The axonemal dynein heavy chain 10 gene is essential for monocilia motility and spine alignment in zebrafish.

Adolescent idiopathic scoliosis (AIS) is a common pediatric musculoskeletal disorder worldwide, characterized by atypical spine curvatures in otherwise healthy children. Human genetic studies have identified candidate genes associated with AIS, however, only a few of these have been shown to recapitulate adult-viable scoliosis in animal models. Using an F0 CRISPR screening approach in zebrafish, we demonstrate that disruption of the dynein axonemal heavy chain 10 (dnah10) gene results in recessive adult-viable scoliosis in zebrafish. Using a stably segregating dnah10 mutant zebrafish, we showed that the ependymal monocilia lining the hindbrain and spinal canal displayed reduced beat frequency, which was correlated with the disassembly of the Reissner fiber and the onset of body curvatures. Taken together, these results suggest that monocilia function in larval zebrafish contributes to the polymerization of the Reissner fiber and straightening of the body axis.

Mitochondrial function and Aß in Alzheimer's disease postmortem brain.

INTRODUCTION: Mitochondrial dysfunction is observed in Alzheimer's disease (AD). However, the relationship between functional mitochondrial deficits and AD pathologies is not well established in human subjects. METHODS: Post-mortem human brain tissue from 11 non-demented (ND) and 12 AD subjects was used to examine mitochondrial electron transport chain (ETC) function. Data were analyzed by neuropathology diagnosis and Apolipoprotein E (APOE) genotype. Relationships between AD pathology and mitochondrial function were determined. RESULTS: AD subjects had reductions in brain cytochrome oxidase (COX) function and complex II Vmax. APOE ε4 carriers had COX, complex II and III deficits. AD subjects had reduced expression of Complex I-III ETC proteins, no changes were observed in APOE ε4 carriers. No correlation between p-Tau Thr 181 and mitochondrial outcomes was observed, although brains from non-demented subjects demonstrated positive correlations between Aß concentration and COX Vmax. DISCUSSION: These data support a dysregulated relationship between brain mitochondrial function and Aß pathology in AD.

Cell type and sex specific mitochondrial phenotypes in iPSC derived models of Alzheimer's disease.

Introduction: Mitochondrial dysfunction is observed in Alzheimer's disease (AD). Altered mitochondrial respiration, cytochrome oxidase (COX) Vmax, and mitophagy are observed in human subjects and animal models of AD. Models derived from induced pluripotent stem cells (iPSCs) may not recapitulate these phenotypes after reprogramming from differentiated adult cells. Methods: We examined mitochondrial function across iPSC derived models including cerebral organoids, forebrain neurons, and astrocytes. iPSCs were reprogrammed from fibroblasts either from the University of Kansas Alzheimer's Disease Research Center (KU ADRC) cohort or purchased from WiCell. A total of four non-demented and four sporadic AD iPSC lines were examined. Models were subjected to mitochondrial respiration analysis using Seahorse XF technology, spectrophotometric cytochrome oxidase (COX) Vmax assays, fluorescent assays to determine mitochondrial mass, mitochondrial membrane potential, calcium, mitochondrial dynamics, and mitophagy levels. AD pathological hallmarks were also measured. Results: iPSC derived neurons and cerebral organoids showed reduced COX Vmax in AD subjects with more profound defects in the female cohort. These results were not observed in astrocytes. iPSC derived neurons and astrocytes from AD subjects had reduced mitochondrial respiration parameters with increased glycolytic flux. iPSC derived neurons and astrocytes from AD subjects showed sex dependent effects on mitochondrial membrane potential, mitochondrial superoxide production, and mitochondrial calcium. iPSC derived neurons from AD subjects had reduced mitochondrial localization in lysosomes with sex dependent effects on mitochondrial mass, while iPSC derived astrocytes from female AD subjects had increased mitochondrial localization to lysosomes. Both iPSC derived neurons and astrocytes from AD subjects showed altered mitochondrial dynamics. iPSC derived neurons had increased secreted Aß, and sex dependent effects on total APP protein expression. iPSC derived astrocytes showed sex dependent changes in GFAP expression in AD derived cells. Conclusion: Overall, iPSC derived models from AD subjects show mitochondrial phenotypes and AD pathological hallmarks in a cell type and sex dependent manner. These results highlight the importance of sex as a biological variable in cell culture studies.

Apolipoprotein E and Alzheimer's disease.

Genetic variation in apolipoprotein E (APOE) influences Alzheimer's disease (AD) risk. APOE ε4 alleles are the strongest genetic risk factor for late onset sporadic AD. The AD risk is dose dependent, as those carrying one APOE ε4 allele have a 2-3-fold increased risk, while those carrying two ε4 alleles have a 10-15-fold increased risk. Individuals carrying APOE ε2 alleles have lower AD risk and those carrying APOE ε3 alleles have neutral risk. APOE is a lipoprotein which functions in lipid transport, metabolism, and inflammatory modulation. Isoform specific effects of APOE within the brain include alterations to Aß, tau, neuroinflammation, and metabolism. Here we review the association of APOE with AD, the APOE isoform specific effects within brain and periphery, and potential therapeutics.

