Predicting progression to Alzheimer's disease with human hippocampal progenitors exposed to serum.

Adult hippocampal neurogenesis is important for learning and memory and is altered early in Alzheimer's disease. As hippocampal neurogenesis is modulated by the circulatory systemic environment, evaluating a proxy of how hippocampal neurogenesis is affected by the systemic milieu could serve as an early biomarker for Alzheimer's disease progression. Here, we used an in vitro assay to model the impact of systemic environment on hippocampal neurogenesis. A human hippocampal progenitor cell line was treated with longitudinal serum samples from individuals with mild cognitive impairment, who either progressed to Alzheimer's disease or remained cognitively stable. Mild cognitive impairment to Alzheimer's disease progression was characterized most prominently with decreased proliferation, increased cell death and increased neurogenesis. A subset of 'baseline' cellular readouts together with education level were able to predict Alzheimer's disease progression. The assay could provide a powerful platform for early prognosis, monitoring disease progression and further mechanistic studies.

Microbiota from Alzheimer's patients induce deficits in cognition and hippocampal neurogenesis.

Alzheimer's disease is a complex neurodegenerative disorder leading to a decline in cognitive function and mental health. Recent research has positioned the gut microbiota as an important susceptibility factor in Alzheimer's disease by showing specific alterations in the gut microbiome composition of Alzheimer's patients and in rodent models. However, it is unknown whether gut microbiota alterations are causal in the manifestation of Alzheimer's symptoms. To understand the involvement of Alzheimer's patient gut microbiota in host physiology and behaviour, we transplanted faecal microbiota from Alzheimer's patients and age-matched healthy controls into microbiota-depleted young adult rats. We found impairments in behaviours reliant on adult hippocampal neurogenesis, an essential process for certain memory functions and mood, resulting from Alzheimer's patient transplants. Notably, the severity of impairments correlated with clinical cognitive scores in donor patients. Discrete changes in the rat caecal and hippocampal metabolome were also evident. As hippocampal neurogenesis cannot be measured in living humans but is modulated by the circulatory systemic environment, we assessed the impact of the Alzheimer's systemic environment on proxy neurogenesis readouts. Serum from Alzheimer's patients decreased neurogenesis in human cells in vitro and were associated with cognitive scores and key microbial genera. Our findings reveal for the first time, that Alzheimer's symptoms can be transferred to a healthy young organism via the gut microbiota, confirming a causal role of gut microbiota in Alzheimer's disease, and highlight hippocampal neurogenesis as a converging central cellular process regulating systemic circulatory and gut-mediated factors in Alzheimer's.

Artificial intelligence for biomarker discovery in Alzheimer's disease and dementia.

With the increase in large multimodal cohorts and high-throughput technologies, the potential for discovering novel biomarkers is no longer limited by data set size. Artificial intelligence (AI) and machine learning approaches have been developed to detect novel biomarkers and interactions in complex data sets. We discuss exemplar uses and evaluate current applications and limitations of AI to discover novel biomarkers. Remaining challenges include a lack of diversity in the data sets available, the sheer complexity of investigating interactions, the invasiveness and cost of some biomarkers, and poor reporting in some studies. Overcoming these challenges will involve collecting data from underrepresented populations, developing more powerful AI approaches, validating the use of noninvasive biomarkers, and adhering to reporting guidelines. By harnessing rich multimodal data through AI approaches and international collaborative innovation, we are well positioned to identify clinically useful biomarkers that are accurate, generalizable, unbiased, and acceptable in clinical practice. HIGHLIGHTS: Artificial intelligence and machine learning approaches may accelerate dementia biomarker discovery. Remaining challenges include data set suitability due to size and bias in cohort selection. Multimodal data, diverse data sets, improved machine learning approaches, real-world validation, and interdisciplinary collaboration are required.

Characterisation of immune cell function in fragment and full-length Huntington's disease mouse models.

