Michael Lardelli

Background: Early endolysosomal and autophagic defects are among the earliest cellular alterations observed in Alzheimer’s disease (AD). However, the molecular mechanisms linking amyloid precursor protein (APP) metabolism to vesicle trafficking dysfunction remain incompletely understood. The APP-derived fragment C99 has emerged as a potential upstream mediator of intracellular toxicity, but its impact on organelle homeostasis and its modulation by metabolic interventions remain unclear. Methods: To investigate these mechanisms, we expressed human C99 in Drosophila neurons and examined intracellular pathology using ultrastructural analysis, fluorescent reporters of autophagy and mitochondrial turnover, and proteomic interactome mapping. The effects of the ketone body β-hydroxybutyrate (BHB) were evaluated to assess the impact of metabolic intervention. Results: Neuronal C99 expression induced pronounced vesicular abnormalities, impaired autophagic turnover, and disrupted mitochondrial quality control. Transmission electron microscopy revealed extensive accumulation of enlarged vesicular compartments, accompanied by reduced mitochondrial turnover and accumulation of aged mitochondria. BHB treatment restored autophagic cargo clearance, improved mitochondrial turnover, and normalized vesicular ultrastructure. These protective effects required neuronal ketone transport, indicating a neuron-intrinsic metabolic mechanism. Proteomic analysis of the C99-associated interactome revealed that ketone treatment remodels networks enriched for vesicle trafficking and proteostasis pathways. Network prioritization identified the retromer component VPS35 as a candidate regulatory hub. Functional analyses demonstrated that depletion of VPS35 abolished the BHB-dependent restoration of autophagy, mitochondrial turnover, and vesicle morphology. Conclusions: Ketone treatment restores mitochondrial quality control and autophagic homeostasis through a VPS35-dependent mechanism in C99-induced neurodegeneration. These findings provide mechanistic insight into how metabolic interventions may restore intracellular homeostasis in Alzheimer’s disease.

Despite the relative maturity of bulk RNA-sequencing, compared to more recent developments such as single-cell and spatial RNA-sequencing, biases which impact data analysis continue to surface. One such bias, termed “Differential Allelic Representation” (DAR), is particularly evident when experimental samples are taken from non-isogenic genetic backgrounds. DAR is an uneven distribution of polymorphic loci between groups of experimental samples undergoing differential gene expression (DGE) analysis. When unequally represented polymorphic loci are also expression quantitative trait loci (eQTLs), DAR can lead to differences in gene expression which are not directly relevant to the primary research objectives. To mitigate DAR in both new and existing datsets, we introduce tadar , a Bioconductor package designed to facilitate transcriptome analysis by accounting for differential allelic representation. tadar implements a methodology that calculates a DAR metric at each polymorphic locus across the genome, which then serves as a predictive measure of a locus’ potential to cause eQTL-driven expression differences. This metric is then used to reduce eQTL noise in bulk RNA-Sequencing data.

The reproducibility crisis in bioscience, characterized by inconsistent study results, impedes our understanding of biological processes and global collaborative studies offer a unique solution. This study is the first global collaboration using the zebrafish (Danio rerio) novel tank test, a behavioral assay for anxiety-like responses. We analyzed data from 20 laboratories worldwide, focusing on housing conditions and experimental setups. Our study included 488 adult zebrafish, tested for 5 min, focusing on a variety of variables. Key findings show females exhibit more anxiety-like behavior than males, underscoring sex as a critical variable. Housing conditions, including higher stocking densities and specific feed types, influenced anxiety levels. Optimal conditions (5 fish/L) and nutritionally rich feeds (e.g., rotifers), mitigated anxiety-like behaviors. Environmental stressors, like noise and transportation, significantly impacted behavior. We recommend standardizing protocols to account for sex differences, optimal stocking densities, nutritionally rich feeds, and minimizing stressors to improve zebrafish behavioral study reliability.

In comparisons between mutant and wild-type genotypes, transcriptome analysis can reveal the direct impacts of a mutation, together with the homeostatic responses of the biological system. Recent studies have highlighted that, when the effects of homozygosity for recessive mutations are studied in non-isogenic backgrounds, genes located proximal to the mutation on the same chromosome often appear over-represented among those genes identified as differentially expressed (DE). One hypothesis suggests that DE genes chromosomally linked to a mutation may not reflect functional responses to the mutation but, instead, result from an unequal distribution of expression quantitative trait loci (eQTLs) between sample groups of mutant or wild-type genotypes. This is problematic because eQTL expression differences are difficult to distinguish from genes that are DE due to functional responses to a mutation. Here we show that chromosomally co-located differentially expressed genes (CC-DEGs) are also observed in analyses of dominant mutations in heterozygotes. We define a method and a metric to quantify, in RNA-sequencing data, localised differential allelic representation (DAR) between those sample groups subjected to differential expression analysis. We show how the DAR metric can predict regions prone to eQTL-driven differential expression, and how it can improve functional enrichment analyses through gene exclusion or weighting-based approaches. Advantageously, this improved ability to identify probable eQTLs also reveals examples of CC-DEGs that are likely to be functionally related to a mutant phenotype. This supports a long-standing prediction that selection for advantageous linkage disequilibrium influences chromosome evolution. By comparing the genomes of zebrafish (Danio rerio) and medaka (Oryzias latipes), a teleost with a conserved ancestral karyotype, we find possible examples of chromosomal aggregation of CC-DEGs during evolution of the zebrafish lineage. Our method for DAR analysis requires only RNA-sequencing data, facilitating its application across new and existing datasets.