Mitochondrial Membrane Potential Influences Amyloid-ß Protein Precursor Localization and Amyloid-ß Secretion.

BACKGROUND: Amyloid-ß (Aß), which derives from the amyloid-ß protein precursor (AßPP), forms plaques and serves as a fluid biomarker in Alzheimer's disease (AD). How Aß forms from AßPP is known, but questions relating to AßPP and Aß biology remain unanswered. AD patients show mitochondrial dysfunction, and an Aß/AßPP mitochondria relationship exists. OBJECTIVE: We considered how mitochondrial biology may impact AßPP and Aß biology. METHODS: SH-SY5Y cells were transfected with AßPP constructs. After treatment with FCCP (uncoupler), Oligomycin (ATP synthase inhibitor), or starvation Aß levels were measured. ß-secretase (BACE1) expression was measured. Mitochondrial localized full-length AßPP was also measured. All parameters listed were measured in ρ0 cells on an SH-SY5Y background. iPSC derived neurons were also used to verify key results. RESULTS: We showed that mitochondrial depolarization routes AßPP to, while hyperpolarization routes AßPP away from, the organelle. Mitochondrial AßPP and cell Aß secretion inversely correlate, as cells with more mitochondrial AßPP secrete less Aß, and cells with less mitochondrial AßPP secrete more Aß. An inverse relationship between secreted/extracellular Aß and intracellular Aß was observed. CONCLUSION: Our findings indicate mitochondrial function alters AßPP localization and suggest enhanced mitochondrial activity promotes Aß secretion while depressed mitochondrial activity minimizes Aß secretion. Our data complement other studies that indicate a mitochondrial, AßPP, and Aß nexus, and could help explain why cerebrospinal fluid Aß is lower in those with AD. Our data further suggest Aß secretion could serve as a biomarker of cell or tissue mitochondrial function.

Coding Variants Coupled With Rapid Modeling in Zebrafish Implicate Dynein Genes, dnaaf1 and zmynd10, as Adolescent Idiopathic Scoliosis Candidate Genes.

Adolescent idiopathic scoliosis (AIS) is the most common pediatric spine disorder affecting ∼3% of children worldwide. Human genetic studies suggest a complex polygenic disease model for AIS with large genetic and phenotypic heterogeneity. However, the overall genetic etiology of AIS remains poorly understood. To identify additional AIS susceptibility loci, we performed whole-exome sequencing (WES) on a cohort of 195 Southern Chinese AIS patients. Bioinformatics analysis identified 237 novel rare variants associated with AIS, located in 232 new susceptibility loci. Enrichment analysis of these variants revealed 10 gene families associated with our AIS cohort. We screened these gene families by comparing our candidate gene list with IS candidate genes in the Human Phenotype Ontology (HPO) database and previous reported studies. Two candidate gene families, axonemal dynein and axonemal dynein assembly factors, were retained for their associations with ciliary architecture and function. The damaging effects of candidate variants in dynein genes dnali1, dnah1, dnaaf, and zmynd10, as well as in one fibrillin-related gene tns1, were functionally analyzed in zebrafish using targeted CRISPR/Cas9 screening. Knockout of two candidate genes, dnaaf1 or zmynd10, recapitulated scoliosis in viable adult zebrafish. Altogether, our results suggest that the disruption of one or more dynein-associated factors may correlate with AIS susceptibility in the Southern Chinese population.

The Reissner Fiber Is Highly Dynamic In Vivo and Controls Morphogenesis of the Spine.

Cerebrospinal fluid (CSF) physiology is important for the development and homeostasis of the central nervous system, and its disruption has been linked to scoliosis in zebrafish [1, 2]. Suspended in the CSF is an extracellular structure called the Reissner fiber, which extends from the brain through the central canal of the spinal cord. Zebrafish scospondin-null mutants are unable to assemble a Reissner fiber and fail to form a straight body axis during embryonic development [3]. Here, we describe hypomorphic missense mutations of scospondin, which allow Reissner fiber assembly and extension of a straight axis. However, during larval development, these mutants display progressive Reissner fiber disassembly, which is concomitant with the emergence of axial curvatures and scoliosis in adult animals. Using a scospondin-GFP knockin zebrafish line, we demonstrate several dynamic properties of the Reissner fiber in vivo, including embryonic fiber assembly, the continuous rostral to caudal movement of the fiber within the brain and central canal, and subcommissural organ (SCO)-spondin-GFP protein secretion from the floor plate to merge with the fiber. Finally, we show that disassembly of the Reissner fiber is also associated with the progression of axial curvatures in distinct scoliosis mutant zebrafish models. Together, these data demonstrate a critical role for the Reissner fiber for the maintenance of a straight body axis and spine morphogenesis in adult zebrafish. Our study establishes a framework for future investigations to address the cellular effectors responsible for Reissner-fiber-dependent regulation of axial morphology. VIDEO ABSTRACT.