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.
The ATP-binding cassette, subfamily A (ABC1), member 7 (ABCA7) gene is associated with Alzheimer’s disease (AD) risk in populations of African, Asian, and European ancestry1-5. Numerous ABCA7 ...mutations contributing to risk have been identified, including a 44 base pair deletion (rs142076058) specific to individuals of African ancestry and predicted to cause a frameshift mutation (p.Arg578Alafs) (Cukier et al., 2016). The UMi043-A human induced pluripotent stem cell line was derived from an African American individual with AD who is heterozygous for this deletion and is a resource to further investigate ABCA7 and how this African-specific deletion may influence disease pathology.
A missense variant in the tetratricopeptide repeat domain 3 (TTC3) gene (rs377155188, p.S1038C, NM_003316.4:c 0.3113C>G) was found to segregate with disease in a multigenerational family with ...late-onset Alzheimer’s disease. This variant was introduced into induced pluripotent stem cells (iPSCs) derived from a cognitively intact individual using CRISPR genome editing, and the resulting isogenic pair of iPSC lines was differentiated into cortical neurons. Transcriptome analysis showed an enrichment for genes involved in axon guidance, regulation of actin cytoskeleton, and GABAergic synapse. Functional analysis showed that the TTC3 p.S1038C iPSC-derived neuronal progenitor cells had altered 3-dimensional morphology and increased migration, while the corresponding neurons had longer neurites, increased branch points, and altered expression levels of synaptic proteins. Pharmacological treatment with small molecules that target the actin cytoskeleton could revert many of these cellular phenotypes, suggesting a central role for actin in mediating the cellular phenotypes associated with the TTC3 p.S1038C variant.
Background
Greater than 20 genetic loci have been consistently associated with Alzheimer disease (AD). Accumulating evidence has implicated the endolysosomal pathway in disease pathology. The ...sortilin‐related receptor 1 (SORL1) gene has been associated with AD through multiple studies and has been suggested to function as a guide for amyloid precursor protein (APP) to the endocytic pathway. We identified a family with multiple AD affected individuals that have a single base pair deletion (g.4292delG) resulting in a frameshift alteration and premature termination of the protein (p.Cys143Cys*1). Although previous studies have implicated SORL1 in AD pathogenesis, it remains unclear how this deletion impacts SORL1 protein function in AD development.
Method
Patient‐specific induced pluripotent stem cells (iPSC) lines were derived to examine the molecular consequences of this SORL1 deletion in neuronal and glial cells (astrocytes and microglia), which can otherwise only be collected from autopsy samples. Each iPSC line generated was validated for pluripotency through immunocytochemical staining and karyotypic stability. Patient iPSC lines and control lines were differentiated into cortical neurons and glia for functional characteristics, including amyloid beta production and uptake, vesicular trafficking, and neurite outgrowth.
Result
We have derived iPSC lines bearing the SORL1 deletion and have confirmed pluripotency by immunocytochemistry. These iPSC lines were differentiated into cortical neurons whose identity was confirmed by immunocytochemical staining for neuronal markers. The SORL1 variant bearing cortical neurons showed enlarged endosomes, as measured by RAB5 staining and fluorescent microscopy, compared to the neurons derived from control iPSCs. In addition, the SORL1 deletion bearing neurons showed an upregulation in the ratio of amyloid beta 42:amyloid beta 40 compared to control neurons as measured by ELISA. Finally, SORL1 deletion bearing neurons had reduced neurite outgrowth and branch point numbers compared to controls.
Conclusion
In summary, our results support that the p.Cys143Ser*1 variant is pathogenic and alters the functionality of cells from the central nervous system. iPSC‐derived neurons and glia cells bearing the deletion in SORL1 p.Cys143Ser*1 variant shows cellular deficits associated with AD pathology, particularly in endolysomal trafficking defects. Studies are continuing to determine if this effect is due to a gain or loss of function.
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.
Abstract
Background
We identified a rare, nonsynonymous variant in the tetratricopeptide repeat domain 3 (TTC3) gene that segregated in a non‐Hispanic white late onset Alzheimer disease (LOAD) family ...(Kohli, et al, 2016). This missense alteration, rs377155188 (p.S1038C), is predicted to be deleterious and is extremely rare. Studies have reported that cortical TTC3 expression is reduced in LOAD patients and negatively correlated with AD neuropathology.
