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Parkinson’s disease (PD) is a movement disorder characterized by the early loss of nigrostriatal dopaminergic pathways producing significant network changes impacting motor coordination. Recently three motor stages of PD have been proposed (a silent period when nigrostriatal loss begins, a prodromal motor period with subtle focal manifestations, and clinical PD) with evidence that motor cortex abnormalities occur to produce clinical PD[8]. We directly assess structural changes in the primary motor cortex and corticospinal tract using parallel analyses of longitudinal clinical and cross-sectional pathological cohorts thought to represent different stages of PD. 18F-FP-CIT positron emission tomography and subtle motor features identified patients with idiopathic rapid-eye-movement sleep behaviour disorder (n = 8) that developed prodromal motor signs of PD. Longitudinal diffusion tensor imaging before and after the development of prodromal motor PD showed higher fractional anisotropy in motor cortex and corticospinal tract compared to controls, indicating adaptive structural changes in motor networks in concert with nigrostriatal dopamine loss. Histological analyses of the white matter underlying the motor cortex showed progressive disorientation of axons with segmental replacement of neurofilaments with α-synuclein, enlargement of myelinating oligodendrocytes and increased density of their precursors. There was no loss of neurons in the motor cortex in early or late pathologically confirmed motor PD compared to controls, although there were early cortical increases in neuronal neurofilament light chain and myelin proteins in association with α-synuclein accumulation. Our results collectively provide evidence of a direct impact of PD on primary motor cortex and its output pathways that begins in the prodromal motor stage of PD with structural changes confirmed in early PD. These adaptive structural changes become considerable as the disease advances potentially contributing to motor PD.

Many genetic risk factors for Parkinson’s disease have lipid-related functions and lipid-modulating drugs such as statins may be protective against Parkinson’s disease. Moreover, the hallmark Parkinson’s disease pathological protein, α-synuclein, has lipid membrane function and pathways dysregulated in Parkinson’s disease such as the endosome–lysosome system and synaptic signalling rely heavily on lipid dynamics. Despite the potential role for lipids in Parkinson’s disease, most research to date has been protein-centric, with large-scale, untargeted serum and CSF lipidomic comparisons between genetic and idiopathic Parkinson’s disease and neurotypical controls limited. In particular, the extent to which lipid dysregulation occurs in mutation carriers of one of the most common Parkinson’s disease risk genes, LRRK2, is unclear. Further, the functional lipid pathways potentially dysregulated in idiopathic and LRRK2 mutation Parkinson’s disease are underexplored. To better determine the extent of lipid dysregulation in Parkinson’s disease, untargeted high-performance liquid chromatography–tandem mass spectrometry was performed on serum (n = 221) and CSF (n = 88) obtained from a multi-ethnic population from the Michael J. Fox Foundation LRRK2 Clinical Cohort Consortium. The cohort consisted of controls, asymptomatic LRRK2 G2019S carriers, LRRK2 G2019S carriers with Parkinson’s disease and Parkinson’s disease patients without a LRRK2 mutation. Age and sex were adjusted for in analyses where appropriate. Approximately 1000 serum lipid species per participant were analysed. The main serum lipids that distinguished both Parkinson’s disease patients and LRRK2 mutation carriers from controls included species of ceramide, triacylglycerol, sphingomyelin, acylcarnitine, phosphatidylcholine and lysophosphatidylethanolamine. Significant alterations in sphingolipids and glycerolipids were also reflected in Parkinson’s disease and LRRK2 mutation carrier CSF, although no correlations were observed between lipids identified in both serum and CSF. Pathway analysis of altered lipid species indicated that sphingolipid metabolism, insulin signalling and mitochondrial function were the major metabolic pathways dysregulated in Parkinson’s disease. Importantly, these pathways were also found to be dysregulated in serum samples from a second Parkinson’s disease cohort (n = 315). Results from this study demonstrate that dysregulated lipids in Parkinson’s disease generally, and in LRRK2 mutation carriers, are from functionally and metabolically related pathways. These findings provide new insight into the extent of lipid dysfunction in Parkinson’s disease and therapeutics manipulating these pathways may be beneficial for Parkinson’s disease patients. Moreover, serum lipid profiles may be novel biomarkers for both genetic and idiopathic Parkinson’s disease.

Genetic alterations in the LRRK2 gene, encoding leucine-rich repeat kinase 2, are a common risk factor for Parkinson’s disease. How LRRK2 alterations lead to cell pathology is an area of ongoing investigation, however, multiple lines of evidence suggest a role for LRRK2 in lipid pathways. It is increasingly recognized that in addition to being energy reservoirs and structural entities, some lipids, including neural lipids, participate in signaling cascades. Early investigations revealed that LRRK2 localized to membranous and vesicular structures, suggesting an interaction of LRRK2 and lipids or lipid-associated proteins. LRRK2 substrates from the Rab GTPase family play a critical role in vesicle trafficking, lipid metabolism and lipid storage, all processes which rely on lipid dynamics. In addition, LRRK2 is associated with the phosphorylation and activity of enzymes that catabolize plasma membrane and lysosomal lipids. Furthermore, LRRK2 knockout studies have revealed that blood, brain and urine exhibit lipid level changes, including alterations to sterols, sphingolipids and phospholipids, respectively. In human LRRK2 mutation carriers, changes to sterols, sphingolipids, phospholipids, fatty acyls and glycerolipids are reported in multiple tissues. This review summarizes the evidence regarding associations between LRRK2 and lipids, and the functional consequences of LRRK2-associated lipid changes are discussed.

Parkinson’s disease (PD) and Multiple System Atrophy (MSA) are progressive and unremitting neurological diseases that are neuropathologically characterized by α-synuclein inclusions. Increasing evidence supports the aggregation of α-synuclein in specific brain areas early in the disease course, followed by the spreading of α-synuclein pathology to multiple brain regions. However, little is known about how the structure of α-synuclein fibrils influence its ability to seed endogenous α-synuclein in recipient cells. Here, we aggregated α-synuclein by seeding with homogenates of PD- and MSA-confirmed brain tissue, determined the resulting α-synuclein fibril structures by cryo-electron microscopy, and characterized their seeding potential in mouse primary oligodendroglial cultures. The combined analysis shows that the two patient material-amplified α-synuclein fibrils share a similar protofilament fold but differ in their inter-protofilament interface and their ability to recruit endogenous α-synuclein. Our study indicates that the quaternary structure of α-synuclein fibrils modulates the seeding of α-synuclein pathology inside recipient cells. It thus provides an important advance in the quest to understand the connection between the structure of α-synuclein fibrils, cellular seeding/spreading, and ultimately the clinical manifestations of different synucleinopathies.

Parkinson's disease (PD) is a movement disorder characterized by the early loss of nigrostriatal dopaminergic pathways producing significant network changes impacting motor coordination. Recently three motor stages of PD have been proposed (a silent period when nigrostriatal loss begins, a prodromal motor period with subtle focal manifestations, and clinical PD) with evidence that motor cortex abnormalities occur to produce clinical PD[8]. We directly assess structural changes in the primary motor cortex and corticospinal tract using parallel analyses of longitudinal clinical and cross-sectional pathological cohorts thought to represent different stages of PD. 18F-FP-CIT positron emission tomography and subtle motor features identified patients with idiopathic rapid-eye-movement sleep behaviour disorder (n = 8) that developed prodromal motor signs of PD. Longitudinal diffusion tensor imaging before and after the development of prodromal motor PD showed higher fractional anisotropy in motor cortex and corticospinal tract compared to controls, indicating adaptive structural changes in motor networks in concert with nigrostriatal dopamine loss. Histological analyses of the white matter underlying the motor cortex showed progressive disorientation of axons with segmental replacement of neurofilaments with α-synuclein, enlargement of myelinating oligodendrocytes and increased density of their precursors. There was no loss of neurons in the motor cortex in early or late pathologically confirmed motor PD compared to controls, although there were early cortical increases in neuronal neurofilament light chain and myelin proteins in association with α-synuclein accumulation. Our results collectively provide evidence of a direct impact of PD on primary motor cortex and its output pathways that begins in the prodromal motor stage of PD with structural changes confirmed in early PD. These adaptive structural changes become considerable as the disease advances potentially contributing to motor PD.

