diabetic-rat brain. Remarkably, cerebral pantothenate was almost entirely localized to myelin-containing structures in both experimental groups. Diabetes did not modify levels or disposition of cerebral pantothenate. These findings are consistent with physiological localization of pantothenate in myelinated white-matter structures, where it could serve to support myelin synthesis. Further investigation of cerebral pantothenate is warranted in neurodegenerative diseases such as HD and Alzheimer's disease, where myelin loss is a known characteristic of pathogenesis.

Brain urea increase is an early Huntington's disease pathogenic event observed in a prodromal transgenic sheep model and HD cases.

The neurodegenerative disorder Huntington's disease (HD) is typically characterized by extensive loss of striatal neurons and the midlife onset of debilitating and progressive chorea, dementia, and psychological disturbance. HD is caused by a CAG repeat expansion in the Huntingtin (HTT) gene, translating to an elongated glutamine tract in the huntingtin protein. The pathogenic mechanism resulting in cell dysfunction and death beyond the causative mutation is not well defined. To further delineate the early molecular events in HD, we performed RNA-sequencing (RNA-seq) on striatal tissue from a cohort of 5-y-old OVT73-line sheep expressing a human CAG-expansion HTT cDNA transgene. Our HD OVT73 sheep are a prodromal model and exhibit minimal pathology and no detectable neuronal loss. We identified significantly increased levels of the urea transporter SLC14A1 in the OVT73 striatum, along with other important osmotic regulators. Further investigation revealed elevated levels of the metabolite urea in the OVT73 striatum and cerebellum, consistent with our recently published observation of increased urea in postmortem human brain from HD cases. Extending that finding, we demonstrate that postmortem human brain urea levels are elevated in a larger cohort of HD cases, including those with low-level neuropathology (Vonsattel grade 0/1). This elevation indicates increased protein catabolism, possibly as an alternate energy source given the generalized metabolic defect in HD. Increased urea and ammonia levels due to dysregulation of the urea cycle are known to cause neurologic impairment. Taken together, our findings indicate that aberrant urea metabolism could be the primary biochemical disruption initiating neuropathogenesis in HD.

Plasma metals as potential biomarkers in dementia: a case-control study in patients with sporadic Alzheimer's disease.

Sporadic Alzheimer's disease (AD) is a neurodegenerative disorder that causes the most prevalent form of age-related dementia but its pathogenesis remains obscure. Altered regulation of metals, particularly pan-cerebral copper deficiency, and more regionally-localized perturbation of other metals, are prominent in AD brain although data on how these CNS perturbations are reflected in the peripheral bloodstream are inconsistent to date. To assess the potential use of metal dysregulation to generate biomarkers in AD, we performed a case-control study of seven essential metals and selenium, measured by inductively coupled plasma mass-spectrometry, in samples from AD and matched control cases. Metals were sodium, potassium, calcium, magnesium, iron, zinc, and copper. In the whole study-group and in female participants, plasma metal levels did not differ between cases and controls. In males by contrast, there was moderate evidence that zinc levels trended towards increase in AD [10.8 (10.2-11.5)] µmol/L, mean (± 95% CI; P = 0.021) compared with controls [10.2 (9.6-10.4)]. Thus alterations in plasma zinc levels differed between genders in AD. In correlational analysis, there was evidence for an increased number of 'strong' metal co-regulations in AD cases and differential co-modulations of metal pairs: copper-sodium (Rcontrol = - 0.03, RAD = 0.65; P = 0.009), and copper-calcium (Rcontrol = - 0.01, RAD = 0.65; P = 0.01) were significant in AD males, potentially consistent with reported evidence for dysregulation of copper in severely damaged brain regions in AD. In conclusion, our data suggest that the measurement of metals co-regulation in plasma may provide a useful representation of those metal perturbations taking place in the AD brain and therefore might be useful as plasma-based biomarkers.

