Case 2 - mechanisms of neurodegeneration

Question 1

microglia hyperreactivity

Answer 1

• Aberrations in Microglial Functions:
  o Aberrations in microglial functions can lead to neurodegeneration, and their activation is detected in all NDDs
• Microglial Activation in NDDs:
  o Microglia respond to danger signals such as protein aggregates, misfolded proteins, damaged synapses, Ca2+ influx, or mitochondrial ROS, contributing to cytokine/chemokine production, phagocytosis activation, and dysregulation of physiological functions.
• Chronic Inflammation in NDDs:
  o Failure to resolve microglial activation results in chronic inflammation, a contributing factor to the neurodegenerative process in NDDs.
• Protein Aggregation and Microglial Activation:
  o Aggregated NDD proteins, including Aβ, tau, α-synuclein, PrP fibrils, or SOD1, induce microglial activation.
• Microglial Populations in NDDs:
  o Different microglial populations, such as disease-associated microglia (DAM) or microglial neurodegenerative phenotype (MgND), play unique roles in regulating cytokine production, phagocytosis, ROS production, and astroglial interactions.

Question 2

inflammasome activation

Answer 2

1. PRRs are activated by a PAMP/DAMP which leads to the upregulation of protein that make up the inflammasome, these proteins include: NLRP-3 (sensor), ASC (adaptor) and caspase 1 (inactive)
   * Note that potassium influx, viral DNA, ROS and crystalline can also induce this pathway not only PAMPs and DAMPs binding to PRRs –> secondary signal
   * DAMPS/PAMPS also lead to activation of other caspases which leads to cleavage of gasdermin-D –> N-terminals form pores  pyroptosis and spreading of cytokines
2. The proteins assemble into inflammasomes which now produces active caspase 1
3. Active caspase 1 can cleave pro-IL-1beta into active IL-1beta which is a pro inflammatory cytokine

Question 3

inflammasome in neurodegeneration

Answer 3

1. Chronic Inflammation: Persistent activation of the inflammasome and the resulting chronic inflammation in the brain are associated with neurodegenerative diseases. This chronic inflammatory state can contribute to the progression of diseases like Alzheimer’s, Parkinson’s, and multiple sclerosis.
2. Microglial Activation: Microglia, the primary immune cells in the brain, play a crucial role in inflammasome activation. Dysregulation of microglial activation and the inflammasome has been observed in neurodegenerative conditions.
3. Amyloid-Beta (Aβ) in Alzheimer’s Disease: In Alzheimer’s disease, the accumulation of Aβ plaques is a key pathological feature. Aβ has been shown to activate the NLRP3 inflammasome, leading to the release of pro-inflammatory cytokines and contributing to neuroinflammation.
4. Alpha-Synuclein in Parkinson’s Disease: In Parkinson’s disease, the aggregation of alpha-synuclein in neurons is linked to inflammasome activation. This process contributes to the inflammatory response and neuronal cell death observed in the disease.

Question 4

ROS

Answer 4

• A molecule (containing oxygen) with a single unpaired electron:
• Nitric oxide (RNS)  ONOO- (peroxynitrite)
• Superoxide
• Hydroxyl radical
• Hydrogen peroxide
• Damage macromolecules:
  o DNA –> 8-oxoguanine (altered guanine), deamination (CU), single strand breaks, etc. (>25 types of lesions)
  o Protein –> protein carbonyl groups
  o Lipids –> Lipid peroxyl radical
• Sources:
  o Enzyme systems
  o Organelles