Rare, yet biologically critical, lipids that contain very long chain fatty acids (VLCFA-lipids) are synthesized in the brain by the enzyme ELOVL4. High levels of VLCFA-lipids are toxic to cells and excess VLCFA-lipids are actively removed by ABCD1 in an ATP-dependent manner. Virtually nothing is known about the impact of VLCFA-lipids in neurodegenerative diseases. Here, we investigated the possible role of VLCFA-lipids in frontotemporal dementia (FTD), which is a leading cause of younger-onset dementia. Using quantitative discovery lipidomics, we identified three VLCFA-lipid species that were significantly increased in FTD brain compared to controls, with strong correlations with ELOVL4. Increases in ELOVL4 expression correlated with significant decreases in the membrane-bound synaptophysin in FTD brain. Furthermore, increases in ABCD1 expression correlated with increases in VLCFA-lipids. We uncovered a new pathomechanism that is pertinent to understanding the pathogenesis of FTD.

Mutations in lysosomal genes increase the risk of neurodegenerative diseases, as is the case for Parkinson’s disease. Here, we found that pathogenic and protective mutations in arylsulfatase A (ARSA), a gene responsible for metachromatic leukodystrophy, a lysosomal storage disorder, are linked to Parkinson’s disease. Plasma ARSA protein levels were changed in Parkinson’s disease patients. ARSA deficiency caused increases in α-synuclein aggregation and secretion, and increases in α-synuclein propagation in cells and nematodes. Despite being a lysosomal protein, ARSA directly interacts with α-synuclein in the cytosol. The interaction was more extensive with protective ARSA variant and less with pathogenic ARSA variant than wild-type. ARSA inhibited the in vitro fibrillation of α-synuclein in a dose-dependent manner. Ectopic expression of ARSA reversed the α-synuclein phenotypes in both cell and fly models of synucleinopathy, the effects correlating with the extent of the physical interaction between these molecules. Collectively, these results suggest that ARSA is a genetic modifier of Parkinson’s disease pathogenesis, acting as a molecular chaperone for α-synuclein.

The propagation and accumulation of pathological α-synuclein protein is thought to underlie the clinical symptoms of the neurodegenerative movement disorder Parkinson's disease (PD). Consequently, there is significant interest in identifying the mechanisms that contribute to α-synuclein pathology, as these may inform therapeutic targets for the treatment of PD. One protein that appears to contribute to α-synuclein pathology is the innate immune pathogen recognition receptor, toll-like receptor 2 (TLR2). TLR2 is expressed on neurons, and its activation results in the accumulation of α-synuclein protein; however, the precise mechanism by which TLR2 contributes to α-synuclein pathology is unclear. Herein we demonstrate using human cell models that neuronal TLR2 activation acutely impairs the autophagy lysosomal pathway and markedly potentiates α-synuclein pathology seeded with α-synuclein preformed fibrils. Moreover, α-synuclein pathology could be ameliorated with a novel small molecule TLR2 inhibitor, including in induced pluripotent stem cell-derived neurons from a patient with PD. These results provide further insight into how TLR2 activation may promote α-synuclein pathology in PD and support that TLR2 may be a potential therapeutic target for the treatment of PD.

Synucleinopathies, such as Parkinson’s disease (PD) and Multiple System Atrophy (MSA) are progressive and unremitting neurological diseases. For both PD and MSA, α-synuclein fibril inclusions inside brain cells are neuropathological hallmarks. In addition, amplification of α-synuclein fibrils from body fluids is a potential biomarker distinguishing PD from MSA. However, little is known about the structure of α-synuclein fibrils amplified from human samples and its connection to α-synuclein fibril structure in the human brain. Here we amplified α-synuclein fibrils from PD and MSA brain tissue, characterized its seeding potential in oligodendroglia, and determined the 3D structures by cryo-electron microscopy. We show that the α-synuclein fibrils from a MSA patient are more potent in recruiting the endogenous α-synuclein and evoking a redistribution of TPPP/p25α protein in mouse primary oligoden-droglial cultures compared to those amplified from a PD patient. Cryo-electron microscopy shows that the PD- and MSA-amplified α-synuclein fibrils share a similar protofilament fold but differ in their inter-protofilament interface. The structures of the brain-tissue amplified α-synuclein fibrils are also similar to other in vitro and ex vivo α-synuclein fibrils. Together with published data, our results suggest that αSyn fibrils differ between PD and MSA in their quaternary arrangement and could further vary between different forms of PD and MSA.

Background: The identification of reliable biomarkers in Parkinson’s disease (PD) would provide much needed diagnostic accuracy, a means of monitoring progression, objectively measuring treatment response, and potentially allowing patient stratification within clinical trials. Whilst the assessment of total alpha-synuclein in biofluids has been identified as a promising biomarker, conflicting trends in these levels across patient plasma samples relative to controls has limited its use. Different commercially available assay platforms that have been used to measure alpha-synuclein may contribute to different study outcomes. Objective: To compare different platform immunoassays for the measurement of total alpha-synuclein using the same plasma samples from 49 PD patients and 47 controls. Methods: Total plasma alpha-synuclein concentrations were assessed using the BioLegend, MesoScale Discovery, and Quanterix platform in plasma samples from PD patients and matched controls. Results: A significant increase in total plasma alpha-synuclein was observed in PD patients using the Biolegend (10%), Mesoscale Discovery (13%) and Quanterix (39%) assays. The Mesoscale Discovery and Quanterix assays showed the strongest correlations (r = 0.78, p

ATP-binding cassette (ABC) transporters are one of the largest groups of transporter families in humans. ABC transporters mediate the translocation of a diverse range of substrates across cellular membranes, including amino acids, nucleosides, lipids, sugars and xenobiotics. Neurodegenerative diseases are a group of brain diseases that detrimentally affect neurons and other brain cells and are usually associated with deposits of pathogenic proteins in the brain. Major neurodegenerative diseases include Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. ABC transporters are highly expressed in the brain and have been implicated in a number of pathological processes underlying neurodegenerative diseases. This review outlines the current understanding of the role of ABC transporters in neurodegenerative diseases, focusing on some of the most important pathways, and also suggests future directions for research in this field.

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are neurodegenerative diseases that are considered to be on the same disease spectrum because of overlapping genetic, pathological and clinical traits. Changes in serum proteins in FTD and ALS are poorly understood, and currently no definitive biomarkers exist for diagnosing or monitoring disease progression for either disease. Here we applied quantitative discovery proteomics to analyze protein changes in FTD (N = 72) and ALS (N = 28) patient serum compared to controls (N = 22). Twenty three proteins were significantly altered in FTD compared to controls (increased—APOL1, C3, CTSH, EIF5A, MYH2, S100A8, SUSD5, WDR1; decreased—C1S, C7, CILP2, COMP, CRTAC1, EFEMP1, FBLN1, GSN, HSPG2, IGHV1, ITIH2, PROS1, SHBG, UMOD, VASN) and 14 proteins were significantly altered in ALS compared to controls (increased—APOL1, CKM, CTSH, IGHG1, IGKC, MYH2; decreased—C7, COMP, CRTAC1, EFEMP1, FBLN1, GSN, HSPG2, SHBG). There was substantial overlap in the proteins that were altered in FTD and ALS. These results were validated using western blotting. Gene ontology tools were used to assess functional pathways potentially dysregulated in the two diseases, and calcium ion binding and innate immunity pathways were altered in both diseases. When put together, these results suggest significant overlap in pathophysiological peripheral changes in FTD and ALS. This study represents the first proteomics side-by-side comparison of serum changes in FTD and ALS, providing new insights into under-recognized perturbed pathways and an avenue for biomarker development for FTD and ALS.