Inflammation is a growing area of research in neurodegeneration. In Huntington's disease (HD), a fatal inherited neurodegenerative disease caused by a CAG-repeat expansion in the gene encoding huntingtin, patients have increased plasma levels of inflammatory cytokines and circulating monocytes that are hyper-responsive to immune stimuli. Several mouse models of HD also show elevated plasma levels of inflammatory cytokines. To further determine the degree to which these models recapitulate observations in HD patients, we evaluated various myeloid cell populations from different HD mouse models to determine whether they are similarly hyper-responsive, as well as measuring other aspects of myeloid cell function. Myeloid cells from each of the three mouse models studied, R6/2, HdhQ150 knock-in and YAC128, showed increased cytokine production when stimulated. However, bone marrow CD11b(+) cells did not show the same hyper-responsive phenotype as spleen and blood cells. Furthermore, macrophages isolated from R6/2 mice show increased levels of phagocytosis, similar to findings in HD patients. Taken together, these results show significant promise for these mouse models to be used to study targeting innate immune pathways identified in human cells, thereby helping to understand the role the peripheral immune system plays in HD progression.

Antibodies against phosphorylcholine are not altered in plasma of patients with Alzheimer's disease.

BACKGROUND: Phosphorylcholine is one of the major epitopes of oxidised low density lipoprotein. Low levels of IgM antibodies against phosphorylcholine (anti-PC) are associated with development of myocardial infarction and stroke. It has been shown that patients with Alzheimer's disease and other dementias have significantly lower serum anti-PC levels compared to controls, suggesting that low levels of atheroprotective anti-PC may play a role in AD and dementia. METHODS: We quantified levels of anti-PC levels using an ELISA in plasma from 176 controls, 125 patients with Alzheimer's disease, 19 patients with vascular dementia and 63 patients with other dementias. RESULTS: We observed similar plasma anti-PC levels in controls, patients with Alzheimer's disease, and other dementias. CONCLUSIONS: Our data suggests that anti-PC is not useful as a biomarker for Alzheimer's disease.

Flt3 ligand does not differentiate between Parkinsonian disorders.

BACKGROUND: Differential diagnosis of parkinsonian disorders is challenging because of overlapping symptoms, especially during early stages of disease. No validated biomarkers are available for early and accurate diagnosis of multiple system atrophy and other parkinsonian disorders. It has been reported that flt3 ligand levels in cerebrospinal fluid could clearly differentiate patients with Parkinson's disease from patients with multiple system atrophy, with 99% sensitivity and 95% specificity. METHODS: We measured flt3 ligand levels in cerebrospinal fluid of subjects with Parkinson's disease (n = 37), multiple system atrophy (n = 30), and progressive supranuclear palsy (n = 19). RESULTS: In our cohort, no significant difference was found in flt3 ligand levels between Parkinson's disease, multiple system atrophy, and progressive supranuclear palsy. CONCLUSIONS: Our results suggest that cerebrospinal fluid flt3 ligand levels do not differentiate between parkinsonian disorders.

Plasma melatonin is reduced in Huntington's disease.

This study was undertaken to determine whether the production of melatonin, a hormone regulating sleep in relation to the light/dark cycle, is altered in Huntington's disease. We analyzed the circadian rhythm of melatonin in a 24-hour study of cohorts of control, premanifest, and stage II/III Huntington's disease subjects. The mean and acrophase melatonin concentrations were significantly reduced in stage II/III Huntington's disease subjects compared with controls. We also observed a nonsignificant trend toward reduced mean and acrophase melatonin in premanifest Huntington's disease subjects. Onset of melatonin rise was significantly more temporally spread in both premanifest and stage II/III Huntington's disease subjects compared with controls. A nonsignificant trend also was seen for reduced pulsatile secretion of melatonin. Melatonin concentrations are reduced in Huntington's disease. Altered melatonin patterns may provide an explanation for disrupted sleep and circadian behavior in Huntington's disease, and represent a biomarker for disease state. Melatonin therapy may help the sleep disorders seen in Huntington's disease.

A secreted proteomic footprint for stem cell pluripotency.

