Neurodegenerative 3

Question 1

Classic signs of Parkinson’s

Answer 1

Bradykinesia
Rigidity
Resting tremor
Disturbed gait
Postural instability

Question 2

Clinical signs of Parkinson’s correlate with

Answer 2

Loss of dopamine containing neurons of the substantia nigra pars compacta

Question 3

His top anthology of Parkinson’s

Answer 3

Presence of intra-neuronal cytoplasmic aggregates called Lewy bodies

Over 90 proteins have
been identified in Lewy bodies, but misfolded α-synuclein appears to
provide the filamentous matrix of the inclusions and exhibits the
typical cross-beta sheet conformation of amyloids in other NDDs. The
other constituents with functional significance include numerous
components of the ubiquitin-proteasome system, chaperone proteins
(HSPs and crystallin) and mitochondrial proteins

Question 4

5-15% of Parkinson’s are attributed to …

Answer 4

Familial Parkinson’s disease FPD
The remainder are idiopathic

Question 5

Genetic factors and protein player’s in Parkinson’s

Answer 5

1) Autosomal dominant FPD is caused by mutations in the SNCA gene that codes for the protein α-synuclein, also mutations in the leucine-rich repeat kinase-2 (LRRK2, aka PARK8). LRRK2 is the most frequent genetic cause of PD, accounting for 4% of familial PD and 1% of sporadic PD across all populations.
2) Autosomal recessive FPD is notably associated with the genes encoding PINK1, Parkin and DJ-1 (PARK7 gene), although many more genes have been implicated in rare cases of PD.
3) Mutations in LRRK2 and GBA1, the gene coding for the lysosomal hydrolase glucocerebrosidase are the most common genetic risk factors for sporadic/idiopathic PD (iPD). Notably, both are involved in autophagy and lysosomal function.
4) The microtubule-associated protein Tau is associated with many different NDDs: Aggregation and deposition of hyperphosphorylated Tau has been identified in over 50% of PD brains at post-mortem.

Question 6

Describe alpha - synuclein in PD

Answer 6

A 140 AA protein encoded by SNCA gene
1st a=identified as the major non-amyloid component of amyloid plaques in AD - Abeta is likewise colocalisd with alpha-syn in Lewy bodies of PD suggesting common pathology
Assoc. with Parkinson’s in 97 by studies linking point mutations in SNCA gene to familial autosomal-dominant forms of PD
6 missense mutations in SNCA now associated with autosomal dominant PD
Certain AA substitutions result in earlier onset and increased speed of disease progression
Disease related mutations affect aggregation dynamics

Question 7

Pre-aggregation oligomers of alpha syn sho

Answer 7

Greater cellular toxicity suggesting that Lewy body’s may act as protective aggressomes
Oligomers bind to lipids thereby increasing membrane permeability in mitochondria, lysosomes etc

Question 8

Dysfunction associated with alpha synuclein oligomers is related to …

Answer 8

the affinity of normal α-Synuclein for cell membranes. α-
Synuclein is highly enriched in pre-synaptic membranes throughout the
CNS, consistent with it’s normal regulatory functions in endocytosis
and exocytosis, particularly the membrane fusion events of synaptic
vesicles mediated by the SNARE protein complex

Question 9

Mutations in the protease DJ-1 cause

Answer 9

Autosomal recessive FPD because the protein serves as a chaperone role that prevents aggregation of alpha synuclein

Question 10

Describe LRRK2 in PD

Answer 10

1) Most frequent genetic cause of PD, accounting for 4% of familial PD and 1% of
sporadic PD across all populations.
2) Late onset autosomal dominant with age-dependent incomplete penetrance, so
many carriers may never experience the consequences of the mutation during
their lifetime (Cookson 2015).
3) Identified in 2002 and linked to the PARK8 locus on chromosome-12
4) LRRK2 is a 286kDa multidomain protein with both kinase and GTPase domains,
but paradoxically the kinase activity of the protein appears independent of GTP
binding.
5) As it’s descriptive title, leucine-rich repeat kinase suggests LRRK2 appears to
exert its major effects through phosphorylation of a wide array of protein targets,
some notable examples of which the Rab family of small GTPases that regulate
membrane traffic and vesicle sorting in cells. This aspect of LRRK2 biology
implicates it in autophagy and lysosomal function, topics of special interest in the
pathogenesis of several NDDS, including PD.
6) Interestingly, the significant mutations of LRRK2 tend to increase the kinase
activity of the protein (Smith 2006) and experimental studies with inhibitors of
the kinase activity have proven neuroprotective in animal models, an effect that
appeared to be mediated by improved lysosomal function (Rocha 2020).