Objective Physiological changes potentially influence disease progression and survival along the Amyotrophic Lateral Sclerosis (ALS)‐Frontotemporal dementia (FTD) spectrum. The peripheral peptides that regulate eating and metabolism may provide diagnostic, metabolic, and progression biomarkers. The current study aimed to examine the relationships and biomarker potential of hormonal peptides. Methods One hundred and twenty‐seven participants (36 ALS, 26 ALS‐ cognitive, patients with additional cognitive behavioral features, and 35 behavioral variant FTD (bvFTD) and 30 controls) underwent fasting blood analyses of leptin, ghrelin, neuropeptide Y (NPY), peptide YY (PYY), and insulin levels. Relationships between endocrine measures, cognition, eating behaviors, and body mass index (BMI) were investigated. Biomarker potential was evaluated using multinomial logistic regression for diagnosis and correlation to disease duration. Results Compared to controls, ALS and ALS‐cognitive had higher NPY levels and bvFTD had lower NPY levels, while leptin levels were increased in all patient groups. All groups had increased insulin levels and a state of insulin resistance compared to controls. Lower NPY levels correlated with increasing eating behavioral change and BMI, while leptin levels correlated with BMI. On multinomial logistic regression, NPY and leptin levels were found to differentiate between diagnosis. Reduced Neuropeptide Y levels correlated with increasing disease duration, suggesting it may be useful as a potential marker of disease progression. Interpretation ALS‐FTD is characterized by changes in NPY and leptin levels that may impact on the underlying regional neurodegeneration as they were predictive of diagnosis and disease duration, offering the potential as biomarkers and for the development of interventional treatments.

Alzheimer's disease is characterized by abnormal amyloid-β (Aβ) peptide accumulation beginning decades before symptom onset. An effective prophylactic treatment aimed at arresting the amyloidogenic pathway would therefore need to be initiated prior to the occurrence of Aβ pathology. The SIGMAR1 gene encodes a molecular chaperone that modulates processing of the amyloid-β protein precursor (AβPP). Fluvoxamine is a selective serotonin reuptake inhibitor and a potent SIGMAR1 agonist. We therefore hypothesized that fluvoxamine treatment would reduce Aβ production and improve cognition. We firstly investigated the impact of SIGMAR1 on AβPP processing, and found that overexpression and knockdown of SIGMAR1 significantly affected γ-secretase activity in SK-N-MC neuronal cells. We then tested the impact of fluvoxamine on Aβ production in an amyloidogenic cell model, and found that fluvoxamine significantly reduced Aβ production by inhibiting γ-secretase activity. Finally, we assessed the efficacy of long-term treatment (i.e., ∼8 months) of 10 mg/kg/day fluvoxamine in the J20 amyloidogenic mouse model; the treatment was initiated prior to the occurrence of predicted Aβ pathology. Physical examination of the animals revealed no overt pathology or change in weight. We conducted a series of behavioral tests to assess learning and memory, and found that the fluvoxamine treatment significantly improved memory function as measured by novel object recognition task. Two other tests revealed no significant change in memory function. In conclusion, fluvoxamine has a clear impact on γ-secretase activity and AβPP processing to generate Aβ, and may have a protective effect on cognition in the J20 mice.

BACKGROUND Patients with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) exhibit changes in eating behavior that could potentially affect lipid levels. OBJECTIVE This study aimed to document changes in lipid metabolism across the ALS-FTD spectrum to identify potential relationships to eating behavior (including fat intake), cognitive change, body mass index (BMI), and effect on survival. METHODS One hundred and twenty-eight participants were recruited: 37 ALS patients, 15 ALS patients with cognitive and behavioral change (ALS-Plus), 13 ALS-FTD, 31 behavioral variant FTD, and 32 healthy controls. Fasting total cholesterol, low density lipoprotein cholesterol (LDL), high density lipoprotein cholesterol (HDL) and triglyceride levels were measured and correlated to eating behavior (caloric, fat intake), cognitive change, and BMI; effect on survival was examined using cox regression analyses. RESULTS There was a spectrum of lipid changes from ALS to FTD with increased triglyceride (p

ATP-binding cassette A7 (ABCA7) is a genetic risk factor for late-onset Alzheimer’s disease (AD). It belongs to a group of transporter genes that specializes in regulating lipid transport in the periphery as well as in the brain. ABCA7 has been implicated in a number of roles relating to AD pathology, including phagocytic clearance of amyloid-β peptides. We have discovered that deletion of ABCA7 in mouse causes a dramatic reduction in white adipose tissue (WAT) in female mice. WAT is important in AD context because it is the primary producer of leptin, which is a hormone that is known to modulate AD neuropathology. WAT in male Abca7–/– mice was not altered. The pathological link between ABCA7 and WAT that impacts on AD is unknown. Our transcription analysis revealed that lipin-1 expression was significantly upregulated in female Abca7–/– mice, indicating that ABCA7 affects WAT development. The circulating leptin level was significantly reduced in female Abca7–/– mice without any change in WAT leptin mRNA or protein expression, indicating that ABCA7 does not affect leptin production, but alters the circulating leptin level indirectly by affecting WAT development. Insulin is a key hormone that regulates WAT development, i.e., adipogenesis, and it was significantly reduced in female Abca7–/– mice. These data when put together suggest that ABCA7 plays a role in regulating WAT development and consequently circulating leptin levels, which are known to modulate AD neuropathology.

Multiple system atrophy (MSA) is a devastating neurodegenerative disease characterized by the clinical triad of parkinsonism, cerebellar ataxia and autonomic failure, impacting on striatonigral, olivopontocerebellar and autonomic systems. At early stage of the disease, the clinical symptoms of MSA can overlap with those of Parkinson’s disease (PD). The key pathological hallmark of MSA is the presence of glial cytoplasmic inclusions (GCI) in oligodendrocytes. GCI comprise insoluble proteinaceous filaments composed chiefly of α-synuclein aggregates, and therefore MSA is regarded as an α-synucleinopathy along with PD and dementia with Lewy bodies. The etiology of MSA is unknown, and the pathogenesis of MSA is still largely speculative. Much data suggests that MSA is a sporadic disease, although some emerging evidence suggests rare genetic variants increase susceptibility. Currently, there is no general consensus on the susceptibility genes as there have been differences due to geographical distribution or ethnicity. Furthermore, many of the reported studies have been conducted on patients that were only clinically diagnosed without pathological verification. The purpose of this review is to bring together available evidence to cross-examine the susceptibility genes and genetic pathomechanisms implicated in MSA. We explore the possible involvement of the SNCA, COQ2, MAPT, GBA1, LRRK2 and C9orf72 genes in MSA pathogenesis, highlight the under-explored areas of MSA genetics, and discuss future directions of research in MSA.

Parkinson’s disease (PD) and Multiple System Atrophy (MSA) are clinically distinctive diseases that feature a common neuropathological hallmark of aggregated α-synuclein. Little is known about how differences in α-synuclein aggregate structure affect disease phenotype. Here, we amplified α-synuclein aggregates from PD and MSA brain extracts and analyzed the conformational properties using fluorescent probes, NMR spectroscopy and electron paramagnetic resonance. We also generated and analyzed several in vitro α-synuclein polymorphs. We found that brain-derived α-synuclein fibrils were structurally different to all of the in vitro polymorphs analyzed. Importantly, there was a greater structural heterogeneity among α-synuclein fibrils from the PD brain compared to those from the MSA brain, possibly reflecting on the greater variability of disease phenotypes evident in PD. Our findings have significant ramifications for the use of non-brain-derived α-synuclein fibrils in PD and MSA studies, and raise important questions regarding the one disease-one strain hypothesis in the study of α-synucleinopathies., Parkinson’s disease (PD) and Multiple System Atrophy (MSA) are characterized by the pathological accumulation of α-synuclein. Here the authors employ fluorescent probes, electron microscopy and NMR spectroscopy to study the properties of α-synuclein aggregates that were amplified from patient brain extracts and observe a greater structural diversity among PD patients compared to MSA patients.

