Yingleong Chan

Viral infections leading to inflammation have been implicated in several common diseases, such as Alzheimer’s disease (AD) and type 1 diabetes (T1D). Of note, herpes simplex virus 1 (HSV-1) has been reported to be associated with AD. We sought to identify the transcriptomic changes due to HSV-1 infection and anti-viral drug (acyclovir, ACV) treatment of HSV-1 infection in dissociated cells from human cerebral organoids (dcOrgs) versus stem cell-derived pancreatic islets (sc-islets) to gain potential biological insights into the relevance of HSV-1-induced inflammation in AD and T1D. We observed that differentially expressed genes (DEGs) in HSV-1-infected sc-islets were enriched for genes associated with several autoimmune diseases, most significantly, T1D, but also rheumatoid arthritis, psoriasis, Crohn’s disease, and multiple sclerosis, whereas DEGs in HSV-1-infected dcOrgs were exclusively enriched for genes associated with AD. The ACV treatment of sc-islets was not as effective in rescuing transcript perturbations of autoimmune disease-associated genes. Finally, we identified gene ontology categories that were enriched for DEGs that were in common across, or unique to, viral treatment of dcOrgs and sc-islets, such as categories involved in the transferase complex, mitochondrial, and autophagy function. In addition, we compared transcriptomic signatures from HSV-1-infected sc-islets with sc-islets that were infected with the coxsackie B virus (CVB) that had been associated with T1D pathogenesis. Collectively, this study provides tissue-specific insights into the molecular effects of inflammation in AD and T1D.

Viral infections leading to inflammation have been implicated in several common diseases such as Alzheimer’s disease (AD) and type 1 diabetes (T1D). Of note, herpes simplex virus 1 (HSV-1) has been reported to be associated with AD. We sought to identify the transcriptomic changes due to HSV-1 infection and anti-viral drug (acyclovir, ACV) treatment of HSV-1 infection in dissoci-ated cells from human cerebral organoids (dcOrgs) versus stem cell-derived pancreatic islets (sc-islets) to gain potential biological insights into the relevance of HSV-1-induced inflammation in AD and T1D. We observed that differentially expressed genes (DEGs) in HSV-1-infected sc-islets were enriched for genes associated with several autoimmune diseases, most significantly T1D but also rheumatoid arthritis, psoriasis, Crohn’s disease, and multiple sclerosis, whereas DEGs in HSV-1-infected dcOrgs were exclusively enriched for genes associated with AD. ACV treatment of sc-islets did not rescue transcript expression of autoimmune disease-associated genes. Finally, we identified gene ontology categories that were enriched for DEGs that were in common across, or unique to, viral treatment of dcOrgs and sc-islets, such as categories involved in the transferase complex, mitochondrial and autophagy function. Collectively, this studies pro-vide insight to the molecular effects of inflammation in AD and T1D.

To comprehensively investigate the intrathecal antibody profile of multiple sclerosis (MS), we examined the cerebrospinal fluid of 195 patients (92 MS and 103 non-MS) for antibodies using a multiplex unbiased bacteria peptide library. We first tested against Epstein-Barr nuclear antigen 1 (EBNA1) for epitope sites enriched in MS and found a significant enrichment at position 407-419. We then used the data to perform a high-throughput screen against a library of 129 viruses known to infect humans. We discovered several additional epitopes from viruses such as Hantaan virus, Human Herpesvirus 6A and Human respiratory syncytial virus B associated with MS. Besides viral epitopes, we also screened for potential autoantigens of the central nervous system (CNS). We discovered several autoantigenic epitopes in proteins such as ADRB3, HTR3A and MPO that were significantly enriched for MS. Because of previous associations of Toxoplasma gondii infection with MS, we also performed a Toxoplasma gondii specific analysis and discovered additional epitopes enriched for MS. We further assessed epitope-epitope correlations within the patient samples and found distinct patterns of association between these microbial and autoantigenic epitopes. Finally, we performed machine-learning to determine if these epitopes are predictive for MS and found that the model incorporating all the epitopes could most effectively discriminate between MS and non-MS (ROC-AUC score = 0.91). Our results demonstrate the effectiveness of multiplex unbiased screens to detect the identity of potentially cross-reactive antibodies targeting MS CNS epitopes and they can also be used as effective biomarkers for MS. One Sentence Summary We performed an unbiased multiplex bacteria peptide antibody library screen on cerebrospinal fluid samples of patients with multiple sclerosis (MS) as well as non-MS controls and detected multiple viral and autoantigenic epitopes that are significantly enriched in MS patient samples.

