Generation of two iPSC lines (UMi038-A & UMi039-A) from siblings bearing an Alzheimer’s disease-associated variant in SORL1

Alzheimer’s disease (AD) is the leading cause of dementia among older adults. SORL1, a top AD risk gene, encodes an endocytic receptor involved amyloid precursor protein (APP) trafficking and processing. Rare loss-of-function SORL1 variants are a strong genetic determinant of AD, and protein-truncating mutations have been found to be causal. We derived iPSCs from two siblings affected with early-onset AD who carry a rare protein-truncating deletion in SORL1 (c.4293delC) (Kunkle et al., 2017). The iPSC lines were characterized for pluripotency, differentiation potential, and genomic stability. These lines are a valuable resource for studying pathogenic mechanisms underlying AD

Examining the impact of a rare protein‐truncating SORL1 variant on AD pathology

Background Recent analyses of rare variants using whole exome sequencing have found an enrichment of SORL1 loss‐of‐function (LoF) variants in early onset Alzheimer’s disease (EOAD). This makes SORL1 one of the highest risk factors for AD, along with other candidate EOAD genes such as APP, PS1, and PS2. SORL1 encodes an endocytic receptor involved in the trafficking of amyloid‐β precursor protein (APP) and the secretion of amyloid‐β. We identified a family with multiple individuals affected with EOAD carrying a single base pair deletion (c.4293del) in SORL1, resulting in a frameshift and premature termination of the protein (p.Cys1431fs; Kunkle et al. JAMA Neurol. 2017). Several recent studies have demonstrated that haploinsufficiency of SORL1 can induce AD‐related phenotypes in cultured neurons. However, the functional consequences of specific variants remain largely undefined. In this study, we used patient‐specific induced pluripotent stem cell (iPSC)‐derived neurons to evaluate the effect of a rare LoF SORL1 variant on AD‐related pathology. Method Patient‐specific iPSC lines were derived from two related heterozygous p.Cys1431fs carriers with EOAD. Each line was validated for pluripotency through immunocytochemical staining and RT‐PCR. Karyotypic stability was assessed by G‐banding. To evaluate the functional consequences of the SORL1 deletion, patient and control iPSC lines were differentiated into cortical neurons and assessed for AD‐related phenotypes. Result The iPSC lines were successfully differentiated into cortical neurons, characterized by immunostaining for various neuronal markers. Our data show that neurons bearing the p.Cys1431fs SORL1 mutation have increased levels of APP accumulated in EEA1+ endosomes compared to neurons from cognitively intact individuals. Additionally, analysis of synaptic density revealed that neurons carrying the p.Cys1431fs variant show a significant reduction of SYN1+ puncta compared to cognitively intact individuals. Conclusion Our results indicate that patient‐derived neurons carrying the p.Cys1431fs variant have cellular defects associated with AD pathology while replicating, at least in part, previous in vitro findings on SORL1 haploinsufficiency

Derivation of autism spectrum disorder-specific induced pluripotent stem cells from peripheral blood mononuclear cells

► Induced pluripotent stem cells (iPSCs) were generated from autistic patients. ► The iPSCs lines had traits similar to naturally occurring stem cells populations. ► Embryoid bodies with germ layer characteristics were derived from ASD-specific iPSC. ► Mature GABAergic neurons could be differentiated from the ASD-specific iPSC. ► iPSC provide valuable reagents for the characterization of the pathology of autism. Induced pluripotent stem cells (iPSCs) hold tremendous potential both as a biological tool to uncover the pathophysiology of disease by creating relevant cell models and as a source of stem cells for cell-based therapeutic applications. Typically, iPSCs have been derived by the transgenic overexpression of transcription factors associated with progenitor cell or stem cell function in fibroblasts derived from skin biopsies. However, the need for skin punch biopsies to derive fibroblasts for reprogramming can present a barrier to study participation among certain populations of individuals, including children with autism spectrum disorders (ASDs). In addition, the acquisition of skin punch biopsies in non-clinic settings presents a challenge. One potential mechanism to avoid these limitations would be the use of peripheral blood mononuclear cells (PBMCs) as the source of the cells for reprogramming. In this article we describe, for the first time, the derivation of iPSC lines from PBMCs isolated from the whole blood of autistic children, and their subsequent differentiation in GABAergic neurons