Metabolite mapping reveals severe widespread perturbation of multiple metabolic processes in Huntington's disease human brain.

Huntington's disease (HD) is a genetically-mediated neurodegenerative disorder wherein the aetiological defect is a mutation in the Huntington's gene (HTT), which alters the structure of the huntingtin protein (Htt) through lengthening of its polyglutamine tract, thus initiating a cascade that ultimately leads to premature death. However, neurodegeneration typically manifests in HD only in middle age, and mechanisms linking the causative mutation to brain disease are poorly understood. Brain metabolism is severely perturbed in HD, and some studies have indicated a potential role for mutant Htt as a driver of these metabolic aberrations. Here, our objective was to determine the effects of HD on brain metabolism by measuring levels of polar metabolites in regions known to undergo varying degrees of damage. We performed gas-chromatography/mass spectrometry-based metabolomic analyses in a case-control study of eleven brain regions in short post-mortem-delay human tissue from nine well-characterized HD patients and nine matched controls. In each patient, we measured metabolite content in representative tissue-samples from eleven brain regions that display varying degrees of damage in HD, thus identifying the presence and abundance of 63 different metabolites from several molecular classes, including carbohydrates, amino acids, nucleosides, and neurotransmitters. Robust alterations in regional brain-metabolite abundances were observed in HD patients: these included changes in levels of small molecules that play important roles as intermediates in the tricarboxylic-acid and urea cycles, and amino-acid metabolism. Our findings point to widespread disruption of brain metabolism and indicate a complex phenotype beyond the gradient of neuropathologic damage observed in HD brain.

Graded perturbations of metabolism in multiple regions of human brain in Alzheimer's disease: Snapshot of a pervasive metabolic disorder.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder that displays pathological characteristics including senile plaques and neurofibrillary tangles. Metabolic defects are also present in AD-brain: for example, signs of deficient cerebral glucose uptake may occur decades before onset of cognitive dysfunction and tissue damage. There have been few systematic studies of the metabolite content of AD human brain, possibly due to scarcity of high-quality brain tissue and/or lack of reliable experimental methodologies. Here we sought to: 1) elucidate the molecular basis of metabolic defects in human AD-brain; and 2) identify endogenous metabolites that might guide new approaches for therapeutic intervention, diagnosis or monitoring of AD. Brains were obtained from nine cases with confirmed clinical/neuropathological AD and nine controls matched for age, sex and post-mortem delay. Metabolite levels were measured in post-mortem tissue from seven regions: three that undergo severe neuronal damage (hippocampus, entorhinal cortex and middle-temporal gyrus); three less severely affected (cingulate gyrus, sensory cortex and motor cortex); and one (cerebellum) that is relatively spared. We report a total of 55 metabolites that were altered in at least one AD-brain region, with different regions showing alterations in between 16 and 33 metabolites. Overall, we detected prominent global alterations in metabolites from several pathways involved in glucose clearance/utilization, the urea cycle, and amino-acid metabolism. The finding that potentially toxigenic molecular perturbations are widespread throughout all brain regions including the cerebellum is consistent with a global brain disease process rather than a localized effect of AD on regional brain metabolism.

Identification of elevated urea as a severe, ubiquitous metabolic defect in the brain of patients with Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder wherein the aetiological defect is a mutation in the Huntington's gene (HTT), which alters the structure of the huntingtin protein through the lengthening of a polyglutamine tract and initiates a cascade that ultimately leads to dementia and premature death. However, neurodegeneration typically manifests in HD only in middle age, and processes linking the causative mutation to brain disease are poorly understood. Here, our objective was to elucidate further the processes that cause neurodegeneration in HD, by measuring levels of metabolites in brain regions known to undergo varying degrees of damage. We applied gas-chromatography/mass spectrometry-based metabolomics in a case-control study of eleven brain regions in short post-mortem-delay human tissue from nine well-characterized HD patients and nine controls. Unexpectedly, a single major abnormality was evident in all eleven brain regions studied across the forebrain, midbrain and hindbrain, namely marked elevation of urea, a metabolite formed in the urea cycle by arginase-mediated cleavage of arginine. Urea cycle activity localizes primarily in the liver, where it functions to incorporate protein-derived amine-nitrogen into urea for recycling or urinary excretion. It also occurs in other cell-types, but systemic over-production of urea is not known in HD. These findings are consistent with impaired local urea regulation in brain, by up-regulation of synthesis and/or defective clearance. We hypothesize that defective brain urea metabolism could play a substantive role in the pathogenesis of neurodegeneration, perhaps via defects in osmoregulation or nitrogen metabolism. Brain urea metabolism is therefore a target for generating novel monitoring/imaging strategies and/or therapeutic interventions aimed at ameliorating the impact of HD in patients.