Multiple system atrophy (MSA) is a sporadic neurodegenerative disease. The major pathological hallmark of MSA is the accumulation of α-synuclein in oligodendrocytes. In contrast to Parkinson's disease no definitive familial etiology for MSA has been determined. Yet, there is a growing body of evidence that perturbation of transcriptional processes leads to MSA pathology. Here we present the results of the first ribosomal-depleted strand-specific RNA-sequencing profile of the MSA brain frontal cortex tissue. Among the 123 differentially expressed genes over 50% were categorized as putative long intervening non-coding RNAs (lincRNAs). Along with the dysregulation of the non-coding portion of the transcriptome, the expression of protein coding genes was also affected, including serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3 (SERPINA3), interleukin 1 receptor-like 1 (IL1RL1) and hemoglobin, beta (HBB). Also of interest was the alternative splicing of SNCA, along with the presence of an antisense transcript overlapping the 3' exon of SNCA. Moreover, we demonstrate widespread antisense transcription throughout the frontal cortex that is largely not affected by MSA-specific neurodegenerative process. MSA causes a large disruption of lincRNAs in the human brain along with protein coding genes related to iron metabolism and immune response regulation. Most of the lincRNAs specific for MSA were novel. Hence our study uncovers another level of complexity in transcriptional pathology of MSA.

Ageing is the major risk factor for Alzheimer’s disease (AD), a condition involving brain hypoxia. The majority of early onset familial AD (EOfAD) cases involve dominant mutations in the gene PSEN1. PSEN1 null mutations do not cause EOfAD. We exploited putative hypomorphic and EOfAD-like mutations in the zebrafish psen1 gene to explore the effects of age and genotype on brain responses to acute hypoxia. Both mutations accelerate age-dependent changes in hypoxia-sensitive gene expression supporting that ageing is necessary, but insufficient, for AD occurrence. Curiously, the responses to acute hypoxia become inverted in extremely aged fish. This is associated with an apparent inability to upregulate glycolysis. Wild type PSEN1 allele expression is reduced in post-mortem brains of human EOfAD mutation carriers (and extremely aged fish), possibly contributing to EOfAD pathogenesis. We also observed that age-dependent loss of HIF1 stabilisation under hypoxia is a phenomenon conserved across vertebrate classes.

Frontotemporal dementia (FTD) and Alzheimer’s disease (AD) are the two common forms of dementia. FTD syndromes are characterized by lobar atrophy (frontotemporal lobar degeneration or FTLD) and the presence of either cellular TDP43 (FTLD-TDP), tau (FTLD-tau), or FUS aggregates, while extracellular β-amyloid plaques and hyperphosphorylated tau tangles develop in AD. Oxidative stress can induce these pathological modifications in disease models, and is thought to play a role in these syndromes. Apolipoprotein D (apoD) is a glial-expressed lipocalin known to protect against oxidative stress, with increased levels in AD, supporting a protective role. The expression of apoD has not been studied in FTLD. This study assesses apoD expression in FTLD-TDP and FTLD-tau in comparison to AD and controls. It also analyzes the effect of apoD on TARDBP (TDP43 gene) and β-amyloid precursor protein (APP). The expression of apoD was analyzed by Western blotting in FTLD-TDP, FTLD-tau, AD, and control post-mortem brain tissue. An apoD-overexpressing cell model was used to study the impact of increased apoD on APP and TARDBP expression. We confirm that apoD expression was increased in AD but surprisingly it was not affected in either of the two main pathological forms of FTLD. Under oxidative stress conditions, apoD had no effect on TDP43 expression but it did decrease APP expression. This suggests that apoD does not act as a neuroprotective factor in FTLD in the same way as in AD. This could contribute to the more rapid degeneration observed in FTLD. Electronic supplementary material The online version of this article (10.1007/s12031-018-1217-9) contains supplementary material, which is available to authorized users.

ObjectiveTo investigate the prevalence of clinically relevant multiple system atrophy (MSA) and Lewy body disease (LBD) pathologies in a large frontotemporal lobar degeneration (FTLD) cohort to determine if concomitant pathologies underlie the heterogeneity of clinical features.MethodsAll prospectively followed FTLD-tau and FTLD-TDP cases held by the Sydney Brain Bank (n = 126) were screened for coexisting MSA and LBD (Braak ≥ stage IV) pathology. Relevant clinical (including family history) and genetic associations were determined.ResultsMSA pathology was not identified in this series. Of the FTLD cohort, 9 cases had coexisting LBD ≥ Braak stage IV and were associated with different FTLD subtypes including Pick disease (n = 2), corticobasal degeneration (n = 2), progressive supranuclear palsy (n = 2), and TDP type A (n = 3). All FTLD-TDP cases with coexisting LBD had mutations in progranulin (n = 2) or an abnormal repeat expansion in C9orf72 (n = 1). All FTLD-tau cases with coexisting LBD were sporadic. The H1H1 MAPT haplotype was found in all cases that could be genotyped (n = 6 of 9). Seven cases presented with a predominant dementia disorder, 3 of which developed parkinsonism. Two cases presented with a movement disorder and developed dementia in their disease course. The age at symptom onset (62 ± 11 years) and disease duration (8 ± 5 years) in FTLD cases with coexisting LBD did not differ from pure FTLD or pure LBD cases in the brain bank.ConclusionCoexisting LBD in FTLD comprises a small proportion of cases but has implications for clinical and neuropathologic diagnoses and the identification of biomarkers.

The MAPT gene is a risk locus for multiple neurodegenerative diseases, including idiopathic Parkinson's and Alzheimer's disease. We examined whether altered DNA methylation of the MAPT promoter, with its potential to modulate gene expression, was a common phenomenon in Alzheimer's disease patients with differing aetiologies. We measured MAPT promoter methylation in a brain tissue cohort of early-onset Alzheimer's disease (EOAD) with defined causative mutations in the PSEN1 gene (Normal = 10, PSEN1 AD = 10), and idiopathic late-onset Alzheimer's disease (Normal = 12, LOAD = 12). We found a brain-region-specific decrease in MAPT promoter methylation in PSEN1 AD patients. Overexpression of PSEN1 reduced MAPT promoter activity in an in vitro luciferase study, and led to an increase in methylation of the endogenous MAPT promoter. Overexpression of PSEN1 with a deletion of exon 9 mutation (Δex9) led to a smaller reduction in MAPT promoter activity relative to wild-type PSEN1 in the luciferase assay, consistent with a decreased ability to modulate endogenous MAPT gene methylation. Our study indicates a novel effect of PSEN1 on MAPT methylation, and suggests a mutation-specific effect of the PSEN1 Δex9 mutation.

Pervasive transcription of the genome produces a diverse array of functional non-coding RNAs (ncRNAs). One particular class of ncRNAs, long intervening non-coding RNAs (lincRNAs) are thought to play a role in regulating gene expression and may be a major contributor to organism and tissue complexity. The human brain with its heterogeneous cellular make-up is a rich source of lincRNAs; however, the functions of the majority of lincRNAs are unknown. Recently, by completing RNA sequencing (RNA-Seq) of the human frontal cortex, we identified linc00320 as being highly expressed in the white matter compared to grey matter in multiple system atrophy (MSA) brain. Here, we further investigate the expression patterns of linc00320 and conclude that it is involved in specific brain regions rather than having involvement in the MSA disease process. We also show that the full-length linc00320 is only expressed in human brain tissue and not in other primates, suggesting that it may be involved in improved functional connectivity for higher human brain cognition.

