Table 2.

In vivo mitophagy reporters and mitochondrial dysfunction-induced neurodegeneration models

In vivo model	Promoter and expression pattern	Model description	Characteristics and phenotype	References
Mitophagy Reporter Models
mt-Keima	Hip11 locus (ubiquitous expression) CAG-promoter driven expression	Mitophagy reporter Not amenable to chemical fixation	Mitophagy phenotype [whole body]: considerable heterogeneity in mitophagy within the same tissue. Low levels of mitophagy in the thymus, high in the heart. Mitophagy phenotype [brain]: high anatomic variation. Cortex, striatum, and substantia nigra exhibit modest levels of basal mitophagy. Mitophagy greater in dentate gyrus, lateral ventricle, and Purkinje cell layer within the cerebellum. Reduced mitophagy in dentate gyrus of aged mice (3 versus 21 months; 70% reduction). Pathological insult: age-related decline in mitophagy in dentate gyrus. Expression of mutant human Huntingtin’s transgene reduced mitophagy in dentate gyrus. Low oxygen (10% oxygen) significantly increased hepatic mitophagy.	(105)
Mito-QC (mCherry-GFP- FIS1(aa.101–152)	Rosa26 locus (ubiquitous expression) CAG-promoter driven expression	Mitophagy reporter Amenable to chemical fixation	Mitophagy phenotype [whole body]: considerable heterogeneity of mitophagy within the same tissue. High levels within cortex of adult kidney, differential mitophagy between proximal (high mitophagy) and distal (low mitophagy) convoluted tubules within kidney. Mitophagy phenotype [brain]: pronounced mito-lysosomes within Purkinje cell layer. Significant mitochondrial turnover in the Purkinje somata. Pathological insult: no change in mitophagy in any tissue analyzed with Pink1 knock-out.	(154, 155)
mt-SRAI-CL1-PEST	AAV- expression into right SNc	Mitophagy reporter Amenable to chemical fixation	Mitophagy phenotype [Brain]: numerous infected neurons positive for mitophagy signal in 6-OHDA-injected mice (same route as for viral infection); mitophagy signal in TH-negative (non-DA) neurons only.	(108)
Genetic Neurodegenerative Models of Mitochondrial Origin
Disruption of mtDNA Homeostasis
Mutator (POLγAD257A)	PolγA locus; ubiquitous expression	Homozygous knock-in mutant of PolγA (nucleus-encoded catalytic subunit of mtDNA polymerase) D257 A mutation causes loss of 3′-5′ exonuclease activity necessary for proof-reading newly synthesized mtDNA	Aging phenotype: decreased lifespan and premature onset of age-associated phenotypes (weight loss, reduced subcutaneous fat, alopecia, kyphosis, osteoporosis, anemia, reduced fertility, and cardiac hypertrophy). Neuronal phenotype: No neurodegeneration up to 12 months. Intact nigralstriatal pathway, no astrogliosis. POLγAD257A (Mutator); Parkin−/−: large reduction in TH-positive (DA) neurons in midbrain. Reduced striatal dopamine, decreased DA metabolites. L-DOPA responsive motor phenotype. No neuroinflammation or Lewy body formation. Mitochondrial phenotype: 3-5x increase in mtDNA point mutations, increased mtDNA deletions. Reduced mtDNA copy number. Random point mutations in genes for respiratory chain subunits. Increased apoptosis. Little age-related decline in cardiac mitochondrial fitness. Increased megamitochondria in aged hearts (6 months). Mitophagy phenotype: Increased phospho-Ser65-ubiquitin in cortex (not liver), increased hepatic mitophagy (POLγAD257A; mt-Keima), reduced Parkin protein expression. POLγAD257A (Mutator); Parkin−/−mice: strong inflammatory phenotype (high serum IL-6, IFNβ1, TNF, IL-1β, CCL2, IL-12(p70), IL-13, IL-17, CXCL1 and CCL4). No change in mtDNA mutation frequency compared with POLγAD257A; Parkin+/+, but reduced mtDNA pathogenicity. Reduced ETC complex activity (complex I and III). POLγAD257A (Mutator); Parkin-Tg and POLγAD257A (Mutator); Parkin−/−: Parkin fails to prevent accelerated cardiac aging.	(105, 156, 159, 160, 161, 162, 234)
mitoPARK (DAT-cre x TfamloxP)	DAT promoter: DA neuron expression Homozygous deletion of mitochondrial transcription factor A (Tfam)	TFAM knockout in midbrain DA neurons TFAM knockout leads to mtDNA depletion and abolishes mtDNA expression.	Neuronal phenotype: adult onset of slowly progressive motor impairment, loss of TH-positive neurons and TH-positive terminals in striatum; depletion of nigral and striatal dopamine, age-dependent reduction in soma size and neurite branching in DA neurons. Loss of dopamine in olfactory bulb, intraneuronal inclusions, cognitive dysfunction (preceding motor dysfunction). Gastrointestinal dysfunction, gut inflammation, and gut-microbiome changes. Age-dependent L-DOPA responsive motor phenotype. Mitochondrial phenotype: Severe respiratory chain deficiency, reduced cytochrome oxidase subunit I expression and activity in midbrain DA neurons, fragmentation, large mitochondrial aggregates. Reduced distal axonal mitochondria [dysfunctional axonal mitochondrial transport]. Mitophagy phenotype: Endogenous Parkin recruitment not detected [potential technical limitations or low expression]. TfamloxP/loxP; DAT-cre; AAV-Parkin-mCherry: no Parkin colocalization with mitochondria. TfamloxP/loxP; DAT-cre; Parkin−/−: no Parkin-dependent effect on mitochondrial aggregates, mitochondrial morphology, locomotion, or TH-positive cell loss in SNc.	(163, 164, 166, 235, 236)
