Mitochondrial quality control and Parkinson's

Mitochondrial defects

the principal drivers/cause of Parkinson’s

Ex. defects in Ca2+ buffering

mitochondrial anatomy

Mitochondrial genome

~40 genes → synthesis, trafficking, etc. happens inside (prokaryotic characteristic)

*majority of genes are not mitochondrial

Inner mitochondrial membrane

oxidative phosphorylation → only mitochondria needs O2 to make ATP

Mitochondria (functions)

ATP synthesis, Ca2+ buffering, apoptosis, metabolite synthesis

Warburg effect

tumor cells can survive in the absence of oxidative phosphorylation (no O2) → survives off of 2 ATPs

Mitochondrial dynamics

mitochondria are not static → networks of fusing/dividing mitochondria (dynamically moving b/w 2 states)

• combine = fusion

• separation = fission

*divide like ancient bacteria

Why is dynamic behavior important?

maintaining dynamic shift b/w fusion and fission is critical for healthy/normal cells → if disrupted, it can cause a harmful disease like Parkinson’s

fusion → outer 1st, then inner

fission → both inner/outer changed simultaneously

*can’t start unless there is a cue (Drp1) from the cytoplasm

Fission proteins

Drp1, Fis1, MFF, MID49, MID51, DNM2

• regulated by Drp1 and Fis1 → compress/separate mitochondrial tubules

• OMM and IMM fission occurs simultaneously

Fusion proteins

MFN1, MFN2, OPA1

*regulators

Fis1

located in OMM and prevents fusion by inhibiting Mfn2/Opa1 (activity)

Drp1

in the cytoplasm and translocates to the mitochondria

MFN1 and MFN2

regulate fusion (OMM fusion)

Opa1

regulates inner membrane fusion

How are fission and fusion regulated?

tightly and dynamically (happening all the time)

What happens during fission?

low energy demand → uncoupled respiration

• reduced ATP synthesis

• mitochondrial degradation

*lazy (only work when they have to

What happens during fusion?

energy demands + stress → upregulation of metabolic competence

• repair of damaged mitochondria

*Ex. exercising, stress, starvation

Do fission and fusion always lead to the same consequences/results?

in certain contexts yes, but in others the opposite happens (context-specific)

What happens as mitochondria age?

they collect damage → need fission and fusion

*1/2 life = 14 days (life span ~1 month)

Mitochondrial quality control

damages need to be converted to healthy

• damage = fission (depolymerized) → removed

• healthy part fuses w/ other mitochondria to become healthy

Surveillance mechanisms

• mechanisms in healthy individuals → defects lead to Parkinson’s

1) fusion-mediated complementation

2) mitochondria-derived vesicles

3) mitophagy

What is the first line of defense against mild mitochondroal impairment?

fusion-mediated complementation and MDV

What happens when impairment increases?

damaged compartments are segregated from the mitochondria (fission) and undergo mitophagy

PINK1/Parkin-mediated mitophagy

mediate mitophagy in the face of mitochondrial depolarization (loss of membrane integrity → no repolarization)

• positive feedback loop → completion

Receptor-mediated mitophagy

induced by hypoxia/during erythropoiesis → connection of damaged mitochondria to autophagosome is built by mitochondrial receptors

• NIX, BNIP3, FUNC1, and BCL2L13 (interaction w/ LC3)

replicative fission and mitochondrial biogenesis

• old mitochondria → 2 (each make up new mitochondria)

asymmetric fission and mitochondrial renewal/removal

• new mitochondria will grow old → separate junk/degrade and keep the good parts → used to create new mitochondria

ER-mitochondria contact

sites mark the location where mitochondria will divide (fission) or merge (fusion)

*ER regulates both states

mitochondrial fusion (mechanism)

1. mediated by interaction of MFN1/2 on OMM

2. Opa1 interacts w/ cardiolipin on IMM @ ER-mito contact sites

*initiated by ER

mitochondrial fission (mechanism)

1. initial OMM constriction via ER protein INF2

2. mediated actin polymerization

3. IMM fission by MTFP1 (IMM protein)

4. DNM1L recruited to OMM adaptor proteins to complete final constriction/scission of mitochondria

5. mitophagy (mediated by ER)

*initiated by ER

What happens if ER-mito interaction is interupted?

get hyperfusion

What causes cell death?

too much Ca2+ in the cell (mitochondria is a buffer) → can activate all sorts of pathways