Background: The endo‐lysosomal pathway (ELP) of cells is central to their importation of nutrients and for recycling of cellular components. Much genetic evidence supports the importance of dysfunction of the ELP in Alzheimer’s disease (AD). However, AD is a complex disorder and how AD‐associated genetic variation affects ELP function is unclear. Sanfilippo syndrome childhood dementia is a consequence of extreme ELP dysfunction, where inherited recessive mutations result in the ELP’s inability to properly catabolise a particular polysaccharide. Unlike AD, Sanfilippo is genetically simple. All the causative genes have been identified and the biochemical consequences of their mutations are understood. Animal models of Sanfilippo are considered excellent models of the human disease. Using zebrafish as a model organism, we are dissecting shared disease mechanisms between these two dementia subtypes.Method: Zebrafish models of both (early‐onset familial) AD and Sanfilippo syndrome were generated using CRISPR‐Cas9‐based approaches. The brain transcriptomes (RNA‐seq) and metabolomes (metabolomics/fluxomics) of both models are compared within single zebrafish families. Spatial working memory in both models is assessed using a Y‐maze.Result: Preliminary brain transcriptome analyses have identified changes‐in‐common to both oxidative phosphorylation and ribosome gene expression in both AD and Sanfilippo model zebrafish. Metabolomics and behavioural analyses of these models are ongoing.Conclusion: Neurodegeneration in Sanfilippo syndrome and Alzheimer’s disease may share common mechanisms that are amenable to common therapeutic approaches.

Sanfilippo syndrome (mucopolysaccharidosis type III, MPSIII) causes childhood dementia, while Alzheimer’s disease is the most common type of adult-onset dementia. There is no cure for either of these diseases, and therapeutic options are extremely limited. Increasing evidence suggests commonalities in the pathogenesis of these diseases. However, a direct molecular-level comparison of these diseases has never been performed. Here, we exploited the power of zebrafish reproduction (large families of siblings from single mating events raised together in consistent environments) to conduct sensitive, internally controlled, comparative transcriptome and proteome analyses of zebrafish models of early-onset familial Alzheimer’s disease (EOfAD, psen1 ^Q96_K97del/+ ) and MPSIIIB ( naglu ^{A603fs/A603fs} ) within single families. We examined larval zebrafish (7 days post fertilisation), representing early disease stages. We also examined the brains of 6-month-old zebrafish, which are approximately equivalent to young adults in humans. We identified substantially more differentially expressed genes and pathways in MPS III zebrafish than in EOfAD-like zebrafish. This is consistent with MPS III being a rapidly progressing and earlier onset form of dementia. Similar changes in expression were detected between the two disease models in gene sets representing extracellular matrix receptor interactions in larvae, and the ribosome and lysosome pathways in 6-month-old adult brains. Cell type-specific changes were detected in MPSIIIB brains at 6 months of age, likely reflecting significant disturbances of oligodendrocyte, neural stem cell, and inflammatory cell functions and/or numbers. Our ‘omics analyses have illuminated similar disease pathways between EOfAD and MPS III indicating where efforts to find mutually effective therapeutic strategies can be targeted.

Sanfilippo syndrome childhood dementia, also known as mucopolysaccharidosis type III (MPS III), is a rare inherited lysosomal storage disorder. Subtypes of MPS III are caused by deficiencies in one of four enzymes required for degradation of the glycosaminoglycan heparan sulfate (HS). An inability to degrade HS leads to progressive neurodegeneration and death in the second or third decades of life. Knowledge of MPS III pathogenesis is incomplete, and no effective therapies exist. We generated the hypomorphic mutations sgsh^S387Lfs, naglu^A603Efs and hgsnat^G577Sfsin the endogenous zebrafish genes orthologous to human SGSH, NAGLU, and HGSNAT that are loci for mutations causing MPS III subtypes MPS IIIA, B and C respectively. Our models display the primary MPS III disease signature of significant brain accumulation of HS, while behavioural analyses support hyperactivity phenotypes. Brain transcriptome analysis revealed changes related to lysosomal, glycosaminoglycan, immune system and iron homeostasis biology in all three models but also distinct differences in brain transcriptome state between models. The transcriptome analysis also indicated marked disturbance of the oligodendrocyte cell state in the brains of MPS IIIA, B and C zebrafish, supporting that effects on this cell type are an early and consistent characteristic of MPS III. Overall, our zebrafish models recapture key characteristics of the human disease and phenotypes seen in mouse models. Our models will allow exploitation of the zebrafish’s extreme fecundity and accessible anatomy to dissect the pathological mechanisms both common and divergent between the MPS IIIA, B, and C subtypes.