PD-mito-PstI	DAT promoter-driven tetracycline transactivator protein (tTA) Inducible mito-PstI exclusively in DA neurons	Expression of tetracycline-sensitive mitochondria-targeted restriction enzyme, PstI, in DA neurons Mitochondrial matrix localization—COX8A MTS Mito-targeted restriction enzyme damages mtDNA in DA neurons	Neuronal phenotype: progressive degeneration of the DA population within SNc, striatal dopamine depletion, age-dependent loss of TH-positive neurons, L-DOPA reversible motor deficit. Locomotor deficits precede TH-positive cell loss. Absence of inclusions. Motor phenotypes initially arise from a striatal dysfunction. Mitochondrial phenotype: double strand breaks in mtDNA, mtDNA depletion, mtDNA deletions, ETC dysfunction. Mitophagy phenotype: PD-mito-PstI; Parkin−/−: mild acceleration, but no worsening of motor dysfunction and neuronal degeneration.	(167, 168)
Twinkle-duplication (Twinkle-Tg)	Transgenic expression of Twinkle (in-frame duplication of aa. 353–365) TH promoter: DA neuron expression 4x Twinkle [mRNA] increase in Twinkle-Tg	In-frame duplication of the mitochondrial DNA helicase, Twinkle Disruption of mtDNA replication	Neuronal phenotype: motor impairment, decreased TH-positive neurons, age-dependent neurobehavioral deficits. Mitochondrial phenotype: Age-dependent increase in mtDNA deletions, reduced mtDNA copy number, mild bioenergetic defects. Mitophagy phenotype: reduced Parkin protein expression, increased LC3 protein expression. Twinkledup/+; Parkin−/− (TwinkPark): increased mtDNA deletions, reduced mitochondrial function (complex II activity) and compromised bioenergetics. Reduced membrane potential, neurobehavioral deficits, reduced striatal dopamine and increased TH-positive cell loss by 19 months.	(169, 170)
Disruption of Key Mitochondrial Processes
ΔOTC	TH-Cre; ΔOTC Cre-mediated recombination in DA neurons	Exogenous expression of ΔOTC proposed to induce mitochondrial unfolded protein response (mtUPR) in vivo Ornithine transcarbamylase enzyme transgene (OTC normally restricted to liver) – Deletion mutant Δ30–114 Mitochondrially localized enzyme. Cre-recombination induces mitochondrial unfolded protein response in TH-positive neurons	Neuronal phenotype: mildly reduced motor function, reduced SNc dopamine content, decreased TH-positive neurons, L-DOPA responsive motor phenotype. Mitophagy phenotype: Pink1−/−versus Pink1−/−; ΔOTC: reduced DA neurons or reduction in DA content following ΔOTC expression [additive effects of PINK1 loss unknown—no comparison of ΔOTC alone versus Pink1−/−; ΔOTC], no L-DOPA responsive motor phenotype.	(171)
Ndufs4−/−	Mox2-cre: Ndufs4LoxP: Ubiquitous expression DAT-cre; Ndufs4LoxP: DA neuron expression	Conditional knockout of ETC complex I subunit, NDUFS4 Mitochondrial ETC complex I deficiency (activity and expression)	Neuronal phenotype: Ndufs4−/−: TH-positive cell loss, motor deficits, reduced striatal dopamine. Decreased 20S proteasome activity in SNc, decreased neurofilaments in SNc, increased ubiquitinated protein levels in DA neurons in SNc. DAT-cre; Ndufs4loxP: no motor deficits, slight decrease in TH-positive neurons at 24 months, no loss of DA nerve terminals, no overt neurodegeneration. Slight decrease in dopamine content and alterations to dopamine homeostasis in striatum. Reduced dopamine release. [Conflicting data around TH-positive cell loss, motor deficits, reduced striatal dopamine]. Mitochondrial phenotype: reduced complex I expression and activity. Mitophagy phenotype: small reduction in PINK1 expression, no change in Parkin expression	(237, 238, 239)

AAV, adeno-associated virus; CFP, cyan fluorescent protein; DA, dopaminergic; DAT, dopamine transporter; FRET, Förster resonance energy transfer; GFP, green fluorescent protein; mtDNA, mitochondrial DNA; MTS, mitochondrial targeting sequence; mtUPR, mitochondrial unfolded protein response; OTC, ornithine transcarbamylase; SNc, substantia nigra pars compacta; Tg, transgenic; TH, tyrosine hydroxylase; TOLLES, TOLerance of Lysosomal EnvironmentS; VTA, ventral tegmental area.