Method
To understand the mechanism by which the TTC3 p.S1038C may contribute to LOAD risk, CRISPR/Cas9 genome edited induced pluripotent stem cells (iPSCs) were developed that were homozygous for the variant to examine cellular and transcriptional consequences in iPSC‐derived neuronal cells (Laverde‐Paz, et al, 2021).
Result
Quantitative PCR and western blot analysis demonstrated that TTC3 levels were decreased in edited compared to unedited iPSCs, as well as differentiated neurons. In growing neuronal precursor cells (NPCs), cells with the TTC3 variant recovered more quickly from a scratch wound. Since there is evidence that modulation of TTC3 affects neurite growth, morphological measures of axon formation were assessed using the Incucyte Zoom. Studies demonstrate an increase in neurite outgrowth, which phenotypically corresponds with previous studies of a decrease in TTC3 function. This phenotype was tempered by treatment with Cytochalasin D, an inhibitor of actin polymerization. Additionally, TTC3 ubiquitinates phosphorylated AKT and regulates AKT signaling. The edited cells were found to have an increase in phosphorylated AKT (pAKT) relative to total AKT. RNA‐seq of day 70 neurons identified 979 genes that were differentially expressed (FDR<0.05). This included known AD genes (BACE1) and genes in AD GWAS loci (ADAMTS1, MAF, NCK2). KEGG pathway analysis identified differential expression in PI3K‐Akt signaling pathway components, in which TTC3 has been previously implicated, as well as the axon guidance pathway, the GABAergic synapse pathway, and the Wnt signaling pathway.
Conclusion
Combined, these results suggest that the TTC3 p.S1038C variant causes a loss of function. Utilizing a CRISPR genome edited iPSC carrying a homozygous alteration in TTC3, we were able to identify potential mechanisms by which TTC3 may contribute to LOAD risk.
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.
Abstract
Background
The variant G206A in Presenilin‐1 (
PSEN1
) gene has been identified almost exclusively in Alzheimer Disease (AD) Puerto Rican families. This variant represents a founder effect ...on the African background. The G206A variant associates with extreme variability in age of onset (AOO), ranging from 30 to 90 years. In contrast, other variants at the same amino acid of
PSEN1
(G206N, G206S) have a tighter range of AOO (30‐35 years). We aim to identify the molecular mechanisms involved in the AOO variability between G206A carriers through functional analysis of induced pluripotent stem cells (iPSCs).
Method
Genotyping data were phased using SHAPEIT to identify local ancestry and RFMix to estimate genetic ancestry. p‐Tau181 levels were tested from plasma using Simoa (Quanterix HD‐X). iPSCs of G206A carriers with different AOO were reprogrammed using non‐integrating Sendai virus. These clonal lines were assessed for pluripotency and chromosomal stability Isogenic construction of G206A iPSC lines is currently in progress.
Result
We screened for G206A carriers using whole genome from 896 individuals (182 families) and identified 43 carriers (39 AD and 4 cognitively unimpaired <65 years) from 8 families and 3 isolated cases. 55% of AD G206A carriers had AOO <65. A single African haplotype was identified in all G206Acarriers. We observed higher pTau181 levels in AD G206A carriers with early age of onset, compared to those with late onset. Association analysis did not identify APOE4 or SYNJ1 polymorphisms as contributing to the differences in AOO within G206A carriers. To perform functional studies, we selected three with early (<65) and three with late (>65) AOO AD peripheral blood mononuclear cells (PBMCs)that were reprogrammed into iPSC lines. G‐band Karyotype, factor loss analyses, genetic finger printing, immunocytochemistry (ICC) and qRT‐PCR for intracellular and surface pluripotency markers was confirmed in the iPSC lines.
Conclusion
The
PSEN1
G206A variant, originating in African ancestry haplotype revealing a founder effect, is an important contributor to AD in an underserved population. Understanding the role of the G206A variant in AD pathogenesis and the factors that influence its effect on AOO will provide insight on AD pathogenesis, age of onset variation and identification of potential novel therapeutic targets.
