Chapter 1 Flashcards
How prevalent is PD (worldwide and in Canada)? How has the incidence changed over time?
Worldwide: >6M as of 2016; predicted >9M as of 2020; projected >12M by 2040
2.5-fold increase in incidence (new cases) between 1990-2016
Canada: affects 1 in 500
CA has highest age-standardized worldwide prevalence of PD (2016)
When was PD first described, and what were its key features at the time?
1817, James Parkinson’s “An Essay on the Shaking Palsy” (6 case reports)
Resting tremor, flexed posture, festination (gait disturbance); progressive
What are the 3 “classical” motor symptoms of PD?
Bradykinesia (slowness of movement), resting tremor, and rigidity
List 3-4 motor and nonmotor symptoms associated with PD.
Motor: resting tremor, gait disturbance, bradykinesia, rigidity
Nonmotor: hyposomia (decreased sense of smell), constipation, RBD, depression, anxiety, cognitive decline
Describe how possible, probable, and definite PD are diagnosed, according to standard criteria such as UK Brain Bank, Gelb, and MDS. What are the major updates between 90’s and 2010’s criteria?
UK Brain Bank (1988):
“Parkinsonism” = Bradykinesia and at least one of rigidity, resting tremor, postural instability
“Parkinson’s disease” = 3 or more of unilateral onset, resting tremor, asymmetrical response to L-dopa, L-dopa-induced chorea (uncontrolled movement), L-dopa response for 5+ years, progression for 10+ years
Calne (1992): “Clinically possible,” “clinically probable,” and “clinically definite” PD
Gelb (1999):
“Possible” = Resting tremor or bradykinesia, and either rigidity or asymmetric onset (duration <3 years); response to L-dopa or dopamine agonist
“Probable” = 3 of resting tremor, bradykinesia, rigidity, or asymmetric onset (duration >3 years); response to L-dopa or dopamine agonist
“Definite”: Upon autopsy, neuronal/glial death in SN, LBs in SN or locus ceruleus (noradrenergic nucleus)
MDS (2015):
“Parkinsonism” = bradykinesia with resting tremor and/or rigidity
“Clinically probable” = Lack of exclusion criteria; “red flags” can be counterbalanced
“Clinically established” = Lack of exclusion criteria and “red flags”; >2 supportive criteria (response to DA treatment, unilateral resting tremor, olfactory/cardiac/imaging test result)
Give examples of research scenarios where better sensitivity or specificity would be desired in diagnosing PD.
Sensitivity (catching all possible cases): research into etiology, particularly early-stage
Specificity (correct diagnosis of PD): clinical trials, patient treatment
Describe typical PD progression using Hoehn and Yahr stages.
Stage I (year 0-3): Unilateral impairment, most of function retained
Stage II (year 3-6): Bilaterial/midline impairment, balance retained
Stage III (year 6-7): Balance impairment begins (loss of righting reflex). Mild-moderate disability.
Stage IV (years 7-9): Disabling and severe PD
Stage V (years 9-14): Confined to bed/wheelchair
Describe typical PD progression through the prodromal, early, and late stages of PD.
Prodromal: Pathology in brainstem, olfactory bulb, and peripheral NS; nonmotor symptoms (RBD, constipation, depression/anxiety, urinary dysfunction)
Early: Tremor, mild slowness; some memory problems
Manifest: Falls, sometimes dementia, L-dopa side effects
List 4-5 clincial features used to classify subtypes of PD.
Age of onset, rate of progression, motor/nonmotor symptoms, response to L-dopa
What are some problems associated with clinical subtyping in PD, and why is the field searching for more biological subtyping?
Symptoms can change over time, so individuals may resemble a different clinical subtype than they did before. Clinical subtypes also have no difference in pathology. Genetic subtypes are being identified now…more consistent, may be able to define mechanisms/treatments
Name 2 hallmark characteristics of PD pathology.
LBs in brainstem, DAn loss in SN
What is Braak staging/how does aSyn pathology progress over the course of PD?
Lewy bodies begin in olfactory bulb and move to brainstem, limbic system, then neocortex
How do “brain-first” and “body-first” PD differ? Describe early indicators, pathological progression, post mortem pathology, and supporting evidence.