Human Organoids for Rapid Validation of Gene Variants Linked to Cochlear Malformations

Developmental anomalies of the hearing organ, the cochlea, are diagnosed in approximately one-fourth of individuals with congenital deafness. Most patients with cochlear malformations remain etiologically undiagnosed due to insufficient knowledge about underlying genes or the inability to make conclusive interpretations of identified genetic variants. We used exome sequencing for genetic evaluation of hearing loss associated with cochlear malformations in three probands from unrelated families. We subsequently generated monoclonal induced pluripotent stem cell (iPSC) lines, bearing patient-specific knockins and knockouts using CRISPR/Cas9 to assess pathogenicity of candidate variants. We detected FGF3 (p.Arg165Gly) and GREB1L (p.Cys186Arg), variants of uncertain significance in two recognized genes for deafness, and PBXIP1 (p.Trp574*) in a candidate gene. Upon differentiation of iPSCs towards inner ear organoids, we observed significant developmental aberrations in knockout lines compared to their isogenic controls. Patient-specific single nucleotide variants (SNVs) showed similar abnormalities as the knockout lines, functionally supporting their causality in the observed phenotype. Therefore, we present human inner ear organoids as a tool to rapidly validate the pathogenicity of DNA variants associated with cochlear malformations.Developmental anomalies of the hearing organ, the cochlea, are diagnosed in approximately one-fourth of individuals with congenital deafness. Most patients with cochlear malformations remain etiologically undiagnosed due to insufficient knowledge about underlying genes or the inability to make conclusive interpretations of identified genetic variants. We used exome sequencing for genetic evaluation of hearing loss associated with cochlear malformations in three probands from unrelated families. We subsequently generated monoclonal induced pluripotent stem cell (iPSC) lines, bearing patient-specific knockins and knockouts using CRISPR/Cas9 to assess pathogenicity of candidate variants. We detected FGF3 (p.Arg165Gly) and GREB1L (p.Cys186Arg), variants of uncertain significance in two recognized genes for deafness, and PBXIP1 (p.Trp574*) in a candidate gene. Upon differentiation of iPSCs towards inner ear organoids, we observed significant developmental aberrations in knockout lines compared to their isogenic controls. Patient-specific single nucleotide variants (SNVs) showed similar abnormalities as the knockout lines, functionally supporting their causality in the observed phenotype. Therefore, we present human inner ear organoids as a tool to rapidly validate the pathogenicity of DNA variants associated with cochlear malformations

