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1.
Front Mol Neurosci ; 16: 1201015, 2023.
Article in English | MEDLINE | ID: mdl-37614699

ABSTRACT

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.

2.
Neurobiol Dis ; 171: 105781, 2022 09.
Article in English | MEDLINE | ID: mdl-35667615

ABSTRACT

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.


Subject(s)
Alzheimer Disease , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Apolipoprotein E4/metabolism , Autopsy , Brain/metabolism , Electron Transport Complex IV/metabolism , Humans , Mitochondria/metabolism
3.
Aging Cell ; 20(5): e13356, 2021 05.
Article in English | MEDLINE | ID: mdl-33939248

ABSTRACT

We examined the impact of an APOE ε4 genotype on Alzheimer's disease (AD) subject platelet and lymphocyte metabolism. Mean platelet mitochondrial cytochrome oxidase Vmax activity was lower in APOE ε4 carriers and lymphocyte Annexin V, a marker of apoptosis, was significantly higher. Proteins that mediate mitophagy and energy sensing were higher in APOE ε4 lymphocytes which could represent compensatory changes and recapitulate phenomena observed in post-mortem AD brains. Analysis of the lipid synthesis pathway found higher AceCSI, ATP CL, and phosphorylated ACC levels in APOE ε4 lymphocytes. Lymphocyte ACC changes were also observed in post-mortem brain tissue. Lymphocyte RNAseq showed lower APOE ε4 carrier sphingolipid Transporter 3 (SPNS3) and integrin Subunit Alpha 1 (ITGA1) expression. RNAseq pathway analysis revealed APOE ε4 alleles activated inflammatory pathways and modulated bioenergetic signaling. These findings support a relationship between APOE genotype and bioenergetic pathways and indicate platelets and lymphocytes from APOE ε4 carriers exist in a state of bioenergetic stress. Neither medication use nor brain-localized AD histopathology can account for these findings, which define an APOE ε4-determined molecular and systemic phenotype that informs AD etiology.


Subject(s)
Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Apolipoproteins E/genetics , Aged , Aged, 80 and over , Alzheimer Disease/enzymology , Apolipoproteins E/metabolism , Blood Platelets/enzymology , Cells, Cultured , Electron Transport Complex IV/metabolism , Energy Metabolism , Female , Heterozygote , Humans , Inflammation Mediators/metabolism , Lymphocytes/metabolism , Male , Phenotype , RNA-Seq
4.
Ann Neurol ; 75(5): 793-798, 2014 May.
Article in English | MEDLINE | ID: mdl-24771548

ABSTRACT

We report a 5-generation family with phenotypically diverse neurodegenerative disease including relentlessly progressive choreoathetoid movements, dysarthria, dysphagia, spastic paralysis, and behavioral dementia in descendants of a 67-year-old woman with amyotrophic lateral sclerosis. Disease onset varied with gender, occurring in male children and adult women. Exome sequence analyses revealed a novel mutation (c.1490C>T, p.P497L) in the ubiquilin-2 gene (UBQLN2) with X-linked inheritance in all studied affected individuals. As ubiquilin-2-positive inclusions were identified in brain, we suggest that mutant peptide predisposes to protein misfolding and accumulation. Our findings expand the spectrum of neurodegenerative phenotypes caused by UBQLN2 mutations.


Subject(s)
Cell Cycle Proteins/genetics , Genetic Heterogeneity , Heredodegenerative Disorders, Nervous System/diagnosis , Heredodegenerative Disorders, Nervous System/genetics , Mutation/genetics , Ubiquitins/genetics , Adaptor Proteins, Signal Transducing , Adolescent , Adult , Aged , Autophagy-Related Proteins , Child, Preschool , Female , Genes, Dominant , Humans , Male , Pedigree , Protein Folding , Young Adult
5.
Mol Ther ; 17(9): 1563-73, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19532137

ABSTRACT

Despite recent advances suggesting new therapeutic targets, Alzheimer's disease (AD) remains incurable. Aberrant production and accumulation of the Abeta peptide resulting from altered processing of the amyloid precursor protein (APP) is central to the pathogenesis of disease, particularly in dominantly inherited forms of AD. Thus, modulating the production of APP is a potential route to effective AD therapy. Here, we describe the successful use of an allele-specific RNA interference (RNAi) approach targeting the Swedish variant of APP (APPsw) in a transgenic mouse model of AD. Using recombinant adeno-associated virus (rAAV), we delivered an anti-APPsw short-hairpin RNA (shRNA) to the hippocampus of AD transgenic mice (APP/PS1). In short- and long-term transduction experiments, reduced levels of APPsw transprotein were observed throughout targeted regions of the hippocampus while levels of wild-type murine APP remained unaltered. Moreover, intracellular production of transfer RNA (tRNA)-valine promoter-driven shRNAs did not lead to detectable neuronal toxicity. Finally, long-term bilateral hippocampal expression of anti-APPsw shRNA mitigated abnormal behaviors in this mouse model of AD. The difference in phenotype progression was associated with reduced levels of soluble Abeta but not with a reduced number of amyloid plaques. Our results support the development of allele-specific RNAi strategies to treat familial AD and other dominantly inherited neurodegenerative diseases.


