Timothy Wu

All Publications

• Longitudinal multi-omics in alpha-synuclein Drosophila model discriminates disease- from age-associated pathologies in Parkinson's disease. NPJ Parkinson's disease Moore, J., Wu, T., Dhindsa, J., El Fadel, O., Le, A., Perez, A., Amoh, B., Tarkunde, A., Zhu, K. F., Avalos, M., Dammer, E. B., Duong, D. M., Seyfried, N. T., Shulman, J. M., Al-Ramahi, I., Botas, J. 2025; 11 (1): 46

  Abstract

  Parkinson's disease (PD) starts decades before symptoms appear, usually in the later decades of life, when age-related changes are occurring. To identify molecular changes early in the disease course and distinguish PD pathologies from aging, we generated Drosophila expressing alpha-synuclein (αSyn) in neurons and performed longitudinal bulk transcriptomics and proteomics on brains at six time points across the lifespan and compared the data to healthy control flies as well as human post-mortem brain datasets. We found that translational and energy metabolism pathways were downregulated in αSyn flies at the earliest timepoints; comparison with the aged control flies suggests that elevated αSyn accelerates changes associated with normal aging. Unexpectedly, single-cell analysis at a mid-disease stage revealed that neurons upregulate protein synthesis and nonsense-mediated decay, while glia drive their overall downregulation. Longitudinal multi-omics approaches in animal models can thus help elucidate the molecular cascades underlying neurodegeneration vs. aging and co-pathologies.

  View details for DOI 10.1038/s41531-025-00899-z

  View details for PubMedID 40069190

  View details for PubMedCentralID PMC11897226

• Mechanisms driving fasting-induced protection from genotoxic injury in the small intestine. American journal of physiology. Gastrointestinal and liver physiology Deans-Fielder, K., Wu, T., Nguyen, T., To, S., Huang, Y. Z., Bark, S. J., Mills, J. C., Shroyer, N. F. 2024; 326 (5): G504-G524

  Abstract

  Genotoxic agents such as doxorubicin (DXR) can cause damage to the intestines that can be ameliorated by fasting. How fasting is protective and the optimal timing of fasting and refeeding remain unclear. Here, our analysis of fasting/refeeding-induced global intestinal transcriptional changes revealed metabolic shifts and implicated the cellular energetic hub mechanistic target of rapamycin complex 1 (mTORC1) in protecting from DXR-induced DNA damage. Our analysis of specific transcripts and proteins in intestinal tissue and tissue extracts showed that fasting followed by refeeding at the time of DXR administration reduced damage and caused a spike in mTORC1 activity. However, continued fasting after DXR prevented the mTORC1 spike and damage reduction. Surprisingly, the mTORC1 inhibitor, rapamycin, did not block fasting/refeeding-induced reduction in DNA damage, suggesting that increased mTORC1 is dispensable for protection against the initial DNA damage response. In Ddit4-/- mice [DDIT4 (DNA-damage-inducible transcript 4) functions to regulate mTORC1 activity], fasting reduced DNA damage and increased intestinal crypt viability vs. ad libitum-fed Ddit4-/- mice. Fasted/refed Ddit4-/- mice maintained body weight, with increased crypt proliferation by 5 days post-DXR, whereas ad libitum-fed Ddit4-/- mice continued to lose weight and displayed limited crypt proliferation. Genes encoding epithelial stem cell and DNA repair proteins were elevated in DXR-injured, fasted vs. ad libitum Ddit4-/- intestines. Thus, fasting strongly reduced intestinal damage when normal dynamic regulation of mTORC1 was lost. Overall, the results confirm that fasting protects the intestines against DXR and suggests that fasting works by pleiotropic - including both mTORC1-dependent and independent - mechanisms across the temporally dynamic injury response.NEW & NOTEWORTHY New findings are 1) DNA damage reduction following a 24-h fast depends on the timing of postfast refeeding in relation to chemotherapy initiation; 2) fasting/refeeding-induced upregulation of mTORC1 activity is not required for early (6 h) protection against DXR-induced DNA damage; and 3) fasting increases expression of intestinal stem cell and DNA damage repair genes, even when mTORC1 is dysregulated, highlighting fasting's crucial role in regulating mTORC1-dependent and independent mechanisms in the dynamic recovery process.