With a view to developing a much-needed non-invasive method for monitoring the healthy pluripotent state of human stem cells in culture, we undertook proteomic analysis of the waste medium from cultured embryonic (Man-13) and induced (Rebl.PAT) human pluripotent stem cells (hPSCs). Cells were grown in E8 medium to maintain pluripotency, and then transferred to FGF2 and TGFß deficient E6 media for 48 hours to replicate an early, undirected dissolution of pluripotency. We identified a distinct proteomic footprint associated with early loss of pluripotency in both hPSC lines, and a strong correlation with changes in the transcriptome. We demonstrate that multiplexing of four E8- against four E6- enriched secretome biomarkers provides a robust, diagnostic metric for the pluripotent state. These biomarkers were further confirmed by Western blotting which demonstrated consistent correlation with the pluripotent state across cell lines, and in response to a recovery assay.

Restoring miR-132 expression rescues adult hippocampal neurogenesis and memory deficits in Alzheimer's disease.

Neural stem cells residing in the hippocampal neurogenic niche sustain lifelong neurogenesis in the adult brain. Adult hippocampal neurogenesis (AHN) is functionally linked to mnemonic and cognitive plasticity in humans and rodents. In Alzheimer's disease (AD), the process of generating new neurons at the hippocampal neurogenic niche is impeded, yet the mechanisms involved are unknown. Here we identify miR-132, one of the most consistently downregulated microRNAs in AD, as a potent regulator of AHN, exerting cell-autonomous proneurogenic effects in adult neural stem cells and their progeny. Using distinct AD mouse models, cultured human primary and established neural stem cells, and human patient material, we demonstrate that AHN is directly affected by AD pathology. miR-132 replacement in adult mouse AD hippocampus restores AHN and relevant memory deficits. Our findings corroborate the significance of AHN in mouse models of AD and reveal the possible therapeutic potential of targeting miR-132 in neurodegeneration.

Formation of Mature Nephrons by Implantation of Human Pluripotent Stem Cell-Derived Progenitors into Mice.

In the future, stem cell-based technologies may be harnessed to replace conventional dialysis and transplantation in patients with diabetic nephropathy. Recently, there has been considerable effort to improve methods for the differentiation of human pluripotent stem cells (hPSCs) into kidney cells in culture. Here, we present a protocol for obtaining more advanced kidney structures than have currently been possible in vitro, including vascularized glomeruli and tubular elements. HPSCs are first differentiated in 2D culture to a kidney progenitor stage. These cells are then dissociated and injected subcutaneously into immunocompromised mice. Twelve weeks later, the cells have developed into mature kidney structures and are excised for further characterization. This method constitutes a significant improvement on protocols that involve either exclusively a 2D culture or placing the cells in 3D organoid culture at the air-liquid interface in vitro.

Development of novel mass spectrometric methods for identifying HOCl-induced modifications to proteins.

Protein oxidation is thought to contribute to a number of inflammatory diseases, hence the development of sensitive and specific analytical techniques to detect oxidative PTMs (oxPTMs) in biological samples is highly desirable. Precursor ion scanning for fragment ions of oxidized amino acid residues was investigated as a label-free MS approach to mapping specific oxPTMs in a complex mixture of proteins. Using HOCl-oxidized lysozyme as a model system, it was found that the immonium ions of oxidized tyrosine and tryptophan formed in MS(2) analysis could not be used as diagnostic ions, owing to the occurrence of isobaric fragment ions from unmodified peptides. Using a double quadrupole linear ion trap mass spectrometer, precursor ion scanning was combined with detection of MS(3) fragment ions from the immonium ions and collisionally-activated decomposition peptide sequencing to achieve selectivity for the oxPTMs. For chlorotyrosine, the immonium ion at 170.1 m/z fragmented to yield diagnostic ions at 153.1, 134.1, and 125.1 m/z, and the hydroxytyrosine immonium ion at 152.1 m/z gave diagnostic ions at 135.1 and 107.1 m/z. Selective MS(3) fragment ions were also identified for 2-hydroxytryptophan and 5-hydroxytryptophan. The method was used successfully to map these oxPTMs in a mixture of nine proteins that had been treated with HOCl, thereby demonstrating its potential for application to complex biological samples.

In vitro analysis of glial cell function in ganglioside-deficient mice.