Mitochondrial proteins

SIRT5, NRF2, SLP2

• purpose - mitigate stress

*upregulated when the cell is under stress → accelerate fusion (hyperfusion)

SIRT5

removes acetyl, succinyl, and malonyl groups from lysine residues in proteins (inhibits Drp1)

NRF2

TF that regulates stress protein

SLP2

mitochondrial protein

Stress responses

mitigation → normal mitochondria

mild → hyperfusion

prolonged → hyperfission (fragmented mitochondria)

*will inhibit fission

Cellular stress response signaling

phosphorylates elF2a → stops all protein synthesis except proteins that help restore homeostasis

Cellular stress

drug induced, starvation, oxidative or proteostatic

What happens during Parkin-induced mitophagy?

1) PINK1 phosphorylates ubiquitin → attracts double membrane → autophagosome

2) parkin recruited to mitochondria and activates UPS → degrade mitochondrial membrane proteins

3) parkin promotes recruitment of autophagy adaptors to damaged mitochondria (OPTN, NDP52)

4) mitochondrial Rab GTPase activating protein

What is the importance of parkin-induced mitophagy?

principally defective process

• specific indicator that mitochondria is damaged

• depolarization, DNA mutations, increased ROS, misfolded proteins (tau, amyloid beta, alpha-synuclein → Alzheimer’s, Parkinson’s)

What is the role of PINK1 in healthy mitochondria?

used and degraded by the ubiquitin pathway (tight regulation)

What is the role of PINK1 in damaged mitochondria?

more PINK1 tagged to the outer membrane → attaches Parkin → forms complex in damaged mitochondria → mitophagy (autophagy and proteosomal degradation)

What happens if PINK1 is damaged?

stabilize on damaged mitochondria → phosphorylate ubiquitin → inactive parkin transferred to the outer membrane → parkin activated

*PINK1 is ubiquitin kinase

Parkinson’s (symptoms)

• stooped posture

• back rigidity

• flexed elbows and wrists

• tremors in the legs

• shuffling, short steps (obvious sign)

• slightly flexed hips and knees

• hand tremor (uncontrolled movements)

• reduced arm swing

• forward tilt of trunk

• masked face

*motor functions affected most significantly (Ex. Michael J Fox)

Parkinson’s (non-motor skill symptoms)

• mental/behavioral issues (depression, anxiety, fatigue, personality changes, etc.)

• sense of smell

• sweating and melanoma

• GI issues (sexual, urinary, weight loss)

• pain

Parkinson’s (motor skill symptoms)

• vocal symptoms

• rigidity

• tremors

• walking difficulties

• dystonia (repetitive movements make body parts twist)

• bradykinesia (mask-like face)

Parkinson’s (RFs)

• toxins

• genetic factors

• pesticide exposure

• water pollutants

• air pollutants

• aging

• family history

• male

• chemicals

• hydrocarbon solvent exposure

*thought as disease of old age, lifetime exposure to these factors increase risk of diagnosis (exact mechanism unknown)

Rotenone

*pesticide → mitochondrial complex I inhibitor (activity decreased in many cases of idiopathic PD)

produces Parkinsonism in rats

What did the epidemiological and animal studies show?

associations w/ altered risk → farming (herbicides, pesticides), welding (Fe, Mg), well water (toxins), smoking, coffee, etc.

• if you could reproduce a diseased model w/ some RF, there is an association

*smokers had a lower risk of being diagnosed w/ Parkinson’s

Potential role of environmental factors (Parkinson’s)

1. MPTP metabolized to MPP+

2. taken up through dopamine transporter

3. affected mitochondria = mitochondrial defect

4. disrupts complex I of ETC

5. neuronal cell death (RF)

*took an analgesic and tested it in animals → caused Parkinson’s-like disease

chemicals linked to Parkinson’s → disruption of mitochondrial function

• treatment - dopamine agonists (bring levels close to physiological) → manage systems (no cure)

Positive association (Parkinson’s disease risk)

• medication

• coffee

• plasma urate levels

• exercise

• cigarettes

Negative association (Parkinson’s disease risk)

• heavy metals

• solvents

• TBI

• psychological state

• dairy consumption

• pesticides

• medication

Dopamine-producing neurons

cells primarily affected by mitochondrial disfunction (in basal ganglia → substantia nigra)

• produce dopamine → neurotransmitter for movement control (motor symptoms of disease)

*maximum loss of function

How do astrocytes relate to PD?

mitochondrial disfunction of supporting cells of brain also play a role in PD progression

*glial cells also affected

Familial PD

associated w/ several genetic mutations linked to mitochondrial disfunction

• PINK1 and Parkin → important for segregating damaged mitochondria and recreating healthy mitochondria

increased damage

decreased removal

What are key features of PD?