  View details for DOI 10.1152/ajpgi.00126.2023

  View details for PubMedID 38349111

  View details for PubMedCentralID PMC11376978

• Tau polarizes an aging transcriptional signature to excitatory neurons and glia ELIFE Wu, T., Deger, J. M., Ye, H., Guo, C., Dhindsa, J., Pekarek, B. T., Al-Ouran, R., Liu, Z., Al-Ramahi, I., Botas, J., Shulman, J. M. 2023; 12

  Abstract

  Aging is a major risk factor for Alzheimer's disease (AD), and cell-type vulnerability underlies its characteristic clinical manifestations. We have performed longitudinal, single-cell RNA-sequencing in Drosophila with pan-neuronal expression of human tau, which forms AD neurofibrillary tangle pathology. Whereas tau- and aging-induced gene expression strongly overlap (93%), they differ in the affected cell types. In contrast to the broad impact of aging, tau-triggered changes are strongly polarized to excitatory neurons and glia. Further, tau can either activate or suppress innate immune gene expression signatures in a cell-type-specific manner. Integration of cellular abundance and gene expression pinpoints nuclear factor kappa B signaling in neurons as a marker for cellular vulnerability. We also highlight the conservation of cell-type-specific transcriptional patterns between Drosophila and human postmortem brain tissue. Overall, our results create a resource for dissection of dynamic, age-dependent gene expression changes at cellular resolution in a genetically tractable model of tauopathy.

  View details for DOI 10.7554/eLife.85251

  View details for Web of Science ID 001071628100001

  View details for PubMedID 37219079

  View details for PubMedCentralID PMC10259480

• Functional screening of lysosomal storage disorder genes identifies modifiers of alpha-synuclein neurotoxicity. PLoS genetics Yu, M., Ye, H., De-Paula, R. B., Mangleburg, C. G., Wu, T., Lee, T. V., Li, Y., Duong, D., Phillips, B., Cruchaga, C., Allen, G. I., Seyfried, N. T., Al-Ramahi, I., Botas, J., Shulman, J. M. 2023; 19 (5): e1010760

  Abstract

  Heterozygous variants in the glucocerebrosidase (GBA) gene are common and potent risk factors for Parkinson's disease (PD). GBA also causes the autosomal recessive lysosomal storage disorder (LSD), Gaucher disease, and emerging evidence from human genetics implicates many other LSD genes in PD susceptibility. We have systemically tested 86 conserved fly homologs of 37 human LSD genes for requirements in the aging adult Drosophila brain and for potential genetic interactions with neurodegeneration caused by α-synuclein (αSyn), which forms Lewy body pathology in PD. Our screen identifies 15 genetic enhancers of αSyn-induced progressive locomotor dysfunction, including knockdown of fly homologs of GBA and other LSD genes with independent support as PD susceptibility factors from human genetics (SCARB2, SMPD1, CTSD, GNPTAB, SLC17A5). For several genes, results from multiple alleles suggest dose-sensitivity and context-dependent pleiotropy in the presence or absence of αSyn. Homologs of two genes causing cholesterol storage disorders, Npc1a / NPC1 and Lip4 / LIPA, were independently confirmed as loss-of-function enhancers of αSyn-induced retinal degeneration. The enzymes encoded by several modifier genes are upregulated in αSyn transgenic flies, based on unbiased proteomics, revealing a possible, albeit ineffective, compensatory response. Overall, our results reinforce the important role of lysosomal genes in brain health and PD pathogenesis, and implicate several metabolic pathways, including cholesterol homeostasis, in αSyn-mediated neurotoxicity.