Gangliosides are a family of sialic acid-containing glycosphingolipids highly enriched in neuronal and glial membranes, where they play pleiotropic roles in nervous system function. In this glial cell biological study, we used mice deficient in glycosyltransferases involved in ganglioside biosynthesis to gain insights into the possible role of ganglioside overexpression or deficiency on glial cell proliferation, migration, and differentiation in vitro. Primary cultures of olfactory ensheathing cells, oligodendrocyte lineage cells, and Schwann cells isolated from beta1,4-N-acetylgalactosaminyl (beta1,4-GalNAc) transferase- and alpha-2,8-sialyltransferase-deficient mice demonstrated subtle differences in their behavior when compared with wild-type glia. Oligodendrocyte-axonal interactions were investigated in dissociated embryonic mixed spinal cord cultures in which axonal ensheathment with myelin internodes and organized nodes of Ranvier form. In these myelinating cultures, deficiency of complex gangliosides, as found in beta1,4-GalNAc T(-/-) mice, resulted in the temporal disorganization of K(v) and Na(+) channels at the nodes of Ranvier, similar to that seen in beta1,4-GalNAc T(-/-) mice in vivo. These data show that glycosyltransferase deficiency and the consequent ganglioside imbalance has subtle effects on a range of glial cell functions and that in vitro systems can be used to explore these in ways that complement whole animal physiology. Our results are also consistent with the absence of gross neurodevelopmental dysfunction in mice lacking a variety of different gangliosides, suggesting that ganglioside redundancy and substitution are mechanisms that compensate for the lack of a full complement of complex gangliosides.

A Critical Evaluation of Wet Biomarkers for Huntington's Disease: Current Status and Ways Forward.

There is an unmet clinical need for objective biomarkers to monitor disease progression and treatment response in Huntington's disease (HD). The aim of this review is, therefore, to provide practical advice for biomarker discovery and to summarise studies on biofluid markers for HD. A PubMed search was performed to review literature with regard to candidate saliva, urine, blood and cerebrospinal fluid biomarkers for HD. Information has been organised into tables to allow a pragmatic approach to the discussion of the evidence and generation of practical recommendations for future studies. Many of the markers published converge on metabolic and inflammatory pathways, although changes in other analytes representing antioxidant and growth factor pathways have also been found. The most promising markers reflect neuronal and glial degeneration, particularly neurofilament light chain. International collaboration to standardise assays and study protocols, as well as to recruit sufficiently large cohorts, will facilitate future biomarker discovery and development.

Generation of Functioning Nephrons by Implanting Human Pluripotent Stem Cell-Derived Kidney Progenitors.

Human pluripotent stem cells (hPSCs) hold great promise for understanding kidney development and disease. We reproducibly differentiated three genetically distinct wild-type hPSC lines to kidney precursors that underwent rudimentary morphogenesis in vitro. They expressed nephron and collecting duct lineage marker genes, several of which are mutated in human kidney disease. Lentiviral-transduced hPSCs expressing reporter genes differentiated similarly to controls in vitro. Kidney progenitors were subcutaneously implanted into immunodeficient mice. By 12 weeks, they formed organ-like masses detectable by bioluminescence imaging. Implants included perfused glomeruli containing human capillaries, podocytes with regions of mature basement membrane, and mesangial cells. After intravenous injection of fluorescent low-molecular-weight dextran, signal was detected in tubules, demonstrating uptake from glomerular filtrate. Thus, we have developed methods to trace hPSC-derived kidney precursors that formed functioning nephrons in vivo. These advances beyond in vitro culture are critical steps toward using hPSCs to model and treat kidney diseases.

A Metabolic Study of Huntington's Disease.