Neuroinflammation is a central process in the pathogenesis of several neurodegenerative diseases such as Alzheimer’s disease (AD), and there are active efforts to target pathways involved in neuroinflammation for molecular biomarker discovery and therapeutic development in neurodegenerative diseases. It was also proposed that there may be an infectious etiology in AD that is associated with viruses such as herpes simplex virus (HSV-1) and influenza A virus (IAV), leading to neuroinflammation-induced AD pathogenesis or disease progression. We sought to develop high-throughput, quantitative molecular biomarker assays using dissociated cells from human cerebral organoids (dcOrgs), that can used for screening compounds to reverse AD-associated neuroinflammation. We found that HSV-1 infection, but not IAV infection, in dcOrgs led to increased intracellular Aβ42 and phosphorylated Tau-Thr212 (pTau-212) expression, lower ratios of secreted Aβ42/40, as well as neuronal loss, and increased proportions of astrocytes and microglia, which are hallmarks of AD. Among the glia cell-type markers, Iba1 (microglia) and GFAP (astrocyte) expression were most strongly correlated with HSV-1 expression, which further supported that these biomarkers are perturbed by glia-mediated neuroinflammation. By performing large-scale RNA sequencing, we observed that differentially expressed transcripts in HSV-1 infected dcOrgs were specifically enriched for AD-associated GWAS genes, but not for genes associated with other common neurodegenerative, neuropsychiatric or autoimmune diseases. Immediate treatment of HSV-1 infected dcOrgs with anti-herpetic drug acyclovir (ACV) rescued most of the cellular and transcriptomic biomarkers in a dosage-dependent manner, indicating that it is possible to use our high-throughput platform to identify compounds or target genes that can reverse these neuroinflammation-induced biomarkers associated with AD.

Mitochondrial (MT) dysfunction has been associated with several neurodegenerative diseases including Alzheimer’s disease (AD). While MT-copy number differences have been implicated in AD, the effect of MT heteroplasmy on AD has not been well characterized. Here, we analyzed over 1800 whole genome sequencing data from four AD cohorts in seven different tissue types to determine the extent of MT heteroplasmy present. While MT heteroplasmy was present throughout the entire MT genome for blood samples, we detected MT heteroplasmy only within the MT control region for brain samples. We observed that an MT variant 10398A>G (rs2853826) was significantly associated with overall MT heteroplasmy in brain tissue while also being linked with the largest number of distinct disease phenotypes of all annotated MT variants in MitoMap. Using gene-expression data from our brain samples, our modeling discovered several gene networks involved in mitochondrial respiratory chain and Complex I function associated with 10398A>G. The variant was also found to be an expression quantitative trait loci (eQTL) for the gene MT-ND3. We further characterized the effect of 10398A>G by phenotyping a population of lymphoblastoid cell-lines (LCLs) with and without the variant allele. Examination of RNA sequence data from these LCLs reveal that 10398A>G was an eQTL for MT-ND4. We also observed in LCLs that 10398A>G was significantly associated with overall MT heteroplasmy within the MT control region, confirming the initial findings observed in post-mortem brain tissue. These results provide novel evidence linking MT SNPs with MT heteroplasmy and open novel avenues for the investigation of pathomechanisms that are driven by this pleiotropic disease associated loci.

