Table 2 Involvement of Tau, Aβ, APP-CTFs, and AD risk factors in mitophagy failure in vitro and in vivo.

From: Mitophagy in Alzheimer’s disease: Molecular defects and therapeutic approaches

	Models	Experimental approaches/methods	Molecular mechanisms	Expected/reported phenotypes	Refs.
Tau species
NH₂hTau	I^ary hippocampal neurons from rats	RT-PCR	↓ mitDNA (mtND2)/gDNA (Htert) ratio	↑ Mitochondrial degradation	[29]
		WB	↓ TOMM20, VDAC1, TIMM23, CytC, ATPB & Mn-SODII proteins	↑ Mitochondrial degradation
			↑ LC3-II protein	↑ Phagophores number
			↑ PINK1 protein & ↑ Parkin protein in mitochondria	↑ PINK1/Parkin activation
			↓ p62 protein	↑ Lysosomal degradation
	Tg2576 mice (6 months)	WB (Mit fraction from purified synaptosomes)	↑ Parkin protein	↑ Parkin recruitment to mitochondria	[30]
hTau	HEK293T cells	WB	↑ TOMM20 & COX IV proteins	↑ Mitochondrial mass	[11]
		RT-PCR	↑ mitDNA (Atp-6)/gDNA (RpI13) ratio	↑ Mitochondrial mass
		WB (Mit fraction)	↓ PINK1 & Parkin proteins ↑ p62 protein	↓ PINK1/Parkin activation ↓ Lysosomal degradation
	Mice I^ary hippo neurons	WB	↑ LC3-II protein	↑ Phagophores number
hTau (P301L)	N2a cells	IF (Parkin transfection ± CCCP) (Mit fraction)	↓ Parkin protein	↓ Parkin activation	[31]
		Co-IP & PLA	↑ hTau (P301L)-Parkin interaction
		IF (Parkin transfection ± CCCP)	↓ Mitochondria ubiquitination	↓ Mitochondria tagged from degradation
	C. elegans (CK12)	IHC & crossing with mito-Rosella worms	↓ Mitochondria degradation in mitolysosomes	↓ Lysosomal degradation
hTau F3ΔK280	C. elegans (BR5270)	IHC ± PQT	↓ LC3-BNIP3/NIX colocalization	↓ Phagophores recruitment	[16]
hTau T231E & K274/281Q	C. elegans	IHC ± PQT	↓ Mitolysosomes number	↓ Mitolysosomes formation	[32]
Aβ species
Aβ_1-42	PC12 cells (5 µM, 24 h)	WB & RT-qPCR	↑ Bcl-1 & ↑ Parkin mRNA & protein	↑ Mitophagy initiation	[34]
	PC12 cells (5 µM, 24 h)	WB	↑ LC3 protein & LC3-II/I ratio	↑ Phagophores number	[34]
	PC12 cells (7 µM, 12 h)	WB	↓ Bcl-1 protein	↓ Mitophagy initiation	[33]
			↓ PINK1 & Parkin proteins	↓ PINK1/Parkin activation
			↓ LC3-II protein	↓ Phagophores number
			↑ p62 protein	↓ Lysosomal degradation
	C. elegans (CL2355)	IHC ± PQT	↓ LC3-BNIP3/NIX colocalization	↓ Phagophores recruitment	[16]
	CA1 injected rats	IHC & WB	↓ Bcl-1 protein	↓ Mitophagy initiation	[37]
			↓ PINK1 & Parkin proteins	↓ PINK1/Parkin activation
			↑ p62 protein	↓ Lysosomal degradation
	Mouse I^ary hippo neurons (5 µM)	WB	↑ Bcl-1 protein	↑ Mitophagy initiation	[35]
			↑ LC3-II protein	↑ Phagophores number
			↑ PINK1 & Parkin proteins ↑ Parkin protein in mitochondria	↑ PINK1/Parkin activation
		IF & mRFP-GFP-LC3 probe	↓ Mitolysosomes number	↓ Mitophagy
oAβ_1-42	HEK293T cells (10 µM)	WB	↑ mitochondria number	Mitochondria accumulation	[36]
		WB (Mit fraction)	↑ Parkin protein	Parkin activation
		WB (Mit fraction)	↑ LC3-II protein	↑ Phagophores recruitment
		TEM	↑ Mitophagosomes number	Mitophagosomes accumulation
		WB (Mit fraction)	↑ p62 protein	↓ Lysosomal degradation
mAβ_1-42	SK-N-BE cells (1 µM)	WB	↑ Bcl-1 protein	↓ Mitophagy initiation	[39]
		Co-IP	↓ Bcl-1 & Bcl-2 protein complex	↓ Mitophagy initiation
		WB	↑ LC3-II protein	↑ Phagophores number
		IF & GFP-RFP-LC3 probe	↑ Autophagosomes number ↑ Autolysosomes number	Autophagosomes accumulation
		WB	↑ p62 protein	Autophagosomes accumulation
		Enzymatic assay	↓ CTSD activity	↓ Lysosomal degradation
APP-CTFs
AICD	HEK293T	WB & RT-qPCR	↑ PINK1 mRNA & protein	↑ PINK1/Parkin activation	[41]
	SH-SY5Y	WB	↑ LC3-II protein	↑ Phagophores number
	SH-SY5Y	WB	↓ P62, TOMM20 & TIMM23 proteins	↑ Lysosomal degradation
C99 and C83	SH-SY5Y APPswe ± γ-sec inhibitor	WB	↑ LC3-II protein	↑ Phagophores number	[45]
		WB	↓ Mature CTSB protein	↓ Lysosomal degradation
		Enzymatic assay	↓ CTSB activity	↓ Lysosomal degradation
	3xTgAD mice ± γ-sec inhibitor	WB	↑ LC3-II protein	↑ Phagophores number
		TEM	↑ Autophagic vesicles number	↑ Phagophores number
		WB	↑ p62 protein	↓ Lysosomal degradation
	AAV-C99 mice	IHC	↑ LAMP1 puncta number & size	Lysosomal failure
	AAV-C99 mice ± γ-sec inhibitor	WB	↑ LC3-II protein	↑ Phagophores number
	AAV-C99 mice ± γ-sec inhibitor	IHC	↑ CTSB puncta number & size	Lysosomal failure