BACKGROUND: Huntington's disease patients have a number of peripheral manifestations suggestive of metabolic and endocrine abnormalities. We, therefore, investigated a number of metabolic factors in a 24-hour study of Huntington's disease gene carriers (premanifest and moderate stage II/III) and controls. METHODS: Control (n = 15), premanifest (n = 14) and stage II/III (n = 13) participants were studied with blood sampling over a 24-hour period. A battery of clinical tests including neurological rating and function scales were performed. Visceral and subcutaneous adipose distribution was measured using magnetic resonance imaging. We quantified fasting baseline concentrations of glucose, insulin, cholesterol, triglycerides, lipoprotein (a), fatty acids, amino acids, lactate and osteokines. Leptin and ghrelin were quantified in fasting samples and after a standardised meal. We assessed glucose, insulin, growth hormone and cortisol concentrations during a prolonged oral glucose tolerance test. RESULTS: We found no highly significant differences in carbohydrate, protein or lipid metabolism markers between healthy controls, premanifest and stage II/III Huntington's disease subjects. For some markers (osteoprotegerin, tyrosine, lysine, phenylalanine and arginine) there is a suggestion (p values between 0.02 and 0.05) that levels are higher in patients with premanifest HD, but not moderate HD. However, given the large number of statistical tests performed interpretation of these findings must be cautious. CONCLUSIONS: Contrary to previous studies that showed altered levels of metabolic markers in patients with Huntington's disease, our study did not demonstrate convincing evidence of abnormalities in any of the markers examined. Our analyses were restricted to Huntington's disease patients not taking neuroleptics, anti-depressants or other medication affecting metabolic pathways. Even with the modest sample sizes studied, the lack of highly significant results, despite many being tested, suggests that the majority of these markers do not differ markedly by disease status.

Analysis of White Adipose Tissue Gene Expression Reveals CREB1 Pathway Altered in Huntington's Disease.

BACKGROUND: In addition to classical neurological symptoms, Huntington's disease (HD) is complicated by peripheral pathology and both the mutant gene and the protein are found in cells and tissues throughout the body. Despite the adipose tissue gene expression alterations described in HD mouse models, adipose tissue and its gene expression signature have not been previously explored in human HD. OBJECTIVE: We investigated gene expression signatures in subcutaneous adipose tissue obtained from control subjects, premanifest HD gene carriers and manifest HD subjects with the aim to identify gene expression changes and signalling pathway alterations in adipose tissue relevant to HD. METHODS: Gene expression was assessed using Affymetrix GeneChip® Human Gene 1.0 ST Array. Target genes were technically validated using real-time quantitative PCR and the expression signature was validated in an independent subject cohort. RESULTS: In subcutaneous adipose tissue, more than 500 genes were significantly different in premanifest HD subjects as compared to healthy controls. Pathway analysis suggests that the differentially expressed genes found here in HD adipose tissue are involved in fatty acid metabolism pathways, angiotensin signalling pathways and immune pathways. Transcription factor analysis highlights CREB1. Using RT-qPCR, we found that MAL2, AGTR2, COBL and the transcription factor CREB1 were significantly upregulated, with CREB1 and AGT also being significantly upregulated in a separate cohort. CONCLUSIONS: Distinct gene expression profiles can be seen in HD subcutaneous adipose tissue, with CREB1 highlighted as a key transcription factor.

A 24-Hour Study of the Hypothalamo-Pituitary Axes in Huntington's Disease.

BACKGROUND: Huntington's disease is an inherited neurodegenerative disorder characterised by motor, cognitive and psychiatric disturbances. Patients exhibit other symptoms including sleep and mood disturbances, muscle atrophy and weight loss which may be linked to hypothalamic pathology and dysfunction of hypothalamo-pituitary axes. METHODS: We studied neuroendocrine profiles of corticotropic, somatotropic and gonadotropic hypothalamo-pituitary axes hormones over a 24-hour period in controlled environment in 15 healthy controls, 14 premanifest and 13 stage II/III Huntington's disease subjects. We also quantified fasting levels of vasopressin, oestradiol, testosterone, dehydroepiandrosterone sulphate, thyroid stimulating hormone, free triiodothyronine, free total thyroxine, prolactin, adrenaline and noradrenaline. Somatotropic axis hormones, growth hormone releasing hormone, insulin-like growth factor-1 and insulin-like factor binding protein-3 were quantified at 06:00 (fasting), 15:00 and 23:00. A battery of clinical tests, including neurological rating and function scales were performed. RESULTS: 24-hour concentrations of adrenocorticotropic hormone, cortisol, luteinizing hormone and follicle-stimulating hormone did not differ significantly between the Huntington's disease group and controls. Daytime growth hormone secretion was similar in control and Huntington's disease subjects. Stage II/III Huntington's disease subjects had lower concentration of post-sleep growth hormone pulse and higher insulin-like growth factor-1:growth hormone ratio which did not reach significance. In Huntington's disease subjects, baseline levels of hypothalamo-pituitary axis hormones measured did not significantly differ from those of healthy controls. CONCLUSIONS: The relatively small subject group means that the study may not detect subtle perturbations in hormone concentrations. A targeted study of the somatotropic axis in larger cohorts may be warranted. However, the lack of significant results despite many variables being tested does imply that the majority of them do not differ substantially between HD and controls.