  View details for DOI 10.1371/journal.pgen.1010760

  View details for PubMedID 37200393

  View details for PubMedCentralID PMC10231792

• Oxytocin signaling is necessary for synaptic maturation of adult-born neurons. Genes & development Pekarek, B. T., Kochukov, M., Lozzi, B., Wu, T., Hunt, P. J., Tepe, B., Hanson Moss, E., Tantry, E. K., Swanson, J. L., Dooling, S. W., Patel, M., Belfort, B. D., Romero, J. M., Bao, S., Hill, M. C., Arenkiel, B. R. 2022; 36 (21-24): 1100-1118

  Abstract

  Neural circuit plasticity and sensory response dynamics depend on forming new synaptic connections. Despite recent advances toward understanding the consequences of circuit plasticity, the mechanisms driving circuit plasticity are unknown. Adult-born neurons within the olfactory bulb have proven to be a powerful model for studying circuit plasticity, providing a broad and accessible avenue into neuron development, migration, and circuit integration. We and others have shown that efficient adult-born neuron circuit integration hinges on presynaptic activity in the form of diverse signaling peptides. Here, we demonstrate a novel oxytocin-dependent mechanism of adult-born neuron synaptic maturation and circuit integration. We reveal spatial and temporal enrichment of oxytocin receptor expression within adult-born neurons in the murine olfactory bulb, with oxytocin receptor expression peaking during activity-dependent integration. Using viral labeling, confocal microscopy, and cell type-specific RNA-seq, we demonstrate that oxytocin receptor signaling promotes synaptic maturation of newly integrating adult-born neurons by regulating their morphological development and expression of mature synaptic AMPARs and other structural proteins.

  View details for DOI 10.1101/gad.349930.122

  View details for PubMedID 36617877

  View details for PubMedCentralID PMC9851403

• Whole blood transcriptome analysis in bipolar disorder reveals strong lithium effect. Psychological medicine Krebs, C. E., Ori, A. P., Vreeker, A., Wu, T., Cantor, R. M., Boks, M. P., Kahn, R. S., Olde Loohuis, L. M., Ophoff, R. A. 2020; 50 (15): 2575-2586

  Abstract

  Bipolar disorder (BD) is a highly heritable mood disorder with complex genetic architecture and poorly understood etiology. Previous transcriptomic BD studies have had inconsistent findings due to issues such as small sample sizes and difficulty in adequately accounting for confounders like medication use.We performed a differential expression analysis in a well-characterized BD case-control sample (Nsubjects = 480) by RNA sequencing of whole blood. We further performed co-expression network analysis, functional enrichment, and cell type decomposition, and integrated differentially expressed genes with genetic risk.While we observed widespread differential gene expression patterns between affected and unaffected individuals, these effects were largely linked to lithium treatment at the time of blood draw (FDR < 0.05, Ngenes = 976) rather than BD diagnosis itself (FDR < 0.05, Ngenes = 6). These lithium-associated genes were enriched for cell signaling and immune response functional annotations, among others, and were associated with neutrophil cell-type proportions, which were elevated in lithium users. Neither genes with altered expression in cases nor in lithium users were enriched for BD, schizophrenia, and depression genetic risk based on information from genome-wide association studies, nor was gene expression associated with polygenic risk scores for BD.These findings suggest that BD is associated with minimal changes in whole blood gene expression independent of medication use but emphasize the importance of accounting for medication use and cell type heterogeneity in psychiatric transcriptomic studies. The results of this study add to mounting evidence of lithium's cell signaling and immune-related mechanisms.