Chronic administration of cholesterol oximes in mice increases transcription of cytoprotective genes and improves transcriptome alterations induced by alpha-synuclein overexpression in nigrostriatal dopaminergic neurons.

Cholesterol-oximes TRO19622 and TRO40303 target outer mitochondrial membrane proteins and have beneficial effects in preclinical models of neurodegenerative diseases leading to their advancement to clinical trials. Dopaminergic neurons degenerate in Parkinson's disease (PD) and are prone to oxidative stress and mitochondrial dysfunction. In order to provide insights into the neuroprotective potential of TRO19622 and TRO40303 for dopaminergic neurons in vivo, we assessed their effects on gene expression in laser captured nigrostriatal dopaminergic neurons of wildtype mice and of mice that over-express alpha-synuclein, a protein involved in both familial and sporadic forms of PD (Thy1-aSyn mice). Young mice were fed the drugs in food pellets or a control diet from 1 to 4months of age, approximately 10months before the appearance of striatal dopamine loss in this model. Unbiased weighted gene co-expression network analysis (WGCNA) of transcriptional changes revealed effects of cholesterol oximes on transcripts related to mitochondria, cytoprotection and anti-oxidant response in wild-type and transgenic mice, including increased transcription of stress defense (e.g. Prdx1, Prdx2, Glrx2, Hspa9, Pink1, Drp1, Trak1) and dopamine-related (Th, Ddc, Gch1, Dat, Vmat2, Drd2, Chnr6a) genes. Even at this young age transgenic mice showed alterations in transcripts implicated in mitochondrial function and oxidative stress (e.g. Bcl-2, Bax, Casp3, Nos2), and both drugs normalized about 20% of these alterations. Young Thy1-aSyn mice exhibit motor deficits that differ from parkinsonism and are established before the onset of treatment; these deficits were not improved by cholesterol oximes. However, high doses of TRO40303 improved olfaction and produced the same effects as dopamine agonists on a challenging beam test, specifically an increase in footslips, an observation congruent with its effects on transcripts involved in dopamine synthesis. High doses of TRO19622 increased alpha-synuclein aggregates in the substantia nigra; this effect, not seen with TRO40303 was inconsistent and may represent a protective mechanism as in other neurodegenerative diseases. Overall, the results suggest that cholesterol oximes, while not improving early effects of alpha-synuclein overexpression on motor behavior or pathology, may ameliorate the function and resilience of dopaminergic neurons in vivo and support further studies of neuroprotection in models with dopaminergic cell loss.

Human dementia with Lewy bodies brain shows widespread urea elevations.