“Brain-first”: classic PD pathology of LBs in brainstem and DAn loss in SN. aSyn pathology progresses according to Braak staging. RBD is seen after onset of motor symptoms.
“Body-first”: LBs begin in PNS/ENS, travel through vagus nerve to brain. RBD is seen prior to onset of motor symptoms. Evidence: protective vagotomy; animal studies showing gut-brain spread of aSyn; LBs in GI nerves prior to PD diagnosis
How does PD medication work, and what are some medications commonly used to treat PD?
Replaces DA lost from striatum.
Early stage: L-dopa (supply DA), DA agonist (activate DAR), COMT (increases DA bioavailability), MAO-B inhibitors (prevent mitochondrial breakdown of DA)
When was a genetic connection to PD first discovered?
In 1997, A53T aSyn mutation (autosomal dominant, 85% penetrant) discovered in Greek and Italian families
List 7 genes where mutations are strongly implicated in familial PD.
SNCA, LRRK2, GBA, DJ-1, VPS35, PRKN, PINK1
What approaches were/are used to identify rare variants associated with PD?
Linkage studies, genome sequencing, exome sequencing
What are some of the main challenges associated with identifying and replicating genes linked to familial PD?
Rare mutations, globally dispersed, high cost of sequencing
Which 4 genes are implicated in PD through both rare variants/monogenic disease and common variants/sporadic disease?
SNCA, LRRK2, GBA, VPS13C
Compare and contrast the symptoms and onset of monogenic and sporadic PD.
Monogenic: highly penetrant, earlier onset (30s to 50s), atypical symptoms.
Sporadic: later onset (60s to 80s), slower progression, typical symptoms/disease course (for instance, cognitive decline is more rare and observed late stage)
List 4 ways in which SNCA variants are implicated in PD.
Rare point mutations in coding regions of the gene, variations in REP1 dinucleotide repeat length, SNPs, whole gene duplication/triplication
Name 5 genes whose SNPs associated with sporadic PD risk in the first GWAS analyses conducted in 2009.
SNCA, MAPT, PARK16, LRRK2, BST1
Summarize the results of the largest PD GWAS to date (2023). How many loci were implicated and what % of PD heritability do they explain?
2019 meta-analysis of 17 European ancestry PD GWAS. 90 loci implicated (including SNCA, GBA, LRRK2), explaining 22% of PD heritability. Enriched for lysosomal genes, genes expressed in brain; SNPs also associated with brain volume, educational attainment, and smoking.
What are 4 major biological pathways implicated in genetic susceptibility for PD?
Lysosome, endosome, immune, mitochondrial
Name 4 reasons why there is thought to be “missing heritability” in sporadic PD not captured by current GWAS approaches.
- Lack of power to detect rare variants with small effect sizes in GWAS
- Limited ability of microarray platform and imputation to capture structural variants
- GxG, GxE interaction effects may be present
- Epigenetic contribution to heritability not fully understood
Describe two ways common genetic variation can be used to estimate sporadic PD risk.
- Polygenic risk score for PD, based on 90 Nalls loci (top decile has 6-fold increased PD risk)
- PD composite score based on genotype, asomia (loss of smell), age, sex, family history (90% specificity, 80% sensitivity)
Besides risk of developing PD, what other two aspects of PD can be estimated by GWAS?
Age of onset, progression (early stage, limited data so far)
Name 3 strategies for inferring causality from PD GWAS.
- Mendelian randomization (estimates the probability that an exposure/trait has a causal relationship with PD phenotype due to influence of genetic variation on the exposure/trait)
- Colocalization (estimates probability that two SNPs/traits affect PD at all, at the same locus, or at different loci)
- Linkage score disequilibrium regression (tests shared genetic etiology between traits; for instance, PD and smoking, education, brain volume in Nalls 2019)
How can understanding the genetic etiology of PD inform precision medicine approaches? Provide two real-life examples.
Personalized targeting of the pathway that lead to PD in an individual. Examples: Increasing GCase production in GBA-mutant individuals, reducing SNCA expression in SNCA mutant individuals.
Name some limitations of our current understanding of the genetic architecture of PD. What knowledge gaps still exist?