  View details for DOI 10.1017/S0033291719002745

  View details for PubMedID 31589133

• Integrated analysis of the aging brain transcriptome and proteome in tauopathy MOLECULAR NEURODEGENERATION Mangleburg, C., Wu, T., Yalamanchili, H. K., Guo, C., Hsieh, Y., Duong, D. M., Dammer, E. B., De Jager, P. L., Seyfried, N. T., Liu, Z., Shulman, J. M. 2020; 15 (1): 56

  Abstract

  Tau neurofibrillary tangle pathology characterizes Alzheimer's disease and other neurodegenerative tauopathies. Brain gene expression profiles can reveal mechanisms; however, few studies have systematically examined both the transcriptome and proteome or differentiated Tau- versus age-dependent changes.Paired, longitudinal RNA-sequencing and mass-spectrometry were performed in a Drosophila model of tauopathy, based on pan-neuronal expression of human wildtype Tau (TauWT) or a mutant form causing frontotemporal dementia (TauR406W). Tau-induced, differentially expressed transcripts and proteins were examined cross-sectionally or using linear regression and adjusting for age. Hierarchical clustering was performed to highlight network perturbations, and we examined overlaps with human brain gene expression profiles in tauopathy.TauWT induced 1514 and 213 differentially expressed transcripts and proteins, respectively. TauR406W had a substantially greater impact, causing changes in 5494 transcripts and 697 proteins. There was a ~ 70% overlap between age- and Tau-induced changes and our analyses reveal pervasive bi-directional interactions. Strikingly, 42% of Tau-induced transcripts were discordant in the proteome, showing opposite direction of change. Tau-responsive gene expression networks strongly implicate innate immune activation. Cross-species analyses pinpoint human brain gene perturbations specifically triggered by Tau pathology and/or aging, and further differentiate between disease amplifying and protective changes.Our results comprise a powerful, cross-species functional genomics resource for tauopathy, revealing Tau-mediated disruption of gene expression, including dynamic, age-dependent interactions between the brain transcriptome and proteome.

  View details for DOI 10.1186/s13024-020-00405-4

  View details for Web of Science ID 000576854700001

  View details for PubMedID 32993812

  View details for PubMedCentralID PMC7526226

• Candidate <i>CSPG4</i> mutations and induced pluripotent stem cell modeling implicate oligodendrocyte progenitor cell dysfunction in familial schizophrenia MOLECULAR PSYCHIATRY de Vrij, F. M., Bouwkamp, C. G., Gunhanlar, N., Shpak, G., Lendemeijer, B., Baghdadi, M., Gopalakrishna, S., Ghazvini, M., Li, T. M., Quadri, M., Olgiati, S., Breedveld, G. J., Coesmans, M., Mientjes, E., de Wit, T., Verheijen, F. W., Beverloo, H., Cohen, D., Kok, R. M., Bakker, P., Nijbur, A., Spijker, A. T., Haffmans, P., Hoencamp, E., Bergink, V., Vorstman, J. A., Wu, T., Loohuis, L., Amin, N., Langen, C. D., Hofman, A., Hoogendijk, W. J., van Duijimn, C. M., Ikram, M., Vernooij, M. W., Tiemeier, H., Uitterlinden, A. G., Elgersma, Y., Distel, B., Gribnau, J., White, T., Bonifati, V., Kushner, S. A., GRP Study Consortium 2019; 24 (5): 757-771