Mitochondrial (MT) dysfunction has been associated with several neurodegenerative diseases including Alzheimer’s disease (AD). While MT-copy number differences have been implicated in AD, the effect of MT heteroplasmy on AD has not been well characterized. Here, we analyzed over 1,800 whole genome sequencing data from four AD cohorts in seven different tissue types to determine the extent of MT heteroplasmy present. While MT-heteroplasmy was present throughout the entire MT genome for blood samples, we detected MT-heteroplasmy only within the MT control region for brain samples. We observed that an MT variant 10398A>G (rs2853826) was significantly associated with overall MT-heteroplasmy in brain tissue while also being linked with the largest number of distinct disease phenotypes of all annotated MT variants in MitoMap . Using gene-expression data from our brain samples, our modeling discovered several gene networks involved in mitochondrial respiratory chain and Complex I function associated with 10398A>G. The variant was also found to be an expression quantitative trait loci (eQTL) for the gene MT-ND3. We further characterized the effect of 10398A>G by phenotyping a population of lymphoblastoid cell-lines (LCLs) with and without the variant allele. Examination of RNA sequence data from these LCLs reveal that 10398A>G was an eQTL for MT-ND4. We also observed in LCLs that 10398A>G was significantly associated with overall MT-heteroplasmy within the MT control region, confirming the initial findings observed in post-mortem brain tissue. These results provide novel evidence linking MT SNPs with MT heteroplasmy and open novel avenues for the investigation of pathomechanisms that are driven by this pleiotropic disease associated loci.

Inducing somatic cells into pluripotent stem cells (iPSCs) provides an excellent model for studying systems in-vitro . Understanding the impact of individual donor genetic backgrounds on reprogramming ability would allow researchers to harness these genetic differences and increase the efficiency of the reprogramming process. To better understand the genetic basis of reprogramming cells into iPSCs, we present Induction of Pluripotency from Pooled Cells (iPPC) - an efficient, scalable, and reliable reprogramming procedure. Using our deconvolution algorithm that employs low-coverage pooled sequencing and single nucleotide polymorphisms (SNPs), we estimate individual donor proportions of cell lines within large cohorts. With iPPC, we concurrently reprogrammed over one hundred donor LCLs into iPSCs and found strong correlations of individual donors’ reprogramming ability across multiple experiments. We note that individual donors’ reprogramming ability remains consistent across both same-day replicates and multiple experimental runs, and that the expression of certain immunoglobulin precursor genes (IGLV10-54, IGLV3-9, IGLV1-17, IGLV1-6, and IGLV3-1) may impact reprogramming ability. Our process enables a multiplex framework to study the reprogramming ability of different donor cells into iPSCs and also provides a reliable method along with a pooled library of donor iPSCs for downstream research and investigation of other in-vitro phenotypes.

Inducing somatic cells into pluripotent stem cells (iPSCs) provides an excellent model for studying systems in-vitro. Understanding the impact of individual donor genetic backgrounds on reprogramming ability would allow researchers to harness these genetic differences and increase the efficiency of the reprogramming process. To better understand the genetic basis of reprogramming cells into iPSCs, we present Induction of Pluripotency from Pooled Cells (iPPC) - an efficient, scalable, and reliable reprogramming procedure. Using our deconvolution algorithm that employs low-coverage pooled sequencing and single nucleotide polymorphisms (SNPs), we estimate individual donor proportions of cell lines within large cohorts. With iPPC, we concurrently reprogrammed over one hundred donor LCLs into iPSCs and found strong correlations of individual donors’ reprogramming ability across multiple experiments. We note that individual donors’ reprogramming ability remains consistent across both same-day replicates and multiple experimental runs, and that the expression of certain immunoglobulin precursor genes (IGLV10-54, IGLV3-9, IGLV1-17, IGLV1-6, and IGLV3-1) may impact reprogramming ability. Our process enables a multiplex framework to study the reprogramming ability of different donor cells into iPSCs and also provides a reliable method along with a pooled library of donor iPSCs for downstream research and investigation of other in-vitro phenotypes. Funding Information: This work was funded by grant NIH/NIDCD R21DC018092 from the National Institute on Deafness and Other Communication and grant NIH/NIA U01AG061835 from the National Institute on Aging. Conflict of Interests: GMC holds leadership positions in many companies related to DNA sequencing technologies. A full list of these companies is available at http://arep.med.harvard.edu/gmc/tech.html. The remaining authors declare that they have no competing interests.