Genetic diversity beyond European-ancestry populations
GxG, GxE interactions
Epigenetic-genetic interactions
Rare/structural variants
List 4 risk factors and 4 protective factors for PD.
Risk factors: pesticide exposure, TBI, dairy intake, melanoma
Protective factors: smoking, caffeine, physical activity, ibuprofen
Name 5 limitations/common pitfalls in studying and interpreting the epidemiology of PD. What are the limitations specifically with respect to studies of PD and pesticide exposure?
General epidemiological limitations:
1. Applies to only sPD
2. Uncertainty in diagnosis of early/mid-stage PD
3. Recall bias
4. “Normative” control populations?
5. Accuracy and completeness in measurement of exposures and other confounders
Pesticide exposure: Inconsistent timing, duration, and dose of exposures; self-reported rather than quantitative data
How was a link between pesticide exposure and PD first established?
In 1983, heroin users of a MPTP-contaminated supply developed parkinsonism. Similar structure to paraquat herbicide. Studies in the 1980s-2000s revealed increased PD prevalence in communities with pesticide and heavy metal-contaminated well water.
What are the mechanisms of action for MPTP, paraquat, and rotenone in causing parkinsonism?
All are mitochondrial toxins, impairing activity of mitochondrial complex I (first enzyme in respiratory chain).
MPTP: immediate cell loss in SN & sustained loss over time, without LBs (rodent & monkey studies)
paraquat: SN cell loss, microglial reactivity, upregulation of aSyn, Ub/protesome dysfunction, with LBs (rodent intraperitoneal injection)
rotenone: SN cell loss, upregulation of aSyn, Ub/protesome dysfunction, with LBs
How does organochlorine insecticide exposure relate to PD pathology?
Ochl concentration in postmortem brain correlates with LB pathology in caudate/putamen, and amount of DA
List 2-3 examples of gene-pesticide interactions in PD risk.
LRRK2 G2019S influences paraquat inflammatory response/microglial reactivity in mice
GSTP1 genotype and herbicide exposure interact to modify PD age of onset
ABCB1 genotype and Ochl exposure interact to modify PD risk
Describe how genetics and exposure influence PD risk and progression through the life course.
Baseline PD risk due to genetics.
Exposure to pollutants/toxicants in early or mid-life may initiate aSyn accumulation in olfactory system or GI tract, with response to exposure depending on genotype.
Further exposures and aging may exacerbate PD progression.
Summarize the hypothesized contributions of early life exposures, injuries, and/or infections to developing PD later in life.
“Two-hit” hypothesis: one exposure or injury early in life may increase inflammation, while a second exposure, injury, or effects of aging might exacerbate this and initiate neurodegeneration
What are the mechanisms of action for smoking and coffee drinking in protecting against PD?
Smoking activates nicotinic ACh receptors, which stimulate DA production and Ca2+ release, have antioxidant activity, and enhance neuronal excitability.
Coffee activates D2 receptors in striatum and blocks A2A receptors. May interact with genotype and estrogen (stronger protective effect dependent on genotype and in males). Also increases SCFA production in gut.
What clues can be used to ascertain whether PD occurring in families is driven more by genetics or environment/exposures
If genetically driven, age of onset will be earlier and similar across generations. If environmentally driven, time of onset cross-sectionally will be more similar within a generation, not across generations.
Describe 3-4 mechanisms behind brain injury and risk for PD.
Breakdown of BBB, increase in microglial activation/release of inflammatory cytokines, resulting in immediate and sustained cell loss. Mitochondrial disruption, increased glutamate release
Is alcohol consumption a risk factor or protective factor for PD?
Mild- to moderate-drinking is protective for PD because of increased urate with alcohol consumption (natural antioxidant, byproduct of purine metabolism). Heavy drinking increases risk for PD.
Which substances/behaviours act on PD risk by modifying urate levels?
Alcohol, fructose consumption, and exercise increase urate levels; dairy consumption decreases urate levels
Describe the evidence for the protective role of exercise in PD and the mechanisms of action.
In prospective studies, playing sports in college or level of daily physical activity in adulthood is associated with reduced PD risk. Exercise can also ameliorate motor symptoms in individuals with PD, and in mice, treadmill exercise reduces cortical aSyn aggregation.