INTRODUCTION: Several recent studies have uncovered the presence of widespread urea elevations in multiple neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease dementia (PDD), vascular dementia (VaD), and Huntington's disease (HD). However, it is currently unknown whether dementia with Lewy bodies also shows these alterations in urea. This study aimed to investigate if and where urea is perturbed in the DLB brain. METHODS: Tissues from ten brain regions were obtained from 20 diagnosed cases of DLB and 19 controls. Urea concentrations were measured using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Case-control differences were assessed by nonparametric Mann-Whitney U tests, and s-values, E-values, effect sizes, and risk ratios were determined for each brain region. The results were compared to those previously obtained for AD, PDD, VaD, and HD. RESULTS: As with other previously investigated dementia diseases, DLB shows widespread urea elevations, affecting all ten regions investigated in the current study; the degree of these elevations is lower than that seen in AD or PDD, similar to that seen in HD, and higher than that observed in VaD. The highest urea fold-change was observed in the pons and the lowest in the primary visual cortex. CONCLUSION: Urea elevations appear to be a shared alterations across at least five neurodegenerative diseases, despite their many differences in clinical and neuropathological presentation. The cause and effects of this perturbation should be the focus of future studies, for its possible contributions to the pathology of these conditions.

Localized Pantothenic Acid (Vitamin B5) Reductions Present Throughout the Dementia with Lewy Bodies Brain.

Background: Localized pantothenic acid deficiencies have been observed in several neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease dementia (PDD), and Huntington's disease (HD), indicating downstream energetic pathway perturbations. However, no studies have yet been performed to see whether such deficiencies occur across the dementia with Lewy bodies (DLB) brain, or what the pattern of such dysregulation may be. Objective: Firstly, this study aimed to quantify pantothenic acid levels across ten regions of the brain in order to determine the localization of any pantothenic acid dysregulation in DLB. Secondly, the localization of pantothenic acid alterations was compared to that previously in AD, PDD, and HD brains. Methods: Pantothenic acid levels were determined in 20 individuals with DLB and 19 controls by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) across ten brain regions. Case-control differences were determined by nonparametric Mann-Whitney U test, with the calculation of S-values, risk ratios, E-values, and effect sizes. The results were compared with those previously obtained in DLB, AD, and HD. Results: Pantothenic acid levels were significantly decreased in six of the ten investigated brain regions: the pons, substantia nigra, motor cortex, middle temporal gyrus, primary visual cortex, and hippocampus. This level of pantothenic acid dysregulation is most similar to that of the AD brain, in which pantothenic acid is also decreased in the motor cortex, middle temporal gyrus, primary visual cortex, and hippocampus. DLB appears to differ from other neurodegenerative diseases in being the only of the four to not show pantothenic acid dysregulation in the cerebellum. Conclusions: Pantothenic acid deficiency appears to be a shared mechanism of several neurodegenerative diseases, although differences in the localization of this dysregulation may contribute to the differing clinical pathways observed in these conditions.

Decreases in a molecule called pantothenic acid (also known as vitamin B5) have been observed in several areas of the brain in multiple dementia disease, including Alzheimer's disease, Parkinson's disease dementia, and Huntington's disease. However, it is unknown whether such changes also occur in another dementia disease, dementia with Lewy bodies, which shows many of the same symptoms and molecular changes as these conditions. As such, this study was performed in order to determine if and where changes in pantothenic acid occur throughout the dementia with Lewy bodies brain. Using a methodology called liquid chromatography­mass spectrometry, which is able to measure pantothenic acid levels in a highly precise manner in brain tissues, we found that several regions of the dementia with Lewy bodies brain show decreases in pantothenic acid, including some involved in movement such as the substantia nigra and motor cortex, as well as regions associated with cognition and memory such as the hippocampus­looking most similar to the pattern of changes already seen in Alzheimer's disease. It is possible that these changes contribute to the progression of dementia with Lewy bodies; however, further studies need to be performed to determine at what point these changes happen during the disease and how they may contribute to the development of symptoms.

Widespread selenium deficiency in the brain of cases with Huntington's disease presents a new potential therapeutic target.