Question 11

Describe LRRK2 and endo lysosome dysfunction in PD

Answer 11

1) LRRK2 has been shown to phosphorylate 14 different Rab family small GTPases disrupting membrane traffic and vesicle sorting in neurons. This aspect of LRRK2 biology implicates it in autophagy and lysosomal function, and defective proteostasis in PD. This is also confirmed by studies showing that inhibition of the kinase stimulates macro-autophagy.
2) The significant mutations of LRRK2 tend to increase the kinase activity of the protein (Smith 2006) and
inhibitors of the kinase activity improve lysosomal function in animal models (Rocha 2020). Importantly, even normal LRRK2 shows increased activity in the substantia nigra of Parkinson’s patients and animals with PD models.
importance of the Rab family of small GTPases in vesicle traffic of the endo-lysosome system.
Rab GTPases act as molecular switches that control multiple events in vesicle traffic, such as targeting and membrane fusion. The multiple Rab family members imparts specificity to the different stages depicted.

Question 12

Describe LRRK2 and axonal transport in PD

Answer 12

1) Studies over the past 10 year have shown that LRRK2 binds to microtubules (MTs).
2) Parkinson’s mutations in LRRK2 increase its binding to MTs.
3) That LRRK2 can form filamentous structures has been known for 10 years, but
recent structural biology demonstrates that LRRK2 can form right-handed helices
around the MTs (see projection below from Watanabe et al 2020)
4) LRRK2 binding to MTs can act as a roadblock for kinesin and dynein MT- based
motor proteins, thereby inhibiting axonal transport.

Question 13

Describe the impact of PINK1/Parkin mutations in PD

Answer 13

PINK1 is a serine/threonine kinase that acts as a molecular sensor
of mitochondrial health, constantly surveying mitochondrial status
until it detects damage and signals for the recruitment and
activation of the E3 ubiquitin ligase Parkin.
2) Parkin/PARK2, is one of a large family of E3 ubiquitin ligases, and
mutations in this gene occurs in 50% of familial cases and 10–20%
in sporadic cases with high penetrance in early-onset PD
3) It has been shown that mitochondrial and lysosomal biogenesis
(renewal) are triggered by PINK1/Parkin mediated mitophagy
(mitochondria-triggered autophagy).
4) Therefore, failure of the PINK1/Parkin system due to mutation in
either protein will result in: 1) Accumulation of dysfunctional
mitochondria that persistently leak damaging reactive oxygen
species (ROS), and 2) Failure to generate new healthy mitochondria.

Question 14

Describe the role of PINK/Parkin in mitochondrial homeostasis

Answer 14

Healthy mitochondria import PINK1 where it is processed and ubiquitinated and subsequently
released for destruction by the proteasome. Damaged mitochondria cannot import PINK1, which
then accumulates on the surface of the outer mitochondrial membrane and recruits the ubiquitin-
ligase Parkin. The collaboration of PINK1 and Parkin rapidly coats the damaged mitochondrion with
special poly-ubiquitin chains that recruit the autophagic apparatus.

Question 15

What is PINK1

Answer 15

Serine/threonine kinase that acts as a molecular sensory of mitochondrial health, constantly surveying mitochondrial status until it detects damage and signals for the recruitment and activation of the E3 ubiquitin ligase Parkin

Question 16

Describe Parkinson’s disease clinical confusion

Answer 16

Although dementia is present is the majority of long-
term PD patients, dementia may occur relatively early in
PD and is regarded as clinically separate from PD and
termed “Parkinson’s Disease Dementia” (PDD).
2) A clinical condition called “Dementia with Lewy Bodies”
(DLB) shows the motor manifestations of PD but with a
more rapid onset of dementia than is usual in PDD.
3) PD dementia (PDD) is distinguished from DLB by the
order of manifestation of motor vs cognitive symptoms.
In PDD, individuals have a PD diagnosis first based on
motor features and subsequently develop dementia. In
DLB, dementia is present before or within one year of
the motor symptoms.

Question 17

Describe the shared dysfunctional proteins of PD and AD

Answer 17

1) Parkinson’s disease is not considered a Tauopathy, but a growing
number of studies have shown Tau involvement in its disease
process.
2) Deposits of aggregated Tau have been observed in around 50% of
PD brains.
3) Alpha-synuclein and LRRK2 promote the hyperphosphorylation of
Tau by various kinases and LRRK2 can directly phosphorylate Tau
when the Tau bound to microtubules.
4) Tau, amyloid-beta and alpha-synuclein pathologies are all found
in “Dementia with Lewy Bodies” (DLB).
5) Amyloid-beta (Aβ) plaques and tau Neurofibrillary Tangles (NFTs)
occur in 30-40% of patients with PD.
6) Up to 50% of patients with PDD develop sufficient A-β plaques
and Tau-NFTs for a secondary diagnosis of Alzheimer’s disease.