Mechanisms: BDNF release, DA release, synaptic plasticity, stabilized antioxidant response, increased urate, upregulation of PGC1a (energy metabolism regulator)
How is “epigenetics” defined, and how has this definition changed over time (1950s-present)?
1957: Waddington’s “epigenetic landscape” (effect of gene regulation on phenotype during development and differentiation)
1996: Riggs, “mitotically heritable” factors other than DNA sequence
2007: Bird, “structural adaptation” of chromosomal regions to register or perpetuate altered activity states
2017: Greally, “cellular reprogramming”
2019: NIH Epigenomics Roadmap, “heritable changes in gene activity and expression, and stable alterations in transcriptional potential”
List 5-10 examples of epigenetic marks.
DNAm, DNAhm, miRNA, lncRNA, histone variants, histone acetylation, histone methylation, histone Ub/SUMOylation, RNA modifications, adenine methylation…
How are epigenetic states (DNAm, DNAhm, histone PTMs) inherited mitotically and/or meiotically? What is the evidence for inheritance across generations?
Mitotic inheritance:
DNAm - DNMT1 recognizes hemi-methylated DNA and copies DNAm from parent strand to daughter strand.
DNAhm - Some mC is oxidized to hmC during S-phase (perhaps those not bound by MeCp2), so overall hmC level is maintained through mitotic divisions
Histone PTMs - repressive PTMs are mitotically inherited (parental histones recruit HMTs), while active PTMs are re-established after mitosis
Meiotic inheritance:
DNAm - all except imprinted DNAm is erased during gamete formation, again after fertilization. In agouti mice, incomplete DNAm erasure at IAP retrotransposon results in meiotic transmission of agouti phenotype (yellow fur, obesity, diabetes). In humans, evidence is more intergenerational than transgenerational…
DNAhm - increases in zygote via TET3 action
Histone PTMs - histone methylation thought to be erased in zygote and re-established during development, similar to DNAm…in amoebas and worms, transcriptional states are inherited via HMTs
What is polycredodism and how is it relevant to studying epigenetics?
A series of cell fate decisions in response to perturbation, that create a population of different cell comprising a tissue (cell type heterogeneity/variation in cell type proportion)
What is biological embedding and how is it relevant to studying epigenetics? Provide an example.
Effect of life experience on biology, which can impact later life health and wellbeing. Example: licking and grooming maternal behaviour in rats alters GR promoter DNAm in hippocampus, leading to higher GR expression in adulthood and impacting stress response.
List 5 examples of major challenges in interpreting epigenome-wide association studies.
- Cohort selection (representative?)
- Statistical approaches to high-dimensional data analysis
- Accounting for technical artefacts
- Accounting for genetic variation and cell type proportion
- Reverse causation (transcription –> DNAm)
Where is the methyl group in 5mC found?
5th carbon in pyrimidine ring of cytosine
How many CpGs/CpHs are in the human genome, and how many are methylated?
~5% of cytosines are CpG (28M), 80% methylated
~95% of cytosines are CpH (~530M), 2-6% methylated
What is a CpG island, and where are CGIs found (in general, and when methylated)?
> 500bp stretch of DNA with >50% CpGs. Evolutionarily conserved, found at gene promoters. TET is present to maintain low DNAm.
Methylated CGIs at imprinted genes, inactive Xchr, and genes with germ cell-specific expression.
How is DNAm deposited and removed?
DNMT1: Recognizes hemi-methylated DNA, deposits DNAm on daughter strand during replication.
DNMT3A/B: De novo DNAm, active during early development/after fertilization.
DNMT3L: enhances activity of other DNMTs (no catalytic domain)
TET: oxidation of 5mC –> 5hmC –> 5fC –> 5cac
TDG/BER: excision of 5fC/5cac and replacement w/C
Describe the function of DNAm depending on CpG density and its position in a gene.
CGIs/promoters: transcriptional repression (prevents transcription complex binding, recruits repressive proteins)
CpG shores: tissue-specific gene expression
CpG open seas/enhancers: tissue-specific/dynamic gene expression control (i.e., response to environment)
Gene bodies: may be initiated by transcription; splicing regulation; suppression of repeat transcription
What are the relative abundances of mCG, hmCG, mCH, and C in adult mouse cortex?