  Abstract

  Schizophrenia is highly heritable, yet its underlying pathophysiology remains largely unknown. Among the most well-replicated findings in neurobiological studies of schizophrenia are deficits in myelination and white matter integrity; however, direct etiological genetic and cellular evidence has thus far been lacking. Here, we implement a family-based approach for genetic discovery in schizophrenia combined with functional analysis using induced pluripotent stem cells (iPSCs). We observed familial segregation of two rare missense mutations in Chondroitin Sulfate Proteoglycan 4 (CSPG4) (c.391G > A [p.A131T], MAF 7.79 × 10-5 and c.2702T > G [p.V901G], MAF 2.51 × 10-3). The CSPG4A131T mutation was absent from the Swedish Schizophrenia Exome Sequencing Study (2536 cases, 2543 controls), while the CSPG4V901G mutation was nominally enriched in cases (11 cases vs. 3 controls, P = 0.026, OR 3.77, 95% CI 1.05-13.52). CSPG4/NG2 is a hallmark protein of oligodendrocyte progenitor cells (OPCs). iPSC-derived OPCs from CSPG4A131T mutation carriers exhibited abnormal post-translational processing (P = 0.029), subcellular localization of mutant NG2 (P = 0.007), as well as aberrant cellular morphology (P = 3.0 × 10-8), viability (P = 8.9 × 10-7), and myelination potential (P = 0.038). Moreover, transfection of healthy non-carrier sibling OPCs confirmed a pathogenic effect on cell survival of both the CSPG4A131T (P = 0.006) and CSPG4V901G (P = 3.4 × 10-4) mutations. Finally, in vivo diffusion tensor imaging of CSPG4A131T mutation carriers demonstrated a reduction of brain white matter integrity compared to unaffected sibling and matched general population controls (P = 2.2 × 10-5). Together, our findings provide a convergence of genetic and functional evidence to implicate OPC dysfunction as a candidate pathophysiological mechanism of familial schizophrenia.

  View details for DOI 10.1038/s41380-017-0004-2

  View details for Web of Science ID 000465499600010

  View details for PubMedID 29302076

  View details for PubMedCentralID PMC6755981

• The Alkaline Phosphatase (ALPL) Locus Is Associated with B6 Vitamer Levels in CSF and Plasma. Genes Loohuis, L. M., Albersen, M., de Jong, S., Wu, T., Luykx, J. J., Jans, J. J., Verhoeven-Duif, N. M., Ophoff, R. A. 2018; 10 (1)

  Abstract

  The active form of vitamin B6, pyridoxal phosphate (PLP), is essential for human metabolism. The brain is dependent on vitamin B6 for its neurotransmitter balance. To obtain insight into the genetic determinants of vitamin B6 homeostasis, we conducted a genome-wide association study (GWAS) of the B6 vitamers pyridoxal (PL), PLP and the degradation product of vitamin B6, pyridoxic acid (PA). We collected a unique sample set of cerebrospinal fluid (CSF) and plasma from the same healthy human subjects of Dutch ancestry (n = 493) and included concentrations and ratios in and between these body fluids in our analysis. Based on a multivariate joint analysis of all B6 vitamers and their ratios, we identified a genome-wide significant association at a locus on chromosome 1 containing the ALPL (alkaline phosphatase) gene (minimal p = 7.89 × 10-10, rs1106357, minor allele frequency (MAF) = 0.46), previously associated with vitamin B6 levels in blood. Subjects homozygous for the minor allele showed a 1.4-times-higher ratio between PLP and PL in plasma, and even a 1.6-times-higher ratio between PLP and PL in CSF than subjects homozygous for the major allele. In addition, we observed a suggestive association with the CSF:plasma ratio of PLP on chromosome 15 (minimal p = 7.93 × 10-7, and MAF = 0.06 for rs28789220). Even though this finding is not reaching genome-wide significance, it highlights the potential of our experimental setup for studying transport and metabolism across the blood⁻CSF barrier. This GWAS of B6 vitamers identifies alkaline phosphatase as a key regulator in human vitamin B6 metabolism in CSF as well as plasma. Furthermore, our results demonstrate the potential of genetic studies of metabolites in plasma and CSF to elucidate biological aspects underlying metabolite generation, transport and degradation.