Question 18

Describe Huntington’s disease

Answer 18

1) Progressive fatal neurodegenerative genetic disease,
inherited in an autosomal dominant manner with age-
dependent penetrance (fully penetrant by age 65 years).
2) Prevalence of 4-10 per 100,000 in western world.
3) Mean age of onset is 40 years with death 15-20 years from
time of onset.
4) Clinical features of progressive motor dysfunction, cognitive
decline and psychiatric disturbance caused by neuronal
dysfunction and cell death in affected pathways
5) Neurons of the striatum are most susceptible to death as
well as neurons that project from the cortex to the striatum.
6) Motor signs of chorea, dystonia, bradykinesia and loss of
coordination.

Question 19

Describe the action of HTT genes and Huntingtin in HD

Answer 19

1) HD is caused by a CAG triplet repeat expansion in the HTT gene, which as CAG is
one of 2 codons that specify glutamine in the genetic code, produces an
extension of a poly-glutamine stretch (polyQ) in the Huntingtin protein
2) Huntingtin is a large protein of 3144 amino acids
3) Longer CAG repeats predict earlier onset and account for 50-70% of variance in
age of onset
4) Interestingly CAG repeats correlate less well with speed of progression
5) In non-HD population the CAG sequence is repeated 9-35 times with an mean of
between 17 and 20 repeats. CAG expansions >35 result in HD
6) Age of onset is inversely correlated with length of CAG expansion with juvenile
onset-HD associated with HTT genes bearing 75 or more repeats.
7) Abnormal poly-glutamine (polyQ) expansion has been associated with 8 other
NDs, each affecting a different protein and characterised by loss of specific
neurons(eg Ataxin2 in one form of ALS). This represents strong evidence that
polyQ expansion is the causative factor for dominant inheritance, with the
specific protein in which the expansion occurs determining the cell specificity of
the disease.

Question 20

What is the normal functions of Huntingtin

Answer 20

The HTT gene is alternatively spliced so many different isoforms of
HTT protein are possible. Huntington (HTT) is a large “Scaffold
protein” and coordinates protein-protein interactions in many
different cell processes:
1) Gene transcription, RNA splicing, receptor-mediated
endocytosis, vesicle trafficking, ciliogenesis and metabolic
regulation.
2) Importantly HTT is involved in regulation of autophagy
(NB*deletion of polyQ stretch enhances autophagy and
increases longevity in mice)
3) HTT is an essential protein as gene knockout is embryonically
lethal and causes neurodegeneration if KO’d out in adulthood
4) Over-expression of wt-HTT stimulates the transport of BDNF to
axon terminals but m-HTT does not.

Question 21

Describe Huntingtin protein in HD

Answer 21

1) Significantly, mutant HT (mHT) is subject to various levels of intracellular
proteolysis that generates N-terminal fragments (N-mHtt) containing the
abnormal polyQ stretch. Shorter fragments appear to be more toxic.
2) N-terminal fragments translocate to the nucleus where they interfere
with several aspects of transcription. Certain genes suffer transcriptional
repression by N-mHtt, notably the neuronal survival factor BDNF, and
PGC1α, an important inducer of mitochondrial biogenesis.
3) Mutant HTT also forms aggregates, initially thought to be the toxic
entities in HD, but may actually be protective as they reduce the level of
soluble toxic fragments.
4) C-terminal fragments are also toxic as they inhibit the actions of
Dynamin (essential for vesicle budding in RM-endocytosis.
5) In contrast to above: normal (wild type) HT has not been shown to
undergo intracellular proteolysis.

Question 22

Describe amyotrophic lateral sclerosis (ALS)/ MND

Answer 22

1) Progressive fatal neurodegenerative disease of motor neurons.
2) Prevalence of 4-6 per 100,000 in western world.
3) Mean age of onset is 55 years with death 15-20 years from time of onset.
4) Paralysis begins focally and disseminates in a pattern that suggests that degeneration is
spreading within a pool of contiguous motor neurons.
5) 90% of cases appear sporadic (sALS) with only 10% inherited/familial (fALS)
6) ALS differs from other NDDs in that multiple proteins have shown to be involved; however
most sALS involve the DNA-binding protein TDP43, while fALS involves superoxide dismutase-1
(SOD1) and the RNA regulator FUS.
7) Both TDP43 and FUS contain “prion-like domains” that are rich in hydrophobic amino acids
and prone to aggregation.
TPD-43 aggregates in
ALS motor neurons

Question 23

Describe superoxide dismutase-1 in ALS

Answer 23

1) SOD-1 mutations in about 20% of ALS cases
2) SOD-1 is a free radical scavenging enzyme composed of 153 amino acids
3) >150 mutations of SOD-1 have been reported to be pathogenic. Initially through loss
of normal scavenging of toxic free radicals, but secondarily through misfolding of the
protein and disturbance of proteostasis.
4) Mutant SOD-1 misfolds and is targeted for proteasomal degradation by ubiquitination
5) Misfolded protein escapes proteasomal digestion
6) mSOD-1 appears to impair the Unfolded Protein Response (UPS) and autophagy.
Increases in autophagosomes are prevalent in motor neurons in the spinal chord
during ALS suggesting that the process isn’t being successfully completed.