BACKGROUND: Huntington or Huntington's disease (HD) is an autosomal dominant neurodegenerative disease characterised by both progressive motor and cognitive dysfunction; its pathogenic mechanisms remain poorly understood and no treatment can currently slow, stop, or reverse its progression. There is some evidence of metallomic dysfunction in limited regions of the HD brain; we hypothesised that these alterations are more widespread than the current literature suggests and may contribute to pathogenesis in HD. METHODS: We measured the concentrations of eight essential metals (sodium, potassium, magnesium, calcium, iron, zinc, copper, and manganese) and the metalloid selenium across 11 brain regions in nine genetically confirmed, clinically manifest cases of HD and nine controls using inductively-coupled plasma mass spectrometry. Case-control differences were assessed by non-parametric Mann-Whitney U test (p < 0.05), risk ratios, E-values, and effect sizes. FINDINGS: We observed striking decreases in selenium levels in 11 out of 11 investigated brain regions in HD, with risk ratios and effect sizes ranging 2.3-9.0 and 0.7-1.9, respectively. Increased sodium/potassium ratios were observed in every region (risk ratio = 2.5-8.0; effect size = 1.2-5.8) except the substantia nigra (risk ratio = 0.25; effect size = 0.1). Multiple regions showed increased calcium and/or zinc levels, and localised decreases in iron, copper, and manganese were present in the globus pallidus, cerebellum, and substantia nigra, respectively. INTERPRETATION: The observed metallomic alterations in the HD brain may contribute to several pathogenic mechanisms, including mitochondrial dysfunction, oxidative stress, and blood-brain barrier dysfunction. Selenium supplementation may represent a potential, much-needed therapeutic pathway for the treatment of HD that would not require localised delivery in the brain due to the widespread presence of selenium deficiency in regions that show both high and low levels of neurodegeneration. FUNDING: In Acknowledgments, includes the Lee Trust, the Endocore Research Trust, Cure Huntington's Disease Initiative, the Oakley Mental Health Research Foundation, the Medical Research Council (MRC), the New Zealand Neurological Foundation, and others.

Extensive multiregional urea elevations in a case-control study of vascular dementia point toward a novel shared mechanism of disease amongst the age-related dementias.

Introduction: Vascular dementia (VaD) is one of the most common causes of dementia among the elderly. Despite this, the molecular basis of VaD remains poorly characterized when compared to other age-related dementias. Pervasive cerebral elevations of urea have recently been reported in several dementias; however, a similar analysis was not yet available for VaD. Methods: Here, we utilized ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to measure urea levels from seven brain regions in post-mortem tissue from cases of VaD (n = 10) and controls (n = 8/9). Brain-urea measurements from our previous investigations of several dementias were also used to generate comparisons with VaD. Results: Elevated urea levels ranging from 2.2- to 2.4-fold-change in VaD cases were identified in six out of the seven regions analysed, which are similar in magnitude to those observed in uremic encephalopathy. Fold-elevation of urea was highest in the basal ganglia and hippocampus (2.4-fold-change), consistent with the observation that these regions are severely affected in VaD. Discussion: Taken together, these data not only describe a multiregional elevation of brain-urea levels in VaD but also imply the existence of a common urea-mediated disease mechanism that is now known to be present in at least four of the main age-related dementias.

Widespread Decreases in Cerebral Copper Are Common to Parkinson's Disease Dementia and Alzheimer's Disease Dementia.