Multiple system atrophy (MSA) is a rare, yet fatal neurodegenerative disease that presents clinically with autonomic failure in combination with parkinsonism or cerebellar ataxia. MSA impacts on the autonomic nervous system affecting blood pressure, heart rate and bladder function, and the motor system affecting balance and muscle movement. The cause of MSA is unknown, no definitive risk factors have been identified, and there is no cure or effective treatment. The definitive pathology of MSA is the presence of α-synuclein aggregates in the brain and therefore MSA is classified as an α-synucleinopathy, together with Parkinson's disease and dementia with Lewy bodies. Although the molecular mechanisms of misfolding, fibrillation and aggregation of α-synuclein partly overlap with other α-synucleinopathies, the pathological pathway of MSA is unique in that the principal site for α-synuclein deposition is in the oligodendrocytes rather than the neurons. The sequence of pathological events of MSA is now recognized as abnormal protein redistributions in oligodendrocytes first, followed by myelin dysfunction and then neurodegeneration. Oligodendrocytes are responsible for the production and maintenance of myelin, the specialized lipid membrane that encases the axons of all neurons in the brain. Myelin is composed of lipids and two prominent proteins, myelin basic protein and proteolipid protein. In vitro studies suggest that aberration in protein distribution and lipid transport may lead to myelin dysfunction in MSA. The purpose of this perspective is to bring together available evidence to explore the potential role of α-synuclein, myelin protein dysfunction, lipid dyshomeostasis and ABCA8 in MSA pathogenesis.

Brain cholesterol homeostasis is regulated by a group of proteins called ATP-binding cassette subfamily A (ABCA) transporters. Certain ABCA transporters regulate amyloid-β protein precursor (AβPP) processing to generate amyloid-β peptides (Aβ) and are associated with an increased risk for late-onset Alzheimer's disease (AD). ABCA5 is a little-known member of the ABCA subfamily with no known function. In this study we undertook a comprehensive analysis of ABCA5 expression in the human and mouse brains. We explored the potential role of ABCA5 in AβPP processing associated with AD pathology. ABCA5 was differentially expressed in multiple regions of both human and mouse brains. It was strongly expressed in neurons with only weak expression in microglia, astrocytes, and oligodendrocytes. ABCA5 was able to stimulate cholesterol efflux in neurons. ABCA5 expression was specifically elevated in the hippocampus of AD brains. Using two in vitro cell systems we demonstrated that ABCA5 reduces Aβ production, both Aβ40 and Aβ42, without altering AβPP mRNA and protein levels, indicating that the decrease in the Aβ levels was due to changes in AβPP processing and not AβPP expression. This report represents the first extensive expression and functional study of ABCA5 in the human brain and our data suggest a plausible function of ABCA5 in the brain as a cholesterol transporter associated with Aβ generation, information that may offer a potential new target for controlling Aβ levels in the brain.

Heterozygous mutations in GBA1, the gene encoding lysosomal glucocerebrosidase, are the most frequent known genetic risk factor for Parkinson’s disease. Reduced glucocerebrosidase and α-synuclein accumulation are directly related in cell models of Parkinson’s disease. We investigated relationships between Parkinson’s disease-specific glucocerebrosidase deficits, glucocerebrosidase-related pathways, and α-synuclein levels in brain tissue from subjects with sporadic Parkinson’s disease without GBA1 mutations. Brain regions with and without a Parkinson’s disease-related increase in α-synuclein levels were assessed in autopsy samples from subjects with sporadic Parkinson’s disease (n = 19) and age- and post-mortem delay-matched controls (n = 10). Levels of glucocerebrosidase, α-synuclein and related lysosomal and autophagic proteins were assessed by western blotting. Glucocerebrosidase enzyme activity was measured using a fluorimetric assay, and glucocerebrosidase and α-synuclein messenger RNA expression determined by quantitative polymerase chain reaction. Related sphingolipids were analysed by mass spectrometry. Multivariate statistical analyses were performed to identify differences between disease groups and regions, with non-parametric correlations used to identify relationships between variables. Glucocerebrosidase protein levels and enzyme activity were selectively reduced in the early stages of Parkinson’s disease in regions with increased α-synuclein levels although limited inclusion formation, whereas GBA1 messenger RNA expression was non-selectively reduced in Parkinson’s disease. The selective loss of lysosomal glucocerebrosidase was directly related to reduced lysosomal chaperone-mediated autophagy, increased α-synuclein and decreased ceramide. Glucocerebrosidase deficits in sporadic Parkinson’s disease are related to the abnormal accumulation of α-synuclein and are associated with substantial alterations in lysosomal chaperone-mediated autophagy pathways and lipid metabolism. Our data suggest that the early selective Parkinson’s disease changes are likely a result of the redistribution of cellular membrane proteins leading to a chronic reduction in lysosome function in brain regions vulnerable to Parkinson’s disease pathology.

Parkinson's disease (PD) is a neurodegenerative disorder for which environmental factors influence disease risk and may act via an epigenetic mechanism. The microtubule-associated protein tau (MAPT) is a susceptibility gene for idiopathic PD. Methylation levels were determined by pyrosequencing of bisulfite-treated DNA in a leukocyte cohort (358 PD patients and 1084 controls) and in two brain cohorts (Brain1, comprising 69 cerebellum controls; and Brain2, comprising 3 brain regions from 28 PD patients and 12 controls). In vitro assays involved the transfection of methylated promoter-luciferase constructs or treatment with an exogenous micronutrient. In normal leukocytes, the MAPT H1/H2 diplotype and sex were predictors of MAPT methylation. Haplotype-specific pyrosequencing confirmed that the H1 haplotype had higher methylation than the H2 haplotype in normal leukocytes and brain tissues. MAPT methylation was negatively associated with MAPT expression in the Brain1 cohort and in transfected cells. Methylation levels differed between three normal brain regions (Brain2 cohort, putamen

Members of the ABCA (ATP-binding cassette subfamily A) family are characterized by their ability to transport lipids across cellular membranes and regulate lipid homoeostasis in the brain and peripheral tissues. ABCA8 is a little-known member of this subfamily that was originally cloned from human brain libraries and has no known function. In an effort to elucidate the role of ABCA8 in the brain we first undertook a comprehensive analysis of its expression in the human brain. ABCA8 was differentially expressed in multiple regions of adult human brains with significantly higher expression in oligodendrocyte-enriched white matter regions compared with grey matter cortical regions. We then assessed the impact of ABCA8 on sphingomyelin production in oligodendrocyte and showed that ABCA8 was able to significantly stimulate both sphingomyelin synthase 1 expression and sphingomyelin production. Furthermore, ABCA8 expression in the prefrontal cortex across the human life span correlated strongly with age-associated myelination, and the myelinating gene p25α was significantly up-regulated with ABCA8. The present study represents the first extensive expression and functional study of ABCA8 in the human brain and the results strongly suggest that ABCA8 regulates lipid metabolism in oligodendrocytes and potentially plays a role in myelin formation and maintenance.