Intragenic loci within TOMM40 enhances APOE expression in human microglia

Background Previously, we demonstrated that the ancestry‐related risk for Late Onset Alzheimer’s Disease (LOAD) is driven by a local genomic region (termed Local Ancestry; LA) around APOEε4. Furthermore, we showed that in the brain of individuals bearing European LA there is higher expression of APOEε4 compared to those with African LA. In a follow‐up study, utilizing reporter assays and Capture‐C data we located two intronic regions within the European LA, both in the TOMM40 gene (named B10 and B13), that increased APOE expression in microglia and astrocytes. In this study, we sought to validate their regulatory role in APOE expression using CRISPR interference/activation (CRISPRi/a). Method Human Microglial Clone 3 (HMC3) CRISPRi/a lines were produced by transducing inducible dCas9‐VP64 (Activation), dCas9‐KRAB (Interference) or dCas9 (control) using lentiviral vectors. To direct the dCas9 constructs to our regions of interest, we generated multiplex vectors that encode 4 short‐guide RNAs (sgRNAs) targeting either B10 or B13. We used 4 different sgRNAs in each case to ensure full‐length coverage of the tested regions (∼850bp size). An empty multiplex vector was used as a control. We then transduced either of the multiplex vectors into the HMC3 CRISPRi/a lines. We induced expression of the dCas9 constructs for 2 or 6 days with Doxycycline (2ug/ml). RNA was extracted and the expression of APOE and TOMM40 was measured by qRT‐PCR. Result APOE expression significantly increased when targeting B10 or B13 (p=0.001; p=0.003 respectively) with dCas9‐VP64 after 2 days of Doxycycline treatment. Six days after treatment the significance persisted only when targeting B10 (p=0.01). No significant changes in APOE expression were observed in the cells bearing the dCas9‐KRAB presumably due to low endogenous APOE levels. Expression of TOMM40 did not vary under any treatment. Conclusion These preliminary results support our previous findings that regions B10 and B13 may act as regulators for APOE expression as demonstrated by the elevation of ApoE expression when targeting an activator to these regions. The expression of TOMM40 did not vary across cell lines in the evaluated time points supporting that the effect observed is specific for APOE

Convergent Pathways in Idiopathic Autism Revealed by Time Course Transcriptomic Analysis of Patient-Derived Neurons

Potentially pathogenic alterations have been identified in individuals with autism spectrum disorders (ASDs) within a variety of key neurodevelopment genes. While this hints at a common ASD molecular etiology, gaps persist in our understanding of the neurodevelopmental mechanisms impacted by genetic variants enriched in ASD patients. Induced pluripotent stem cells (iPSCs) can model neurodevelopment in vitro, permitting the characterization of pathogenic mechanisms that manifest during corticogenesis. Taking this approach, we examined the transcriptional differences between iPSC-derived cortical neurons from patients with idiopathic ASD and unaffected controls over a 135-day course of neuronal differentiation. Our data show ASD-specific misregulation of genes involved in neuronal differentiation, axon guidance, cell migration, DNA and RNA metabolism, and neural region patterning. Furthermore, functional analysis revealed defects in neuronal migration and electrophysiological activity, providing compelling support for the transcriptome analysis data. This study reveals important and functionally validated insights into common processes altered in early neuronal development and corticogenesis and may contribute to ASD pathogenesis

Uncovering the Role of an ABCA7 Frameshift Deletion in African American Alzheimer’s Disease Cellular Pathology

Abstract Background The ATP Binding Cassette Subfamily A Member 7 (ABCA7) gene is a risk factor for Alzheimer’s disease (AD). While ABCA7 has been implicated as a genetic determinant of AD across populations, the risk effect is the strongest in African Americans (AAs). We previously identified a common 44 base pair deletion in ABCA7 (p.Arg578Alafs) that is predicted to truncate the protein and is significantly associated with AD in AAs (frequency in cases = 15.2%, cognitively unimpaired (CU) = 9.74%, p = 1.41×10 −5 ). Clinically, deletion homozygotes are similar to deletion heterozygotes, supporting dominant gain‐of‐function as a putative disease‐causing mechanism. We sought to determine if the deletion transcript is stable and if the truncated protein is expressed as steps to understanding the mechanism leading to AD risk. Method RT‐PCR analysis of heterozygotes was conducted to assess the stability of the deletion transcript. FLAG‐tagged Arg578Alafs and wildtype ABCA7 vectors were constructed then overexpressed in HEK293 APPsw cells. Western blot analysis of FLAG‐Arg578Alafs was performed to determine if the deletion produces a truncated protein. Induced pluripotent stem cell (iPSC) lines from six AA individuals with AD bearing the Arg578Alafs mutation (three heterozygous; three homozygous) were created in pair with isogenic CRISPR‐corrected control lines. These iPSC lines were validated for pluripotency, genomic stability, and lack of off‐target editing. The isogenic ABCA7 deletion and CRISPR‐corrected control iPSC lines will be differentiated into microglia and neurons and functionally assessed for cell type‐specific AD phenotypes. Result Initial results comparing AD patients to CU individuals show that patient‐derived microglia have normal rates of phagocytosis but are impaired in the uptake and clearance of fibrillar Aβ. RT‐PCR analysis of the deletion in heterozygotes demonstrates that a stable RNA transcript is expressed from the ABCA7 deletion allele. Western blot analysis of overexpressed FLAG‐Arg578Alafs shows that the truncated protein is expressed, albeit at low levels compared to the wildtype protein. Conclusion This ABCA7 deletion produces a stable transcript which appears to be translated into low levels of protein. This supports a potential gain of function as the disease risk mechanism. Studies in isogenic lines will further elucidate the functional effects of this ABCA7 deletion on AD pathology in AAs