Subject(s)
Alleles , Alzheimer Disease/pathology , Alzheimer Disease/therapy , Amyloid beta-Protein Precursor/genetics , Dependovirus/genetics , RNA Interference/physiology , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Amyloid beta-Protein Precursor/metabolism , Animals , Enzyme-Linked Immunosorbent Assay , Humans , Male , Mice , Mice, Transgenic
6.
J Neurosci ; 27(19): 5163-71, 2007 May 09.
Article in English | MEDLINE | ID: mdl-17494702

ABSTRACT

Little is known about the role of protein quality control in the inner ear. We now report selective cochlear degeneration in mice deficient in Fbx2, a ubiquitin ligase F-box protein with specificity for high-mannose glycoproteins (Yoshida et al., 2002). Originally described as a brain-enriched protein (Erhardt et al., 1998), Fbx2 is also highly expressed in the organ of Corti, in which it has been called organ of Corti protein 1 (Thalmann et al., 1997). Mice with targeted deletion of Fbxo2 develop age-related hearing loss beginning at 2 months. Cellular degeneration begins in the epithelial support cells of the organ of Corti and is accompanied by changes in cellular membrane integrity and early increases in connexin 26, a cochlear gap junction protein previously shown to interact with Fbx2 (Henzl et al., 2004). Progressive degeneration includes hair cells and the spiral ganglion, but the brain itself is spared despite widespread CNS expression of Fbx2. Cochlear Fbx2 binds Skp1, the common binding partner for F-box proteins, and is an unusually abundant inner ear protein. Whereas cochlear Skp1 levels fall in parallel with the loss of Fbx2, other components of the canonical SCF (Skp1, Cullin1, F-box, Rbx1) ubiquitin ligase complex remain unchanged and show little if any complex formation with Fbx2/Skp1, suggesting that cochlear Fbx2 and Skp1 form a novel, heterodimeric complex. Our findings demonstrate that components of protein quality control are essential for inner ear homeostasis and implicate Fbx2 and Skp1 as potential genetic modifiers in age-related hearing loss.


Subject(s)
Cochlear Diseases/metabolism , Deafness/metabolism , F-Box Proteins/genetics , Hair Cells, Auditory/metabolism , Nerve Degeneration/metabolism , Aging/genetics , Aging/metabolism , Aging/pathology , Animals , Cell Membrane/genetics , Cell Membrane/metabolism , Cell Membrane/pathology , Cochlear Diseases/genetics , Cochlear Diseases/physiopathology , Connexin 26 , Connexins/genetics , Connexins/metabolism , Deafness/genetics , Deafness/physiopathology , Glycoproteins/metabolism , Hair Cells, Auditory/pathology , Hearing Loss, Sensorineural/genetics , Hearing Loss, Sensorineural/metabolism , Hearing Loss, Sensorineural/physiopathology , Labyrinth Supporting Cells/metabolism , Labyrinth Supporting Cells/pathology , Macromolecular Substances/metabolism , Mice , Mice, Knockout , Nerve Degeneration/genetics , Nerve Degeneration/physiopathology , Protein Binding/physiology , Protein Subunits/genetics , Protein Subunits/metabolism , SKP Cullin F-Box Protein Ligases/metabolism , Ubiquitin-Protein Ligase Complexes/genetics , Ubiquitin-Protein Ligase Complexes/metabolism
7.
J Neurosci ; 25(40): 9152-61, 2005 Oct 05.
Article in English | MEDLINE | ID: mdl-16207874

ABSTRACT

Huntington's disease (HD) and other polyglutamine (polyQ) neurodegenerative diseases are characterized by neuronal accumulation of the disease protein, suggesting that the cellular ability to handle abnormal proteins is compromised. As both a cochaperone and ubiquitin ligase, the C-terminal Hsp70 (heat shock protein 70)-interacting protein (CHIP) links the two major arms of protein quality control, molecular chaperones, and the ubiquitin-proteasome system. Here, we demonstrate that CHIP suppresses polyQ aggregation and toxicity in transfected cell lines, primary neurons, and a novel zebrafish model of disease. Suppression by CHIP requires its cochaperone function, suggesting that CHIP acts to facilitate the solubility of mutant polyQ proteins through its interactions with chaperones. Conversely, HD transgenic mice that are haploinsufficient for CHIP display a markedly accelerated disease phenotype. We conclude that CHIP is a critical mediator of the neuronal response to misfolded polyQ protein and represents a potential therapeutic target in this important class of neurodegenerative diseases.