ATP-binding cassette transporter A7 (ABCA7) is expressed in the brain and has been detected in macrophages, microglia, and neurons. ABCA7 promotes efflux of lipids from cells to apolipoproteins and can also regulate phagocytosis and modulate processing of amyloid precursor protein (APP) to generate the Alzheimer's disease (AD) amyloid-β (Aβ) peptide. Genome-wide association studies have indicated thatABCA7single nucleotide polymorphisms confer increased risk for late-onset AD; however, the role that ABCA7 plays in the brain in the AD context is unknown. In the present study, we crossed ABCA7-deficient (A7−/−) mice with J20 amyloidogenic mice to address this issue. We show that ABCA7 loss doubled insoluble Aβ levels and thioflavine-S–positive plaques in the brain. This was not related to changes in APP processing (assessed by analysis of full-length APP and the APP β C-terminal fragment). Apolipoprotein E regulates cerebral Aβ homeostasis and plaque load; however, the apolipoprotein E concentration was not altered by ABCA7 loss. Spatial reference memory was significantly impaired in both J20 and J20/A7−/−mice compared with wild-type mice; however, there were no cognitive differences between J20 and J20/A7−/−mice. There were also no major differences detected in hippocampal or plaque-associated microglial/macrophage markers between J20 and J20/A7−/−mice, whereas the capacity for bone marrow-derived macrophages derived from A7−/−mice to take up oligomeric Aβ was reduced by 51% compared with wild-type mice. Our results suggest that ABCA7 plays a role in the regulation of Aβ homeostasis in the brain and that this may be related to altered phagocyte function.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by dementia and abnormal deposits of aggregated amyloid-β in the brain. Recent genome-wide association studies have revealed that ABCA7 is strongly associated with AD. In vitro evidence suggests that the role of ABCA7 is related to phagocytic activity. Deletion of ABCA7 in a mouse model of AD exacerbates cerebral amyloid-β plaque load. However, the biological role of ABCA7 in AD brain pathogenesis is unknown. We show that ABCA7 is highly expressed in microglia and when monocytes are differentiated into macrophages. We hypothesized that ABCA7 plays a protective role in the brain that is related to phagocytic clearance of amyloid-β. We isolated microglia and macrophages from Abca7-/- and wild type mice and tested them for their capacity to phagocytose amyloid-β oligomers. We found that the phagocytic clearance of amyloid-β was substantially reduced in both microglia and macrophages from Abca7-/- mice compared to wild type mice. Consistent with these results, in vivo phagocytic clearance of amyloid-β oligomers in the hippocampus was reduced in Abca7-/- mice. Furthermore, ABCA7 transcription was upregulated in AD brains and in amyloidogenic mouse brains specifically in the hippocampus as a response to the amyloid-β pathogenic state. Together these results indicate that ABCA7 mediates phagocytic clearance of amyloid-β in the brain, and reveal a mechanism by which loss of function of ABCA7 increases the susceptibility to AD.

Multiple system atrophy (MSA) is a rare, yet rapidly-progressive neurodegenerative disease that presents clinically with autonomic failure in combination with parkinsonism or cerebellar ataxia. The definitive neuropathology differentiating MSA from Lewy body diseases is the presence of α-synuclein aggregates in oligodendrocytes (called glial cytoplasmic inclusion or GCI) rather than the fibrillar aggregates in neurons (called Lewy bodies). This makes the pathological pathway(s) in MSA unique in that oligodendrocytes are involved rather than predominantly neurons, as is most other neurodegenerative disorders. MSA is therefore regarded as an oligodendrogliopathy. The etiology of MSA is unknown. No definitive risk factors have been identified, although α-synuclein and other genes have been variably linked to MSA risk. Utilization of postmortem brain tissues has greatly advanced our understanding of GCI pathology and the subsequent neurodegeneration. However, extrapolating the early pathogenesis of MSA from such resource has been difficult and limiting. In recent years, cell and animal models developed for MSA have been instrumental in delineating unique MSA pathological pathways, as well as aiding in clinical phenotyping. The purpose of this review is to bring together and discuss various animal models that have been developed for MSA and how they have advanced our understanding of MSA pathogenesis, particularly the dynamics of α-synuclein aggregation. This review will also discuss how animal models have been used to explore potential therapeutic avenues for MSA, and future directions of MSA modeling.

Pathway analysis is a powerful method for discerning differentially regulated genes and elucidating their biological importance. It allows for the identification of perturbed or aberrantly expressed genes within a biological context from extensive data sets and offers a simplistic approach for interrogating such data sets. With the growing use of microarrays and RNA-Seq, data for genome-wide studies are growing at an alarming rate, and the use of deep sequencing is revealing elements of the genome previously uncharacterised. Through the employment of pathway analysis, mechanisms in complex diseases may be explored and novel causatives found primarily through differentially regulated genes. Further, with the implementation of next generation sequencing, a deeper resolution may be attained, particularly in identification of isoform diversity and SNPs. Here, we look at a broad overview of pathway analysis in the human brain transcriptome and its relevance in teasing out underlying causes of complex diseases. We will outline processes in data gathering and analysis of particular diseases in which these approaches have been successful.

Mutations of the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of Parkinson's disease (PD) and are associated with pleiomorphic neuropathology. We hypothesize that LRRK2 mediates its pathogenic effect through alterna- tive splicing of neurodegeneration genes. Methods used in this study included western blotting analysis of sub- cellular protein fractions, exon-array analysis of RNA from cultured neuroblastoma cells transfected with LRRK2 expression vectors, and reverse-transcription polymerase chain reaction (RT-PCR) of RNA from cul- tured cells and postmortem tissue. Overexpression of the LRRK2 G2019S mutant resulted in a significant (2.6-fold; P 5 0.020) decrease in nuclear transactive response DNA-binding protein 43 levels. Exon-array analyses revealed that wild-type LRRK2 had a signifi- cant effect on the expression of genes with nuclear (P < 10 222 ) and cell-cycle functions (P < 10 215 ). We repli- cated changes in gene expression in 30% of selected genes by quantitative RT-PCR. Overexpression of LRRK2 resulted in the altered splicing of two genes associated with PD, with an increased inclusion of exon 10 of microtubule-associated protein tau (1.7-fold; P 5 0.001) and exon 5 of the alpha-synuclein (SNCA) gene (1.6-fold; P 50.005). Moreover, overexpression of LRRK2 (G2019S) and two mutant genes associated with neurodegeneration, TARDBP (M337V) and FUS (R521H), were associated with decreased inclusion out of the dystonin (DST) 1e precursor exons in SK-N-MC cells. Altered splicing of SNCA (1.9-fold; P < 0.001) and DST genes (log2 2.3-fold; P 5 0.005) was observed in a cohort of PD, compared with neurologically healthy, brains. This suggests that aberrant RNA metabolism is an important contributor to idiopathic PD. V C 2012 Movement Disorder Society

The Niemann-Pick type C1 (NPC1) protein mediates the trafficking of cholesterol from lysosomes to other organelles. Mutations in the NPC1 gene lead to the retention of cholesterol and other lipids in the lysosomal compartment, and such defects are the basis of NPC disease. Several parallels exist between NPC disease and Alzheimer's disease (AD), including altered cholesterol homeostasis, changes in the lysosomal system, neurofibrillary tangles, and increased amyloid-beta generation. How the expression of NPC1 in the human brain is affected in AD has not been investigated so far. In the present study, we measured NPC1 mRNA and protein expression in three distinct regions of the human brain, and we revealed that NPC1 expression is upregulated at both mRNA and protein levels in the hippocampus and frontal cortex of AD patients compared to control individuals. In the cerebellum, a brain region that is relatively spared in AD, no difference in NPC1 expression was detected. Similarly, murine NPC1 mRNA levels were increased in the hippocampus of 12-month-old transgenic mice expressing a familial AD form of human amyloid-beta precursor protein (APP) and presenilin-1 (APP/PS1tg) compared to 12-month-old wild type mice, whereas no change in NPC1 was detected in mouse cerebellum. Immunohistochemical analysis of human hippocampus indicated that NPC1 expression was strongest in neurons. However, in vitro studies revealed that NPC1 expression was not induced by transfecting SK-N-SH neurons with human APP or by treating them with oligomeric amyloid-beta peptide. Total cholesterol levels were reduced in hippocampus from AD patients compared to control individuals, and it is therefore possible that the increased expression of NPC1 is linked to perturbed cholesterol homeostasis in AD.

Previous studies suggest that membrane lipids may regulate proteolytic processing of the amyloid precursor protein (APP) to generate amyloid-beta peptide (Abeta). In the present study, we have assessed the capacity for a series of structurally related synthetic ceramide analogues to modulate APP processing in vitro. The compounds tested are established glucosylceramide synthase (GS) inhibitors based on the d -1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) structure. PDMP and related compounds PPMP and EtDO-P4 inhibited Abeta secretion from Chinese hamster ovary cells expressing human APP (CHO-APP) with approximate IC50 values of 15, 5, and 1 μM, respectively. A trend for reduced secretion of the APP alpha-secretase product, sAPPalpha, was also observed in PDMP-treated cells but not in PPMP- or ETDO-P4-treated cells, whereas levels of the cellular beta-secretase product APP C-terminal fragment, CTFbeta, were increased by both PDMP and PPMP but unaltered with EtDO-P4 treatment. Our data also revealed that EtDO-P4 inhibits endogenous Abeta production by human neurons. In conclusion, this study provides novel information regarding the regulation of APP processing by synthetic ceramide analogues and reveals that the most potent of these compounds is EtDO-P4.