	SH-SY5Y APPswe	WB (Mit fraction)	↑ PINK1 & Parkin proteins	↑ PINK1/Parkin activation	[18]
		WB (Mit fraction)	↑ LC3-II protein	↑ Phagophores number
		GFP- LC3 probe	LC3 puncta	↑ Phagophores number
		IF	↑ HSP60- LC3 colocalization	↑ Mitophagosomes number
		IF	↓ mitochondria-LAMP1 colocalization	↓ Mitolysosomes number
		WB (Mit fraction)	↔ p62 protein	↓ Lysosomal degradation
		WB (Mit fraction)	↑ TOMM20, TIMM23, HSP60 & HSP10 proteins	↓ Lysosomal degradation
	SH-SY5Y APPswe ± γ-sec inhibitor	TEM	↑ Mitochondria number with damaged cristae	Damaged mitochondria accumulation
		WB (Mit fraction)	↔ PINK1 & Parkin proteins	↓ PINK1/Parkin activation
		WB (Mit fraction)	↑ LC3-II/I protein ratio	↑ Phagophores number
		Cox8-EGFP-mCherry probe	↔ Cells number containing mitolysosomes	↔ Mitolysosomes number
	SH-SY5Y C99	WB (Mit fraction)	↔ PINK1 & Parkin proteins	↓ PINK1/Parkin activation
			↔ LC3-II/I protein ratio	↔ Phagophores number
			↑ p62, TOMM20 & HSP10 proteins	↓ Lysosomal degradation
	AAV-C99 mice	WB (Mit fraction)	↑ LC3-II protein	↑ Phagophores number
	AAV-C99 mice	WB (Mit fraction)	↔ p62, TOMM20 & HSP10 proteins	↓ Lysosomal degradation
	Young 3xTgAD mice ± γ-sec inhibitor	TEM	↑ Mitochondria with damaged cristae	Damaged mitochondria accumulation
		WB (Mit fraction)	↔ PINK1 protein ↑ LC3-II/I protein ratio	↓ PINK1/Parkin activation
			↔ p62 protein	↑ Phagophores number
			↑ TIMM23 & HSP10 proteins	↓ Lysosomal degradation
	Old 3xTgAD & 2xTgAD mice	TEM	↑ Mitochondria with damaged cristae	Damaged mitochondria accumulation
		WB (Mit fraction)	↑ PINK1 protein	↑ PINK1/Parkin activation
			↑ LC3-II/I protein ratio	↑ Phagophores number
			↓ p62 & TIMM23 proteins	↑ Lysosomal degradation
	iNSCs from FAD patient (PS1 C737A)-derived fibroblasts	WB ± Baf	↑ LC3-II protein & LC3-II/I ratio	↑ Phagophores recruitment	[57]
		WB ± Baf	↑ p62 protein	↓ Lysosomal degradation
		IF	↓ LC3 & LAMP1 colocalization	↓ Autolysosomes number
		WB ± γ-sec inhibitor	↑ LC3-II protein
			↑ PINK1 & Parkin proteins	↑ PINK1/Parkin activation
			↑ p62 protein
			↑ HSP60 protein
		IF ± γ-sec inhibitor	↓ MitoTracker & LAMP1 colocalization	↓ Lysosomal degradation
		IF ± γ-sec inhibitor		↓ Mitolysosomes number
	PS1/2 KO iNSCs	WB	↑ PINK1 & Parkin proteins	↑ PINK1/Parkin activation
			↑ LC3-II protein	↑ Phagophore recruitment
			↑ p62 protein	↓ Lysosomal degradation
		IF	↓ LC3 & LAMP1 colocalization	↓ Autolysosomes number
		IF	↑ HSP60 & p62, Parkin or Ubiquitin colocalization	↑ Mitophagy initiation
Risk factors
APOE4	T98G cells	Proteomic analysis	↑ TOMM5, ATP5B, CS & SDHA aggregation	Mitochondria accumulation	[58]
		DNA pulldown assay	Competition for TFEB CLEAR motif	↓ Expression of mitophagy actors
		RT-PCR	↓ MAP1LC3B, p62/SQSTM1 & LAMP2 transcription	↓ Expression of mitophagy actors
	Hippocampus from hAPOEε4/ε4 mice	IHC & WB	↑ TOMM40 & COX I proteins	Damaged mitochondria accumulation	[60]
		TEM	↑ Mitochondrial length ↓ Mitochondria cristae density	Damaged mitochondria accumulation
		WB	↓ PINK1₅₂ protein	↑ PINK1/Parkin activation
		IHC & WB	↑ Parkin protein	↑ PINK1/Parkin activation
	Astrocytes from hAPOEε4/ε4 mice	WB	↔ p62 protein ↑ TOMM40 & TOMM20 proteins	↓ Lysosomal degradation Mitochondria accumulation	[61]
		RT-qPCR & WB (Mit fraction)	↑ Parkin mRNA & protein in mitochondria	↑ PINK1/Parkin activation
		Co-IP	↑ Ubiquinated Parkin	↓ Parkin activity
		WB ± CQ (Mit fraction)	↓ LC3-II/I protein ratio	↓ Mitophagosomes number
		IF & Mito-GFP ± CCCP	↓ Mito-GFP and LC3 colocalization	↓ Mitophagosomes number
		TEM ± CCCP	↑ PINK1 & ↓ PINK1₅₂ protein	↓ Lysosomal degradation
		LC3-EGFP-mRFP probe ± CCCP	↓ Mitolysosomes number ↓ Autolysosomes number	↓ Lysosomal degradation
		WB ± CCCP±CQ	↓ LC3-II protein	↓ Lysosomal degradation
High cholesterol	SH-SY5Y cells treated with oAβ_1-42	IF	↑ CYC- LC3-II colocalization ↓ CYC-LAMP2 colocalization	↑ Mitophagosomes number ↓ Mitolysosomes number	[63]
	APP/PS1 mice, SREBF2 mice	Digital PCR	↑ mitDNA copy number	Damaged mitochondria accumulation
		WB (Mit fraction) Phos-Tag SDS-PAGE (Mit fraction)	↑ poly-Ubiquinated K63 ↑ PINK1 & Parkin proteins ↑ Phosphorylated PINK1	↑ PINK1/Parkin activation
		IHC	↓ OPTN translocation to mitochondria	↓ Phagophore recruitment