• dopamine neuron degeneration in midbrain

• lewy bodies (protein aggregates) in neurons

*protein misfolding

What are lewy bodies made of?

alpha-synuclein, ubiquitin, synphilin-1, neurofilaments, amyloid-beta, tau

*alpha-synuclein thought to be primary protein responsible; amyloid-beta and tau used to be unique to Alzheimers

What proteins regulate mitophagy?

LRRK2, PINK1/parkin, alpha-synuclein, DJ-1

Pattern of inheritance

can be autosomal dominant or recessive

*onset is important for cost of treatment (chronic/early = more important) → finding therapeutic targets to prevent initiation of disease

What are the consequences of mitochondrial disfunction in PD?

leads to progressive cellular disfunction = neurodegeneration

1. impairment of mitochondrial biogenesis

2. increased ROS

3. defective mitophagy

4. compromised trafficking

5. ETC disfunction

6. variations to mito dynamics

7. Ca2+ imbalance

*alone or in combination

Impaired biogenesis (mutations)

alpha-synuclein, CHCHD2, parkin, PINK1

Ca2+ imbalance (mutations)

alpha-synuclein, PINK1

Altered mitochondrial dynamics (mutations)

VPS35, CHCHD2, Parkin, PINK1, ATP13A2

ETC disfunction (mutations)

LRRK2, VPS35, CHCHD2, parkin, PINK1

Impaired mitochondrial trafficking

LRRK2, PINK1, parkin

Defective mitophagy (mutations)

parkin, PINK1, ATP13A2 (lysosomal protein)

Oxidative stress

alpha-synuclein, LRRK2, CHCHD2, VPS35, parkin, PINK1, ATP13A2

What happens in PD?

Ca2+ buffering is defected, metabolism is defected, and inflammation is increased

Astrocyte function

#s and functions most abundant in striatum/caudate putanem → where dopaminergic neurons is highest/loss of astrocytic function has most detrimental effect on PD

How is astrocyte disfunction treated?

reversal of mitochondrial dysfunction in the striatum → significant therapeutic benefit

GSDMD pores

protein-formed channels in cell membranes involved in inflammation and cell death

• release pro-inflammatory cytokines → release of IL-1B

Microglia

involved in PD

• skewed towards fission → activate inflammasomes (cause inflammatory disease)

• activate innate response and increase neuronal inflammation

Inflammasome

accumulation of proteins that form a super complex of proteins

multiple mito fission events in enterocytes from small bowel of DKO mice

• 2 genes (mitochondrial defects) involved in etiology of Chron’s → increased fission

abnormal mitochondria in autophagosomes (Avi) and autophagolysosomes (Avd) in SI of DKO mice

• double membrane shows formation of phagophore

• lipids aggregate inside of mitochondria

• isolation membrane forms around mitochondria → form Avi → fuses w/ lysosome (Avd)

*process of autophagy but it doesn’t get cleared out

DKO mice show abnormal mitochondria (M) and accumulation of lipofuscin (ceroid; L)

• lipid droplets (beta-oxidation disrupted) → mitochondrial pathway can’t clear out fat (mitophagy is disrupted)

• mitochondria are rounded = dividing more

*translucent

mitochondria undergoing autophagy

• seeing a lot of vacuoles → the cell has died

• phagophore (rER), autophagic vacuoles (A), lipofuscin (L)

What pathways coordinate metabolism and regulate lifespan?

AMPK, insulin/IGF signaling, mTOR signaling

*responses are highly context-dependent/not uniform (Ex. during exercise, increased energy needs → fission)

What happens during starvation?

• AMPK activated

• insulin/IGF, mTOR inhibited

favors fusion → longevity, insulin sensitivity, glucose tolerance

What happens during nutrient excess?

• activates insulin/IGF, mTOR

• AMPK repressed

favors fission → premature aging, cardiomyopathy, obesity, diabetes

Fusion and fission in cancer

regulates metastasis, migration, apoptosis, autophagy, proliferation, metabolism

• fission increases metastasis/proliferation in cancer cells

• decreased fission inhibits apoptosis

• increased fission increases OXPHOS

What are mitochondrial defects associated with?

Alzheimer’s, NAFLD, cardiac ischemia/myopathy, PD

*not unique to PD (fission/fusion)