94% unmodified C
3% mCG
1% hmCG
1% mCH
5fC, 5cac are removed by DNA repair and are transient - low abundance…
Describe the tissue distribution of DNAhm.
Enriched in brain (17-30% of modC in adult mouse frontal cortex) and in ES cells
What are the functions of DNAhm generally and in the brain?
Keep promoters from accumulating mC (transcriptional activation); recruitment of splicing regulators to DNA; learning and memory, neuronal activity-related gene expression
How is DNAhm maintained during cell divisions?
mC –> hmC during S-phase (higher hmC on parental DNA strand)
How do DNAm and DNAhm change during development and aging?
Development: after fertilization, DNAm is erased and DNAhm deposited (TET3). After implantation, DNAm is re-established, forming tissue specific gene expression. Germline stem cells are passively/actively demethylated.
Aging: Passive/active loss of DNAm, gain of DNAhm (brain region-specific) due to reduced fidelity of DNA(h)m machinery. “Epigenetic drift”: stochastic or environment-related.
- Predictable change in DNAm w/aging = clocks
What is the function and distribution of CpH methylation?
Found in ES cells and neurons (50% of adult mouse and human neuronal modC). Thought to be transcriptionally repressive through MeCP2 recruitment; depleted in bodies, upstream, downstream of transcribed genes. Transcription induces H3K36me3, which prevents DNMT3A from binding and catalyzing mCpH at gene bodies.
Describe how histones and DNA are arranged in a nucleosome.
Nucleosome contains 146bp of DNA wrapped around a histone octamer, with 2 each of H2A, H2B, H3, H4 (2 H2A-H2B dimers and 1 H3-H4 tetramer). H1 is the “linker” histone between nucleosomes.
How many nucleosomes are in a typical cell, and how do they compress DNA?
Histones have a positive charge, attracting negatively charged DNA. Each cell has ~30M nucleosomes, packing 2m of DNA into 5-10uM diameter
What is the “histone code”, and why is it controversial? What are some functions of histone modifications beyond transcription regulation?
The “histone code” hypothesis postulates that transcription is regulated by a combination of different histone marks that are “read” by chromatin remodelers; the balance between recruiting and repelling other proteins and DNA tunes transcription.
Controversy: Reverse causation - histone modifications can be added during transcription or after transcription.
What types of post-translational modifications are deposited on histone tails, and what effects do these have on chromatin? List 3-5 examples.
Acetylation: negative charge, repels DNA
Methylation: promotes or represses transcription via recruitment of methyl-binding protein complexes
Ubiquitination: DNA replication, DNA repair, transcription regulation
Phosphorylation: DNA repair
SUMOylation: recruits HDACs (transcription regulation)
Describe the difference between euchromatin and heterochromatin, and give examples of histone marks found in each context.
Euchromatin: open, transcriptionally active. Marked by H3/H4ac, H3K4me1
Heterochromatin: closed, transcriptionally repressed. Marked by H3K27me3 (facultative), H3K9me3 (constitutive)
What are the six H3 modifications subject to profiling by IHEC, and where are they found?
H3K4me1 - enhancers and downstream of TSS
H3K4me3 - TSS of active/bivalent genes
H3K27me3 - TSS of repressed/bivalent genes
H3K36me3 - gene bodies of active genes
H3K27ac - enhancers and TSS of active genes
H3K9me3 - constitutive heterochromatin
How stable are histone modifications, and how are they inherited mitotically and/or meiotically?
Less stable than DNAm; more involved in dynamic transcription regulation.
During mitosis, repressive modifications are copied from parent strand to daughter strand (new histones) via HMT enzymes. Active modifications are not inherited mitotically, but re-established after mitosis via “seeding” of H4K16ac and recruitment of further HATs.
Most histone methylation is erased during fertilization/gametogenesis (histones are removed from sperm) and re-established in the same manner as DNAm.
What is a typical consequence of deregulation of histone methylase/demethylase enzymes?
Aberrant epigenetic/transcriptional profiles are associated with tumorigenesis and metastasis