Several studies of Parkinson's disease (PD) have reported dysregulation of cerebral metals, particularly decreases in copper and increases in iron in substantia nigra (SN). However, few studies have investigated regions outside the SN, fewer have measured levels of multiple metals across different regions within the same brains, and there are no currently-available reports of metal levels in Parkinson's disease dementia (PDD). This study aimed to compare concentrations of nine essential metals across nine different brain regions in cases of PDD and controls. Investigated were: primary motor cortex (MCX); cingulate gyrus (CG); primary visual cortex (PVC); hippocampus (HP); cerebellar cortex (CB); SN; locus coeruleus (LC); medulla oblongata (MED); and middle temporal gyrus (MTG), thus covering regions with severe, moderate, or low levels of neuronal loss in PDD. Levels of eight essential metals and selenium were determined using an analytical methodology involving the use of inductively-coupled plasma mass spectrometry (ICP-MS), and compared between cases and controls, to better understand the extent and severity of metal perturbations. Findings were also compared with those from our previous study of sporadic Alzheimer's disease dementia (ADD), which employed equivalent methods, to identify differences and similarities between these conditions. Widespread copper decreases occurred in PDD in seven of nine regions (exceptions being LC and CB). Four PDD-affected regions showed similar decreases in ADD: CG, HP, MTG, and MCX. Decreases in potassium and manganese were present in HP, MTG and MCX; decreased manganese was also found in SN and MED. Decreased selenium and magnesium were present in MCX, and decreased zinc in HP. There was no evidence for increased iron in SN or any other region. These results identify alterations in levels of several metals across multiple regions of PDD brain, the commonest being widespread decreases in copper that closely resemble those in ADD, pointing to similar disease mechanisms in both dementias.

Substantively Lowered Levels of Pantothenic Acid (Vitamin B5) in Several Regions of the Human Brain in Parkinson's Disease Dementia.

Pantothenic acid (vitamin B5) is an essential trace nutrient required for the synthesis of coenzyme A (CoA). It has previously been shown that pantothenic acid is significantly decreased in multiple brain regions in both Alzheimer's disease (ADD) and Huntington's disease (HD). The current investigation aimed to determine whether similar changes are also present in cases of Parkinson's disease dementia (PDD), another age-related neurodegenerative condition, and whether such perturbations might occur in similar regions in these apparently different diseases. Brain tissue was obtained from nine confirmed cases of PDD and nine controls with a post-mortem delay of 26 h or less. Tissues were acquired from nine regions that show high, moderate, or low levels of neurodegeneration in PDD: the cerebellum, motor cortex, primary visual cortex, hippocampus, substantia nigra, middle temporal gyrus, medulla oblongata, cingulate gyrus, and pons. A targeted ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) approach was used to quantify pantothenic acid in these tissues. Pantothenic acid was significantly decreased in the cerebellum (p = 0.008), substantia nigra (p = 0.02), and medulla (p = 0.008) of PDD cases. These findings mirror the significant decreases in the cerebellum of both ADD and HD cases, as well as the substantia nigra, putamen, middle frontal gyrus, and entorhinal cortex of HD cases, and motor cortex, primary visual cortex, hippocampus, middle temporal gyrus, cingulate gyrus, and entorhinal cortex of ADD cases. Taken together, these observations indicate a common but regionally selective disruption of pantothenic acid levels across PDD, ADD, and HD.

Severe and Regionally Widespread Increases in Tissue Urea in the Human Brain Represent a Novel Finding of Pathogenic Potential in Parkinson's Disease Dementia.

Widespread elevations in brain urea have, in recent years, been reported in certain types of age-related dementia, notably Alzheimer's disease (AD) and Huntington's disease (HD). Urea increases in these diseases are substantive, and approximate in magnitude to levels present in uraemic encephalopathy. In AD and HD, elevated urea levels are widespread, and not only in regions heavily affected by neurodegeneration. However, measurements of brain urea have not hitherto been reported in Parkinson's disease dementia (PDD), a condition which shares neuropathological and symptomatic overlap with both AD and HD. Here we report measurements of tissue urea from nine neuropathologically confirmed regions of the brain in PDD and post-mortem delay (PMD)-matched controls, in regions including the cerebellum, motor cortex (MCX), sensory cortex, hippocampus (HP), substantia nigra (SN), middle temporal gyrus (MTG), medulla oblongata (MED), cingulate gyrus, and pons, by applying ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Urea concentrations were found to be substantively elevated in all nine regions, with average increases of 3-4-fold. Urea concentrations were remarkably consistent across regions in both cases and controls, with no clear distinction between regions heavily affected or less severely affected by neuronal loss in PDD. These urea elevations mirror those found in uraemic encephalopathy, where equivalent levels are generally considered to be pathogenic, and those previously reported in AD and HD. Increased urea is a widespread metabolic perturbation in brain metabolism common to PDD, AD, and HD, at levels equal to those seen in uremic encephalopathy. This presents a novel pathogenic mechanism in PDD, which is shared with two other neurodegenerative diseases.