Subject(s)
Neural Inhibition/drug effects , Neurons/drug effects , Peptides/metabolism , Ubiquitin-Protein Ligases/pharmacology , Animals , Blotting, Western/methods , Cells, Cultured , Cerebral Cortex/cytology , Chlorocebus aethiops , Disease Models, Animal , Embryo, Mammalian , Embryo, Nonmammalian , Fluorescent Antibody Technique/methods , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/metabolism , Huntington Disease/drug therapy , In Vitro Techniques , Mice , Mice, Transgenic , Microtubule-Associated Proteins/metabolism , Mutation , Neurons/cytology , Peptides/genetics , Radioimmunoassay/methods , Rats , Transfection/methods , Zebrafish
8.
Nucleic Acids Res ; 32(2): 661-8, 2004.
Article in English | MEDLINE | ID: mdl-14754988

ABSTRACT

Tau and amyloid precursor protein (APP) are key proteins in the pathogenesis of sporadic and inherited Alzheimer's disease. Thus, developing ways to inhibit production of these proteins is of great research and therapeutic interest. The selective silencing of mutant alleles, moreover, represents an attractive strategy for treating inherited dementias and other dominantly inherited disorders. Here, using tau and APP as model targets, we describe an efficient method for producing small interfering RNA (siRNA) against essentially any targeted region of a gene. We then use this approach to develop siRNAs that display optimal allele-specific silencing against a well-characterized tau mutation (V337M) and the most widely studied APP mutation (APPsw). The allele-specific RNA duplexes identified by this method then served as templates for constructing short hairpin RNA (shRNA) plasmids that successfully silenced mutant tau or APP alleles. These plasmids should prove useful in experimental and therapeutic studies of Alzheimer's disease. Our results suggest guiding principles for the production of allele-specific siRNA, and the general method described here should facilitate the production of gene-specific siRNAs.


Subject(s)
Alleles , Alzheimer Disease/genetics , Mutation/genetics , RNA Interference , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Amyloid beta-Protein Precursor/genetics , Animals , Base Sequence , COS Cells , DNA-Directed RNA Polymerases/metabolism , Genes, Reporter/genetics , HeLa Cells , Humans , Substrate Specificity , Viral Proteins , tau Proteins/genetics
9.
Proc Natl Acad Sci U S A ; 100(12): 7195-200, 2003 Jun 10.
Article in English | MEDLINE | ID: mdl-12782788

ABSTRACT

Small interfering RNA (siRNA) holds therapeutic promise for silencing dominantly acting disease genes, particularly if mutant alleles can be targeted selectively. In mammalian cell models we demonstrate that allele-specific silencing of disease genes with siRNA can be achieved by targeting either a linked single-nucleotide polymorphism (SNP) or the disease mutation directly. For a polyglutamine neurodegenerative disorder in which we first determined that selective targeting of the disease-causing CAG repeat is not possible, we took advantage of an associated SNP to generate siRNA that exclusively silenced the mutant Machado-Joseph disease/spinocerebellar ataxia type 3 allele while sparing expression of the WT allele. Allele-specific suppression was accomplished with all three approaches currently used to deliver siRNA: in vitro-synthesized duplexes as well as plasmid and viral expression of short hairpin RNA. We further optimized siRNA to specifically target a missense Tau mutation, V337M, that causes frontotemporal dementia. These studies establish that siRNA can be engineered to silence disease genes differing by a single nucleotide and highlight a key role for SNPs in extending the utility of siRNA in dominantly inherited disorders.


Subject(s)
Gene Silencing , Genetic Diseases, Inborn/genetics , Genetic Diseases, Inborn/therapy , RNA, Small Interfering/genetics , RNA, Small Interfering/pharmacology , Alleles , Animals , Base Sequence , COS Cells , Dementia/genetics , Dementia/therapy , Genes, Dominant , Genetic Therapy , HeLa Cells , Humans , Machado-Joseph Disease/genetics , Machado-Joseph Disease/therapy , Peptides/genetics , Point Mutation , Polymorphism, Single Nucleotide , tau Proteins/genetics
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