ATP-binding cassette transporter A1 (ABCA1) reduces amyloid-beta burden in transgenic mouse models of Alzheimer's disease (AD). Associations between ABCA1 polymorphisms and AD risk are also established. Little is known regarding the regulation of ABCA1 expression in the brain and how this may be affected by AD. In the present study we assessed ABCA1 mRNA and protein expression in the hippocampus of AD cases compared to controls. ABCA1 was clearly expressed in hippocampal neurons and expression was increased two- to three-fold in AD cases. The increased hippocampal ABCA1 expression was associated with increased APOE and PUMA gene expression, implying an association with neuronal stress. Consistent with this, treatment of SK-N-SH neurons with amyloid-beta peptide resulted in a 48% loss in survival and a significant upregulation of ABCA1, APOE, and PUMA gene expression. Studies in young (2 month) and old (12 month) transgenic mice expressing a familial AD form of human amyloid-beta protein precursor and presenilin-1 revealed a significant age-dependent upregulation of hippocampal Abca1 compared to wild-type control mice. However, hippocampal Apoe and Puma gene expression were not correlated with increased Abca1 expression in mice. Our data indicate that ABCA1 is upregulated in AD hippocampal neurons potentially via an amyloid-beta-mediated pathway.

Apolipoprotein D (apoD) is a lipid binding protein expressed in the brain where its function is largely unknown. Based on changes in lipid metabolism and deposition that occur in the human brain during postnatal development, we investigated changes in apoD expression in the prefrontal cortex in 69 normal cases ranging in age from 40 days to 49 years utilizing gene microarray, quantitative PCR and western blotting methods. In contrast to the high expression of apolipoprotein E (APOE), low-density lipoprotein receptor-related protein 8 (LRP8) and 3-hydroxy-3-methyl-glutaryl-CoA reducatase (HMGCR) (genes that play a role in lipid-related pathways in brain development) early in life, apoD expression was low in neonates and increased in expression throughout life resulting in six- to eight-fold higher levels at the mRNA and protein levels in adults. Recent studies suggest that apoD has a novel antioxidant function in the brain and we found that the increased apoD expression throughout development and into adulthood was correlated with the expression of antioxidant genes superoxide dismutase 1 (SOD1) and glutathione peroxidase 3 (GPX3) as well as proteins that were modified by the lipid peroxidation end-product 4-hydroxynonenal. These studies reveal that apoD expression is increased throughout life in the human prefrontal cortex and that this is correlated with genetic and biochemical markers of oxidative stress.

Cholesterol is an integral component of neuronal membranes and recent evidence has shown that it regulates amyloid-beta protein precursor processing to form amyloid-beta peptides, which are a major constituent of cerebral amyloid plaques associated with Alzheimer's disease. 27-Hydroxycholesterol (27OHC) is synthesized from cholesterol via sterol 27-hydroxylase (CYP27A1) in the brain and, unlike cholesterol, can cross into the brain through the blood brain barrier from the circulation. Previous studies point toward a potential role for 27OHC in the regulation of neuronal amyloid-beta peptide generation, however, this has not been investigated in primary human neurons. Here we show that 27OHC significantly reduced amyloid-beta peptide detected in cell culture supernatants from primary human neurons. We also show that 27OHC does not affect alpha-, beta- or gamma-secretase activity but does upregulate the liver X receptor (LXR) responsive genes ABCA1, ABCG1 and APOE. These data suggest that 27OHC-mediated reduction in extracellular amyloid-beta peptide levels is potentially due to its action as an LXR ligand.

Neuronal apoptosis is crucial for central nervous system development and also contributes to neurodegenerative disease. Apolipoprotein-E (apoE) regulates brain lipid transport and specific neuronal functions and previous research, investigating non-neuronal cell types, identified an association between apoptosis and increased apoE expression. In the present study we used the human SK-N-SH neuronal cell line to investigate potential changes in apoE expression during apoptosis which occurs as a consequence of extended culture (up to 5 days) without replenishing trophic factors. Standard and real-time PCR analysis indicated a significant 6-fold increase in apoE mRNA after 3 days which was correlated with caspase-3 activation, TUNEL positivity and the formation of apoptotic bodies. ApoE protein levels were low in the absence of apoptosis but increased by 8-fold when apoptosis was induced. Analysis of cellular debris that accumulated in the culture supernatants indicated that apoE levels became progressively concentrated in apoptotic bodies. These data indicate that apoE is up-regulated during neuronal apoptosis and raise the possibility that apoE may play a role in the clearance of apoptotic bodies through apoE-receptor interactions.

The amyloid-β protein precursor (AβPP) has long been linked to Alzheimer’s disease (AD). Using J20 mice, which express human AβPP with Swedish and Indiana mutations, we studied early pathological changes in the olfactory bulb. The presence of AβPP/amyloid-β (Aβ) was examined in mice aged 3 months (before the onset of hippocampal Aβ deposition) and over 5 months (when hippocampal Aβ deposits are present). The number of neurons, non-neurons, and proliferating cells was assessed using the isotropic fractionator method. Our results demonstrate that although AβPP is overexpressed in some of the mitral cells, widespread Aβ deposition and microglia aggregates are not prevalent in the olfactory bulb. The olfactory bulbs of the younger J20 group harbored significantly fewer neurons than those of the age-matched wild-type mice (5.57±0.13 million versus 6.59±0.36 million neurons; p = 0.011). In contrast, the number of proliferating cells was higher in the young J20 than in the wild-type group (i.e., 6617±425 versus 4455±623 cells; p = 0.011). A significant increase in neurogenic activity was also observed in the younger J20 olfactory bulb. In conclusion, our results indicate that (1) neurons participating in the mouse olfactory function overexpress AβPP; (2) the cellular composition of the young J20 olfactory bulb is different from that of wild-type littermates; (3) these differences may reflect altered neurogenic activity and/or delayed development of the J20 olfactory system; and (4) AβPP/Aβ-associated pathological changes that take place in the J20 hippocampus and olfactory bulb are not identical.

Five ATP-binding cassette (ABC) subfamily-A transporters (ABCA1, ABCA2, ABCA3, ABCA7 and ABCA8) are expressed in the brain. These transporters may regulate brain lipid transport; however, their relative expression level in isolated human brain cells is unknown. We developed real-time polymerase chain reaction assays to quantify the expression of these genes in human neurons, astrocytes, oligodendrocytes, microglia and cell lines. Neurons expressed predominantly ABCA1 and ABCA3; astrocytes ABCA1, ABCA2 and ABCA3; microglia ABCA1 and oligodendrocytes ABCA2 and ABCA3. Although ABCA7 and ABCA8 expression was relatively low in all cells, the highest expression occurred in microglia and neurons, respectively. ABCA gene expression in the NTERA-2 and MO3.13 cell lines closely resembled the ABCA expression pattern of primary neurons and oligodendrocytes, respectively.

A transposon (TnNip) encoding nisin production was visualized and stabilized as a plasmid of approximately 60 kb in the transconjugant, LM0230Fus(TnNip), formed from a conjugation between nisin-producing Lactococcus lactis subsp. lactis LL33-1 and a plasmid-free LM0230 derivative. The plasmid did not correspond in size to any of the plasmids resident in the donor. A plasmid-cured derivative of this construct was also able to stabilize the transposon, as a plasmid, from all donors used. Therefore, for the first time, a host has been identified that can maintain the TnNip transposons stably excised out of the chromosome. The plasmid DNA was isolated and characterized by restriction digestion and was used to successfully transform cells, thus linking the nisin production phenotype to the plasmid.