  View details for DOI 10.3390/genes10010008

  View details for PubMedID 30583557

  View details for PubMedCentralID PMC6357176

• Transcriptome analysis in whole blood reveals increased microbial diversity in schizophrenia TRANSLATIONAL PSYCHIATRY Loohuis, L., Mangul, S., Ori, A. P. S., Jospin, G., Koslicki, D., Yang, H., Wu, T., Boks, M. P., Lomen-Hoerth, C., Wiedau-Pazos, M., Cantor, R. M., de Vos, W. M., Kahn, R. S., Eskin, E., Ophoff, R. A. 2018; 8: 96

  Abstract

  The role of the human microbiome in health and disease is increasingly appreciated. We studied the composition of microbial communities present in blood across 192 individuals, including healthy controls and patients with three disorders affecting the brain: schizophrenia, amyotrophic lateral sclerosis, and bipolar disorder. By using high-quality unmapped RNA sequencing reads as candidate microbial reads, we performed profiling of microbial transcripts detected in whole blood. We were able to detect a wide range of bacterial and archaeal phyla in blood. Interestingly, we observed an increased microbial diversity in schizophrenia patients compared to the three other groups. We replicated this finding in an independent schizophrenia case-control cohort. This increased diversity is inversely correlated with estimated cell abundance of a subpopulation of CD8+ memory T cells in healthy controls, supporting a link between microbial products found in blood, immunity and schizophrenia.

  View details for DOI 10.1038/s41398-018-0107-9

  View details for Web of Science ID 000432072000002

  View details for PubMedID 29743478

  View details for PubMedCentralID PMC5943399

• Dupuytren's disease susceptibility gene, <i>EPDR1</i>, is involved in myofibroblast contractility JOURNAL OF DERMATOLOGICAL SCIENCE Staats, K. A., Wu, T., Gan, B. S., O'Gorman, D. B., Ophoff, R. A. 2016; 83 (2): 131-137

  Abstract

  Dupuytren's Disease is a common disorder of the connective tissue characterized by progressive and irreversible fibroblastic proliferation affecting the palmar fascia. Progressive flexion deformity appears over several months or years and although usually painless, it can result in a serious handicap causing loss of manual dexterity. There is no cure for the disease and the etiology is largely unknown. A genome-wide association study of Dupuytren's Disease identified nine susceptibility loci with the strongest genetic signal located in an intron of EPDR1, the gene encoding the Ependymin Related 1 protein.Here, we investigate the role of EPDR1 in Dupuytren's Disease.We research the role of EPDR1 by assessing gene expression in patient tissue and by gene silencing in fibroblast-populated collagen lattice (FPCL) assay, which is used as an in vitro model of Dupuytren's contractures.The three alternative transcripts produced by the EPDR1 gene are all detected in affected Dupuytren's tissue and control unaffected palmar fascia tissue. Dupuytren's tissue also contracts more in the FPCL paradigm. Dicer-substrate RNA-mediated knockdown of EPDR1 results in moderate late stage attenuation of contraction rate in FPCL, implying a role in matrix contraction.Our results suggest functional involvement of EPDR1 in the etiology of Dupuytren's Disease.

  View details for DOI 10.1016/j.jdermsci.2016.04.015

  View details for Web of Science ID 000380735000006

  View details for PubMedID 27245865

• Epigenetic age analysis of children who seem to evade aging AGING-US Walker, R. F., Liu, J., Peters, B. A., Ritz, B. R., Wu, T., Ophoff, R. A., Horvath, S. 2015; 7 (5): 334-339

  Abstract

  We previously reported the unusual case of a teenage girl stricken with multifocal developmental dysfunctions whose physical development was dramatically delayed resulting in her appearing to be a toddler or at best a preschooler, even unto the occasion of her death at the age of 20 years. Her life-long physician felt that the disorder was unique in the world and that future treatments for age-related diseases might emerge from its study. The objectives of our research were to determine if other such cases exist, and if so, whether aging is actually slowed. Of seven children characterized by dramatically slow developmental rates, five also had associated disorders displayed by the first case. All of the identified subjects were female. To objectively measure the age of blood tissue from these subjects, we used a highly accurate biomarker of aging known as "epigenetic clock" based on DNA methylation levels. No statistically significant differences in chronological and epigenetic ages were detected in any of the newly discovered cases.

  View details for DOI 10.18632/aging.100744

  View details for Web of Science ID 000357373200011

  View details for PubMedID 25991677

  View details for PubMedCentralID PMC4468314