Phenotype modifications and/or levels of proteins are depicted as ↑ (increased), ↓ (decreased), or ↔ (unchanged) as compared to respective control models. ∆Ψm Mitochondrial membrane potential. CA3 Cornu ammonis, C. elegans Caenorhabditis elegans. oAβ Oligomeric β amyloid peptide, mAβ Monomeric β amyloid peptide, I^ary Primary, Hippo Hippocampal, mitDNA Mitochondrial DNA, gDNA Genomic DNA, CTSD Cathepsin D, CTSB Cathepsin B, TEM Transmission electron microscopy, WB Western blot, IF Immunofluorescence, IHC Immunohistochemistry, Co-IP Co-immunoprecipitation, PLA Proximity ligation assay, Mit fraction Mitochondrial fraction, CCCP Carbonyl cyanide m-chlorophenylhydrazone, PQT Paraquat, γ-sec γ-secretase, Mito-GFP GFP targeted to mitochondria, mRFP-GFP-LC3 and GFP-RFP-LC3 probes: two tandem fluorescent-tagged LC3 probe monitoring autophagic flux based on different pH stability of EGFP and mRFP, Cox8-EGFP-mCherry probe: a tandem fluorescent-tagged mitochondrial targeting sequence of inner membrane protein COX8 (cytochrome c oxidase subunit 8), 3xTgAD mice (APPswe, TauP301L & PS1 M146V), 2xTgAD mice (APPswe, TauP301L & PS1 WT), CQ Chloroquine.

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Table 2 Involvement of Tau, Aβ, APP-CTFs, and AD risk factors in mitophagy failure in vitro and in vivo.

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