Abstract
Background
Over 20 genetic loci have been consistently associated with Alzheimer disease (AD) with accumulating evidence pointing to alterations of the endolysosomal pathway as playing key ...roles. The
sortilin‐related receptor 1
(
SORL1
) gene has been associated with AD through multiple genetic studies and has been suggested to function by guiding APP to the endocytic pathway. We recently identified a family with multiple AD affected individuals that bear a single base pair deletion (g.4292delG) predicted to result in a frameshift alteration and early termination of the protein (p.Cys143Ser*1). Previous studies have implicated
SORL1
variants in AD pathogenesis, but it remains unclear how this deletion impacts SORL1 protein function and AD development.
Method
Induced pluripotent stem cells (iPSC) lines were developed to examine the cellular and molecular consequences in neuronal and glial cell (astrocytes and microglia) populations, which can otherwise only be collected postmortem. Each iPSC line generated was validated for pluripotency through immunocytochemical staining and shown to be negative for any large‐scale chromosomal abnormalities via karyotyping. The patient iPSC lines and control lines were differentiated into cortical neurons and glia cells for functional analyses, including amyloid beta production and uptake, vesicular trafficking, and neurite outgrowth.
Result
We have generated iPSC lines from two individuals bearing this deletion in
SORL1
and have confirmed their pluripotency by immunocytochemistry. These cells were differentiated into cortical neurons whose identity was confirmed by staining for forebrain markers. The
SORL1
variant bearing cortical neurons showed enlarged endosomes compared to the neurons derived from control iPSCs, as measured by the staining with an anti‐RAB5 antibody and fluorescent microscopy. In addition, the
SORL1
deletion bearing neurons showed an upregulation of pathogenic amyloid beta 42 measured by ELISA. Glial cells have emerged as pivotal players in the maintenance of brain homeostasis. Therefore, we will assess the functional impact of this deletion of
SORL1
in microglial cells and astrocytes.
Conclusion
In summary, our results support that the p.Cys143Ser*1 variant is indeed pathogenic. iPSC‐derived neurons and glia cells bearing the deletion in SORL1 p.Cys143Ser*1 variant shows cellular deficits associated with AD pathology which could serve as new avenues for therapeutic development.
Background
Over 20 genetic loci have been consistently associated with Alzheimer disease (AD) with accumulating evidence pointing to alterations of the endolysosomal pathway as playing key roles. The ...sortilin‐related receptor 1 (SORL1) gene has been associated with AD through multiple genetic studies and has been suggested to function by guiding APP to the endocytic pathway. We recently identified a family with multiple AD affected individuals that bear a single base pair deletion (g.4292delG) predicted to result in a frameshift alteration and early termination of the protein (p.Cys143Ser*1). Previous studies have implicated SORL1 variants in AD pathogenesis, but it remains unclear how this deletion impacts SORL1 protein function and AD development.
Method
Induced pluripotent stem cells (iPSC) lines were developed to examine the cellular and molecular consequences in neuronal and glial cell (astrocytes and microglia) populations, which can otherwise only be collected postmortem. Each iPSC line generated was validated for pluripotency through immunocytochemical staining and shown to be negative for any large‐scale chromosomal abnormalities via karyotyping. The patient iPSC lines and control lines were differentiated into cortical neurons and glia cells for functional analyses, including amyloid beta production and uptake, vesicular trafficking, and neurite outgrowth.
Result
We have generated iPSC lines from two individuals bearing this deletion in SORL1 and have confirmed their pluripotency by immunocytochemistry. These cells were differentiated into cortical neurons whose identity was confirmed by staining for forebrain markers. The SORL1 variant bearing cortical neurons showed enlarged endosomes compared to the neurons derived from control iPSCs, as measured by the staining with an anti‐RAB5 antibody and fluorescent microscopy. In addition, the SORL1 deletion bearing neurons showed an upregulation of pathogenic amyloid beta 42 measured by ELISA. Glial cells have emerged as pivotal players in the maintenance of brain homeostasis. Therefore, we will assess the functional impact of this deletion of SORL1 in microglial cells and astrocytes.
Conclusion
In summary, our results support that the p.Cys143Ser*1 variant is indeed pathogenic. iPSC‐derived neurons and glia cells bearing the deletion in SORL1 p.Cys143Ser*1 variant shows cellular deficits associated with AD pathology which could serve as new avenues for therapeutic development.
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