Cellular glycosphingolipid (GSL) storage is known to promote cholesterol accumulation. Although physical interactions between GSLs and cholesterol are thought to cause intracellular cholesterol "trapping," it is not known whether cholesterol homeostatic mechanisms are also impaired under these conditions. ApoA-I-mediated cholesterol efflux via ABCA1 (ATP-binding cassette transporter A1) is a key regulator of cellular cholesterol balance. Here, we show that apoA-I-mediated cholesterol efflux was inhibited (by up to 53% over 8 h) when fibroblasts were treated with lactosylceramide or the glucocerebrosidase inhibitor conduritol B epoxide. Furthermore, apoA-I-mediated cholesterol efflux from fibroblasts derived from patients with genetic GSL storage diseases (Fabry disease, Sandhoff disease, and GM1 gangliosidosis) was impaired compared with control cells. Conversely, apoA-I-mediated cholesterol efflux from fibroblasts and cholesterol-loaded macrophage foam cells was dose-dependently stimulated (by up to 6-fold over 8 h) by the GSL synthesis inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP). Unexpectedly, a structurally unrelated GSL synthesis inhibitor, N-butyldeoxynojirimycin, was unable to stimulate apoA-I-mediated cholesterol efflux despite achieving similar GSL depletion. PDMP was found to up-regulate ABCA1 mRNA and protein expression, thereby identifying a contributing mechanism for the observed acceleration of cholesterol efflux to apoA-I. This study reveals a novel defect in cellular cholesterol homeostasis induced by GSL storage and identifies PDMP as a new agent for enhancing cholesterol efflux via the ABCA1/apoA-I pathway.

Aims: To develop food-grade cloning and expression vectors for use in genetic modification of Lactococcus lactis. Methods and Results: Two plasmid replicons and three dominant selection markers were isolated from L. lactis and used to construct five food-grade cloning vectors. These vectors were composed of DNA only from L. lactis and contained no antibiotic resistance markers. Three of the vectors (pND632, pND648 and pND969) were based on the same plasmid replicon and carried, either alone or in combination, the three different selectable markers encoding resistance to nisin, cadmium and/or copper. The other two (pND965DJ and pND965RS) were derived from a cadmium resistance plasmid, and carried a constitutive promoter and a copper-inducible promoter, respectively, immediately upstream of a multicloning site. All vectors were stable in L. lactis LM0230 for at least 40 generations without selection pressure. The two groups of vectors were compatible in L. lactis LM0230. The vectors pND648 and pND965RS, as representatives of the two groups, were transferred successfully by electroporation into and maintained in an industrial strain of L. lactis. The usefulness of the vectors was further demonstrated by expressing a phage resistance gene (abiI) in another industrial strain of L. lactis. Conclusions: The five food-grade vectors constructed are potentially useful for industrial strains of L. lactis. Significance and Impact of the Study: These vectors represent a new set of molecular tools useful for food-grade modifications of L. lactis.

Macrophage scavenger class A type I and type II receptors (SR-A) are trimeric, integral membrane glycoproteins that bind an unusually broad array of macromolecular ligands. These ligands include modified proteins and lipoproteins, nucleic acids, and a variety of plant and microbial cell wall constituents, such as fucoidan and lipoteichoic acid. Early studies of SR-A functions indicated that the receptors bound, internalized, and degraded their ligands without provoking any macrophage activating signaling events. More recent studies have provided evidence that several SR-A ligands can activate macrophage gene expression via utilization of a receptor-linked, PI3-kinase pathway. To investigate the role of SR-A in engaging signal transduction events, we employed macrophages taken from mice lacking these receptors. Using either fucoidan or lipoteichoic acid, we confirm that both ligands stimulate tyrosine phosphorylation of PI3-kinase and production of modest levels of the cytokine, TNFα. However, macrophages taken from SR-A null mice did not differ from wild type macrophages in these responses, indicating that these signaling events arise independently of SR-A activity. Employing mice lacking CD14, a GPI anchored receptor that binds bacterial lipopolysaccharide and signals via activation of Toll-like receptors, we show that the fucoidan and lipoteichoic acid responses are largely abrogated when CD14 is absent. These data do not provide support for direct SR-A involvement in signal transduction events and suggest that the early characterization of these receptors as initiators of a non-phlogistic, pathogen clearance pathway was correct.

α-Synuclein is an abundantly expressed neuronal protein that is at the center of focus in understanding a group of neurodegenerative disorders called α-synucleinopathies, which are characterized by the presence of aggregated α-synuclein intracellularly. Primary α-synucleinopathies include Parkinson's disease (PD), dementia with Lewy bodies and multiple system atrophy, with α-synuclein also found secondarily in a number of other diseases, including Alzheimer's disease. Understanding how α-synuclein aggregates form in these different disorders is important for the understanding of its pathogenesis in Lewy body diseases. PD is the most prevalent of the α-synucleinopathies and much of the initial research on α-synuclein Lewy body pathology was based on PD but is also relevant to Lewy bodies in other diseases (dementia with Lewy bodies and Alzheimer's disease). Polymorphism and mutation studies of SNCA, the gene that encodes α-synuclein, provide much evidence for a causal link between α-synuclein and PD. Among the primary α-synucleinopathies, multiple system atrophy is unique in that α-synuclein deposition occurs in oligodendrocytes rather than neurons. It is unclear whether α-synuclein originates from oligodendrocytes or whether it is transmitted somehow from neurons. α-Synuclein exists as a natively unfolded monomer in the cytosol, but in the presence of lipid membranes it is thought to undergo a conformational change to a folded α-helical secondary structure that is prone to forming dimers and oligomers. Posttranslational modification of α-synuclein, such as phosphorylation, ubiquitination and nitration, has been widely implicated in α-synuclein aggregation process and neurotoxicity. Recent studies using animal and cell models, as well as autopsy studies of patients with neuron transplants, provided compelling evidence for prion-like propagation of α-synuclein. This observation has implications for therapeutic strategies, and much recent effort is focused on developing antibodies that target extracellular α-synuclein.

Heat pump dryers were studied as an alternative to freeze drying for dehydration of lactic acid bacteria for starter cultures. Factors investigated were bacterial growth phase, addition of dehydro-protectants, drying conditions, drying media and storage life of the dried bacteria. The main criterion for evaluation was the activity of the dried product. Results indicated that single dehydro-protectants were not able to give high activities, but suitable combinations were found giving results at least equal to freeze-drying. Nitrogen purging was found to give a significant improvement in activity.

When lactate was removed from sucrose fermentation in situ, using the anionic-exchange resin Amberlite IRA-67, by Lactococcus lactis growing in batch culture, nisin production increased by two-fold when compared to the alkali pH-controlled fermentation. In comparison to sucrose, lactate removal increased nisin production 1.5-fold and 0.3-fold when galactose and glucose were used as carbon sources respectively.

α-Synuclein is a neuronal protein that is at the center of focus in understanding the etiology of a group of neurodegenerative diseases called α-synucleinopathies, which includes Parkinson’s disease (PD). Despite much research, the exact physiological function of α-synuclein is still unclear. α-Synuclein has similar biophysical properties as apolipoproteins and other lipid-binding proteins and has a high affinity for cholesterol. These properties suggest a possible role for α-synuclein as a lipid acceptor mediating cholesterol efflux (the process of removing cholesterol out of cells). To test this concept, we “loaded” SK-N-SH neuronal cells with fluorescently-labelled cholesterol, applied exogenous α-synuclein, and measured the amount of cholesterol removed from the cells using a classic cholesterol efflux assay. We found that α-synuclein potently stimulated cholesterol efflux. We found that the process was dose and time dependent, and was saturable at 1.0 µg/mL of α-synuclein. It was also dependent on the transporter protein ABCA1 located on the plasma membrane. We reveal for the first time a novel role of α-synuclein that underscores its importance in neuronal cholesterol regulation, and identify novel therapeutic targets for controlling cellular cholesterol levels.