Aicardi Syndrome (AIC) is a rare neurodevelopmental disorder recognized by the classical triad of agenesis of the corpus callosum, chorioretinal lacunae and infantile epileptic spasms syndrome. The diagnostic criteria of AIC were revised in 2005 to include additional phenotypes that are frequently observed in this patient group. AIC has been traditionally considered as X-linked and male lethal because it almost exclusively affects females. Despite numerous genetic and genomic investigations on AIC, a unifying X-linked cause has not been identified. Here, we performed exome and genome sequencing of 10 females with AIC or suspected AIC based on current criteria. We identified a unique de novo variant, each in different genes: KMT2B, SLF1, SMARCB1, SZT2 and WNT8B, in five of these females. Notably, genomic analyses of coding and non-coding single nucleotide variants, short tandem repeats and structural variation highlighted a distinct lack of X-linked candidate genes. We assessed the likely pathogenicity of our candidate autosomal variants using the TOPflash assay for WNT8B and morpholino knockdown in zebrafish (Danio rerio) embryos for other candidates. We show expression of Wnt8b and Slf1 are restricted to clinically relevant cortical tissues during mouse development. Our findings suggest that AIC is genetically heterogeneous with implicated genes converging on molecular pathways central to cortical development.

Probabilistic and parsimony-based arguments regarding available genetics data are used to propose that Hardy and Higgin’s amyloid cascade hypothesis is valid but is commonly interpreted too narrowly to support, incorrectly, the primacy of the amyloid beta peptide (Aβ) in driving Alzheimer’s disease pathogenesis. Instead, increased activity of the βCTF (C99) fragment of APP is likely the critical pathogenic determinant altered by mutations in the APP gene. This model is consistent with the regulation of APP mRNA translation via its 5’ iron responsive element (IRE). Similar arguments support that the pathological effects of familial Alzheimer’s disease mutations in the genes PSEN1 and PSEN2 are not exerted directly via changes in APP cleavage to produce different ratios of Aβ length. Rather, these mutations likely affect the stability of presenilin holoprotein and/or γ-secretase multimers with consequences for γ-secretase activity and other important cellular functions. All fAD mutations in APP, PSEN1, and PSEN2 likely find unity of pathological mechanism in their actions on endolysosomal acidification and mitochondrial function, with detrimental effects on iron homeostasis and promotion of “pseudo-hypoxia” being of central importance. Aβ production is enhanced and distorted by oxidative stress and accumulates due to decreased lysosomal function. It may act as a disease-associated molecular pattern (DAMP) enhancing oxidative stress-driven neuroinflammation during the cognitive phase of the disease. We also discuss fascinating, but largely ignored, data on presenilin biology that may be important in understanding presenilins’ central role in familial Alzheimer’s disease.

In comparisons between mutant and wild-type genotypes, transcriptome analysis can reveal the direct impacts of a mutation, together with the homeostatic responses of the biological system. Recent studies have highlighted that, when homozygous mutations are studied in non-isogenic backgrounds, genes from the same chromosome as a mutation often appear over-represented among differentially expressed (DE) genes. One hypothesis suggests that DE genes chromosomally linked to a mutation may not reflect true biological responses to the mutation but, instead, result from differences in representation of expression quantitative trait loci (eQTLs) between sample groups selected on the basis of mutant or wild-type genotype. This is problematic when inclusion of spurious DE genes in a functional enrichment study results in incorrect inferences of mutation effect. Here we show that chromosomally co-located differentially expressed genes (CC-DEGs) can also be observed in analyses of dominant mutations in heterozygotes. We define a method and a metric to quantify, in RNA-sequencing data, localised differential allelic representation (DAR) between groups of samples subject to differential expression analysis. We show how the DAR metric can predict regions prone to eQTL-driven differential expression, and how it can improve functional enrichment analyses through gene exclusion or weighting of gene-level rankings. Advantageously, this improved ability to identify probable eQTLs also reveals examples of CC-DEGs that are likely to be functionally related to a mutant phenotype. This supports a long-standing prediction that selection for advantageous linkage disequilibrium influences chromosome evolution. By comparing the genomes of zebrafish ( Danio rerio ) and medaka ( Oryzias latipes ), a teleost with a conserved ancestral karyotype, we find possible examples of chromosomal aggregation of CC-DEGs during evolution of the zebrafish lineage. The DAR metric provides a solid foundation for addressing the eQTL issue in new and existing datasets because it relies solely on RNA-sequencing data.