Novel Regulatory Mechanisms for the SoxC Transcriptional Network Required for Visual Pathway Development

What pathways specify retinal ganglion cell (RGC) fate in the developing retina? Here we report on mechanisms by which a molecular pathway involving Sox4/Sox11 is required for RGC differentiation and for optic nerve formation in mice , and is sufficient to differentiate human induced pluripotent stem cells into electrophysiologically active RGCs. These data place Sox4 downstream of RE1 silencing transcription factor in regulating RGC fate, and further describe a newly identified, Sox4-regulated site for post-translational modification with small ubiquitin-related modifier (SUMOylation) in Sox11, which suppresses Sox11's nuclear localization and its ability to promote RGC differentiation, providing a mechanism for the SoxC familial compensation observed here and elsewhere in the nervous system. These data define novel regulatory mechanisms for this SoxC molecular network, and suggest pro-RGC molecular approaches for cell replacement-based therapies for glaucoma and other optic neuropathies. Glaucoma is the most common cause of blindness worldwide and, along with other optic neuropathies, is characterized by loss of retinal ganglion cells (RGCs). Unfortunately, vision and RGC loss are irreversible, and lead to bilateral blindness in ∼14% of all diagnosed patients. Differentiated and transplanted RGC-like cells derived from stem cells have the potential to replace neurons that have already been lost and thereby to restore visual function. These data uncover new mechanisms of retinal progenitor cell (RPC)-to-RGC and human stem cell-to-RGC fate specification, and take a significant step toward understanding neuronal and retinal development and ultimately cell-transplant therapy

Novel Regulatory Mechanisms for the SoxC Transcriptional Network Required for Visual Pathway Development

What pathways specify retinal ganglion cell (RGC) fate in the developing retina? Here we report on mechanisms by which a molecular pathway involving Sox4/Sox11 is required for RGC differentiation and for optic nerve formation in mice , and is sufficient to differentiate human induced pluripotent stem cells into electrophysiologically active RGCs. These data place Sox4 downstream of RE1 silencing transcription factor in regulating RGC fate, and further describe a newly identified, Sox4-regulated site for post-translational modification with small ubiquitin-related modifier (SUMOylation) in Sox11, which suppresses Sox11's nuclear localization and its ability to promote RGC differentiation, providing a mechanism for the SoxC familial compensation observed here and elsewhere in the nervous system. These data define novel regulatory mechanisms for this SoxC molecular network, and suggest pro-RGC molecular approaches for cell replacement-based therapies for glaucoma and other optic neuropathies. Glaucoma is the most common cause of blindness worldwide and, along with other optic neuropathies, is characterized by loss of retinal ganglion cells (RGCs). Unfortunately, vision and RGC loss are irreversible, and lead to bilateral blindness in ∼14% of all diagnosed patients. Differentiated and transplanted RGC-like cells derived from stem cells have the potential to replace neurons that have already been lost and thereby to restore visual function. These data uncover new mechanisms of retinal progenitor cell (RPC)-to-RGC and human stem cell-to-RGC fate specification, and take a significant step toward understanding neuronal and retinal development and ultimately cell-transplant therapy