Generation of a mouse SWATH-MS spectral library to quantify 10148 proteins involved in cell reprogramming.

Murine models are amongst the most widely used systems to study biology and pathology. Targeted quantitative proteomic analysis is a relatively new tool to interrogate such systems. Recently the need for relative quantification on hundreds to thousands of samples has driven the development of Data Independent Acquisition methods. One such technique is SWATH-MS, which in the main requires prior acquisition of mass spectra to generate an assay reference library. In stem cell research, it has been shown pluripotency can be induced starting with a fibroblast population. In so doing major changes in expressed proteins is inevitable. Here we have created a reference library to underpin such studies. This is inclusive of an extensively documented script to enable replication of library generation from the raw data. The documented script facilitates reuse of data and adaptation of the library to novel applications. The resulting library provides deep coverage of the mouse proteome. The library covers 29519 proteins (53% of the proteome) of which 7435 (13%) are supported by a proteotypic peptide.

A Multi-Omic Huntington's Disease Transgenic Sheep-Model Database for Investigating Disease Pathogenesis.

BACKGROUND: The pathological mechanism of cellular dysfunction and death in Huntington's disease (HD) is not well defined. Our transgenic HD sheep model (OVT73) was generated to investigate these mechanisms and for therapeutic testing. One particular cohort of animals has undergone focused investigation resulting in a large interrelated multi-omic dataset, with statistically significant changes observed comparing OVT73 and control 'omic' profiles and reported in literature. OBJECTIVE: Here we make this dataset publicly available for the advancement of HD pathogenic mechanism discovery. METHODS: To enable investigation in a user-friendly format, we integrated seven multi-omic datasets from a cohort of 5-year-old OVT73 (n = 6) and control (n = 6) sheep into a single database utilising the programming language R. It includes high-throughput transcriptomic, metabolomic and proteomic data from blood, brain, and other tissues. RESULTS: We present the 'multi-omic' HD sheep database as a queryable web-based platform that can be used by the wider HD research community (https://hdsheep.cer.auckland.ac.nz/). The database is supported with a suite of simple automated statistical analysis functions for rapid exploratory analyses. We present examples of its use that validates the integrity relative to results previously reported. The data may also be downloaded for user determined analysis. CONCLUSION: We propose the use of this online database as a hypothesis generator and method to confirm/refute findings made from patient samples and alternate model systems, to expand our understanding of HD pathogenesis. Importantly, additional tissue samples are available for further investigation of this cohort.

Phosphodiesterase 10 inhibition reduces striatal excitotoxicity in the quinolinic acid model of Huntington's disease.

Decreased activity of cAMP responsive element-binding protein (CREB) is thought to contribute to the death of striatal medium spiny neurons in Huntington's disease (HD). Therefore, therapies that increase levels of activated CREB, may be effective in fighting neurodegeneration in HD. In this study, we sought to determine whether the phosphodiesterase type 10 (PDE10A) inhibitor TP10 exerts a neuroprotective effect in an excitotoxic model of HD. Rats were surgically administered with quinolinic acid into striatum and subsequently treated with TP10 daily for two or eight weeks. After 2 weeks of TP10 treatment, striatal lesion size was 52% smaller and the surviving cell number was several times higher than in the vehicle-treated group. These beneficial effects of TP10 were maintained through 8 weeks. TP10 treatment also increased significantly the levels of activated CREB in the striatal spiny neurons, which is hypothesized to be a contributing mechanism for the neuroprotective effect. Our findings suggest PDE10A inhibition as a novel neuroprotective approach to the treatment of HD and confirm the importance of phosphodiesterase inhibition in fighting the disease.