In this project, the Hardy team seeks to decipher long-standing mysteries about the genetic underpinnings of tau pathology in Alzheimer’s disease (AD) using cutting-edge gene sequencing technology. Their work will deepen our understanding of the MAPT locus, tau expression, and splicing across a range of neurodegenerative diseases and laboratory-derived models, generating a core, shareable resource to drive the identification of novel therapeutic targets.
Neurofibrillary tau tangles are the second hallmark pathology of AD, emerging after -amyloid beta plaques are well established and alongside the onset of clinical symptoms. The tau protein is encoded by the MAPT (microtubule-associated protein tau) gene, located on a section of chromosome 17 that also contains approximately 11 other genes. This section is generally inherited as a unit from a parent (a haplotype), and the two most common versions of this haplotype are each associated with different neurodegenerative diseases. The H1 haplotype, for example, is linked to an increased risk of APOE-negative sporadic AD and varies in frequency across different biogeographical ancestries. Disease-associated variants of the gene that produces amyloid beta peptides directly cause familial, early-onset AD. However, while MAPT variants are key drivers of other tauopathies that cause dementia, only a few have been identified as contributors to sporadic (classic) AD risk, and none have been found to drive familial early-onset AD. This evidence supports the role of MAPT as a genetic contributor to AD while also suggesting that the H1 haplotype may harbor additional AD-relevant genetic sequences.
During the transcription and translation process that produces tau proteins from the MAPT gene, alternative splicing determines which portions of the gene are included in the final transcript, yielding different tau isoforms. In humans, neurons produce six major tau isoforms, whereas mouse neurons only produce three. Significantly less than 5% of the 133,000 base pairs transcribed from the MAPT gene will be copied into a tau isoform, highlighting the complexity of this splicing process. These tau isoforms are defined by the number of repeats of specific amino acid sequences they contain. Scientists have observed that these sequences influence the isoform’s ability to promote microtubule assembly and its tendency to misfold and aggregate, but the precise mechanisms underlying these effects remain unclear. The balance of tau isoforms varies across developmental stages in AD and other tauopathies, suggesting that maintaining the proper balance is crucial for neuronal function. Most disease-associated MAPT variants alter the relative production of tau isoforms, yet the mechanisms by which these variants drive tau aggregation, neurodegeneration, and distinct clinical tauopathies remain unknown. Additionally, the influence of the MAPT haplotype on tau expression and splicing remains poorly understood.
Advancements in gene sequencing technology have enabled scientists to identify disease-associated variants. Because specific DNA sequences influence how genes are transcribed and spliced into RNA, sequencing can reveal gene locations (loci) and potential protein isoforms. Genes range from a few hundred to over two million base pairs in length, but early sequencing technology could only analyze small stretches—typically 75 to 400 base pairs at a time. These short sequences had to be reassembled into a full gene sequence by inferring their order and overlap, a process prone to errors. The challenge was further complicated by repetitive sequences, as different versions (alleles) of genes may contain varying numbers of repeats, making accurate reconstruction difficult. In a breakthrough, new long-read sequencing technology can scan 5,000 to 30,000 base pairs of DNA or RNA at a time, significantly decreasing the number of inferences required for sequencing each gene or transcript and revealing potential splice locations and other structural information that may predict novel isoforms. For a gene like MAPT that spans over 150,000 base pairs and sits in a haplotype with poorly understood implications, long-read sequencing offers valuable new insights.
The Hardy lab is pioneering analysis of the MAPT genetic locus and its flanking regions on chromosome 17 using long-read sequencing of DNA and RNA. In aim one, they are using long-read sequencing to capture full-length expressed tau transcripts for the first time. They are also assessing the effects of MAPT mutations and haplotype alleles on tau expression and splicing. In aim two, they are using brain samples from multiple tauopathies to study splicing and allele-specific expression (MAPT variants and haplotype) in different neurodegenerative disease brains and brain regions to determine the impact of genetic sequences on phenotypes. By establishing induced pluripotent stem cell (iPSC) lines and matching long-read DNA and RNA sequencing from them, they can determine how gene architecture affects transcription. With their new sequencing knowledge, they are revisiting the MAPT sequencing data from two existing dementia cohorts to determine whether previously unrecognized variants are associated with different neurodegenerative diseases.
At the conclusion of this first year of funding, the Hardy lab has made significant progress toward both project aims. They established stringent parameters for obtaining high-quality RNA from human brain tissue to ensure reliable long-read sequencing data. To secure an ample supply of brain samples with adequate RNA quality, they expanded their brain collection efforts by collaborating with additional brain banks. For comparative purposes, they analyzed the MAPT locus in control and amyloid mouse models to establish a reference framework for human analyses and assess whether amyloid deposition influences how transcripts are spliced and, thus, what tau protein isoforms are produced. Their findings revealed no alternative splicing or significant effects on splicing between these mouse models, underscoring that tau splicing is meaningfully different between humans and mice. They’ve also begun making progress on their second aim of analyzing neurons and organoids derived from individuals with and without MAPT splicing variants. Moving forward, the team plans to investigate the regulation of tau expression during neuronal development and maturation in cell lines carrying different MAPT variants while validating an in vitro model to test potential therapeutic strategies for modifying tau expression.