Skeletal muscle atrophy in R6/2 mice - altered circulating skeletal muscle markers and gene expression profile changes.

BACKGROUND: In addition to classical neurological symptoms, Huntington's disease (HD) is complicated by peripheral pathology, including progressive skeletal muscle wasting, and common skeletal muscle gene expression changes have been shown in HD mice and human HD. OBJECTIVE: To highlight possible mechanisms underlying muscle wasting in HD, we examined gene expression in pathways governing skeletal muscle contractility, skeletal myogenesis, skeletal muscle wasting, apoptosis and the NFκB signaling pathway in two HD mouse models (the transgenic R6/2 and full-length knock-in Q175). In addition, we assessed circulating markers that increase in response to skeletal muscle injury, skeletal Troponin I (sTnI), fatty acid binding protein 3 (FABP3), and Myosin light chain 3 (Myl3). METHODS: We measured gene expression in muscle tissue as well as in cultured primary myocytes using qPCR. Concentrations of cytokines and muscle proteins were obtained using multiplex ELISA. RESULTS: Circulating markers of muscle injury (sTnI, FABP3, and Myl3) were significantly increased in mouse serum. In skeletal muscle, we observed reduced gene expression of components involved in muscle contractility, with pronounced downregulation of Acta1, Myh2 and Tnni2, among others. Alongside, we found increased expression of caspases (3 and 8) and key elements of the NFκB signaling pathway, p65/RelA, Tradd, and TRAF5. We also found similar gene expression alterations in cultured primary myocytes from R6/2 mice stimulated with TNF-α. CONCLUSIONS: Our results indicate that activation of apoptotic and NFκB pathways occur alongside down-regulation of key compartments of the muscle contractility unit in skeletal muscle of HD mice, and muscle atrophy could possibly be a source of circulating disease progression markers.

A critical evaluation of inflammatory markers in Huntington's Disease plasma.

BACKGROUND: Huntington's Disease (HD) is a hereditary, progressive neurodegenerative disorder characterised by both neurological and systemic symptoms. In HD, immune changes can be observed before the onset of overt clinical features raising the possibility that inflammatory markers in plasma could be used to track disease progression. It has previously been demonstrated that a widespread, progressive innate immune response is detectable in plasma throughout the course of HD. OBJECTIVE: The aim of the present study was to investigate the potential of several components of inflammation and innate immunity as plasma biomarkers in HD. METHODS: We utilised antibody-based detection technologies as well as mass spectrometric quantification, multiple reaction monitoring (MRM-MS). RESULTS: Levels of several markers previously described as altered in HD, such as clusterin, complement component 4, complement component 9 and α-2 macroglobulin did not differ between healthy controls and HD subjects as measured by Luminex, ELISA or MRM-MS. C-reactive protein was decreased in early HD, while the other immune markers tested were unaltered. CONCLUSIONS: Although only C-reactive protein was found to be reduced in early HD, some of the inflammatory markers measured correlated with clinical measures.

No diagnostic value of plasma clusterin in Alzheimer's disease.

There is an urgent need for biomarkers to enable early diagnosis of Alzheimer's disease (AD). It has recently been shown that a variant within the clusterin gene is associated with increased risk of AD and plasma levels of clusterin have been found to be associated with the risk of AD. We, therefore, investigated the diagnostic value of clusterin by quantifying clusterin using an ELISA in plasma from 171 controls, 127 patients with AD, 82 patients with other dementias and 30 patients with depression. We observed similar plasma clusterin levels in controls, AD patients and patients with other dementias, suggesting that plasma clusterin levels have no diagnostic value for AD. There was a slight, but significant, increase in plasma clusterin in patients with depression compared to all other groups tested, which may warrant further investigation.