Question 5

ROS and neurodegeneration

Answer 5

1. Oxidative Stress:
   * Excessive ROS production or insufficient antioxidant defense mechanisms lead to oxidative stress, a condition where there is an imbalance between the production of ROS and the cell’s ability to detoxify them.
   * Oxidative stress damages cellular components, including proteins, lipids, and DNA, which can contribute to neuronal dysfunction and death.
2. Mitochondrial Dysfunction:
   * Mitochondria are a major source of ROS during cellular respiration. Dysfunction in mitochondria, common in neurodegenerative diseases, can lead to increased ROS production.
   * ROS produced in the mitochondria can damage mitochondrial DNA and proteins, further compromising cellular energy production and contributing to neurodegeneration.
3. Protein Aggregation:
   * In some neurodegenerative diseases, misfolded proteins aggregate and form toxic deposits. ROS can contribute to the misfolding of proteins and the formation of aggregates.
   * For example, in Alzheimer’s disease, ROS have been linked to the production and accumulation of amyloid-beta (Aβ) plaques.
4. Lipid Peroxidation:
   * ROS can initiate lipid peroxidation, a process where free radicals attack lipids containing unsaturated fatty acids in cell membranes.
   * Lipid peroxidation disrupts the integrity of cell membranes, leading to increased permeability and potentially neuronal damage.
5. Neuronal Damage and Apoptosis:
   * ROS-induced damage to cellular components, including DNA, can trigger programmed cell death or apoptosis.
   * Neuronal apoptosis is a characteristic feature of neurodegenerative diseases, and ROS-mediated damage contributes to the loss of neurons.
6. Inflammation:
   * ROS can activate inflammatory pathways, contributing to chronic neuroinflammation observed in many neurodegenerative diseases.
   * Inflammatory responses, when sustained, can further enhance ROS production, creating a cycle of oxidative stress and inflammation.
7. Antioxidant Defence System:
   * Cells have an antioxidant defence system that includes enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase, which neutralize ROS.
   * Impairment of the antioxidant defence system, as seen in some neurodegenerative diseases, can exacerbate ROS-induced damage.
8. Vulnerability of Neurons and glial cells:
   * Neurons are particularly susceptible to oxidative stress due to their high metabolic activity, abundant lipid content, and post-mitotic nature.
   * Vulnerability to ROS-induced damage contributes to the selective neuronal loss observed in neurodegenerative diseases
   * Oligodendrocytes produce myelin which is highly energy consuming  ROS production

Question 6

synaptic pruning and neurodegeneration

Answer 6

1. Disruption in Alzheimer’s Disease (AD):
   * In Alzheimer’s disease, the accumulation of beta-amyloid plaques is associated with synaptic dysfunction and loss.
   * Abnormal activation of microglia, the brain’s immune cells, in response to beta-amyloid may contribute to excessive synaptic pruning, leading to the elimination of healthy synapses.
2. Microglial Involvement in Pruning:
   * Microglia, in addition to their roles in immune response, are involved in synaptic pruning during development and adulthood.
   * Dysregulation of microglial activity, as observed in neurodegenerative diseases, can lead to excessive or insufficient synaptic pruning.
3. Synaptic Loss in Parkinson’s Disease (PD):
   * In Parkinson’s disease, degeneration of dopaminergic neurons in the substantia nigra is associated with synaptic loss.
   * The loss of synapses contributes to the motor and cognitive symptoms observed in PD.
4. Tau Pathology and Synaptic Dysfunction:
   * In several neurodegenerative diseases, including Alzheimer’s, tau protein pathology is associated with synaptic dysfunction and loss.
   * Tau aggregates may disrupt the normal functioning of neurons, including synaptic maintenance processes.
5. Role in Huntington’s Disease (HD):
   * In Huntington’s disease, a genetic neurodegenerative disorder, there is evidence of synaptic dysfunction and loss.
   * Mutant huntingtin protein, the cause of HD, may directly or indirectly contribute to disruptions in synaptic pruning processes.

Question 7

apoptosis (general)

Answer 7

• Form of programmed cell death in which a suicide program is activated within an animal cell leading to rapid cell death mediated by intracellular proteolytic enzymes called caspases
• All components for executions are already present in the cell and it requires energy
• Causes:
  o DNA damage (radiation, chemotherapy)
  o Growth factor withdrawal
  o Cytokines (TNF alfa/beta)
  o Lack of survival factors
• Caspases:
  o Cleave between cysteine and aspartic acid
  o Inactive caspase is called a procaspase
  o 2 kinds : initiator and executioner caspases
• Caspases cause:
• Conformational changes in membrane
• Activate proteins that degrade the nucleus (nuclear lamina)
• Degrade Golgi apparatus
• Cause actin bundle contractions to form apoptotic blebs
• DNA degradation
  1. Caspase activated DNase (CAD) gets activated by a caspase though removal of iCAD
  2. CAD fragments the DNA

Question 7

cell death

Answer 7

1. apoptosis (intrinsic and extrinsic)
2. necrosis (oncosis, 2nd, regulated (necorptosis, ferroptosis)
3. paraptosis

Question 8

extrinsic pathway

Answer 8

1. Initiation:
   * The extrinsic pathway is initiated by external signals, such as binding of death ligands to death receptors on the cell surface.
   * Common death ligands include Fas ligand (FasL on NK cells), tumor necrosis factor-alpha (TNF-α), and TNF-related apoptosis-inducing ligand (TRAIL).
2. Death Ligand Binding:
   * Binding of death ligands to their corresponding death receptors (Fas, TNFR1, or TRAIL receptors) induces trimerization of the receptors.
3. Formation of Death-Inducing Signalling Complex (DISC):
   * The trimerized death receptors recruit adaptor proteins, such as FADD (Fas-associated death domain) and TRADD (TNFR1-associated death domain), forming the DISC.
4. Activation of Caspase-8:
   * Within the DISC, procaspase-8 is activated and cleaved into its active form, caspase-8.
   * Caspase-8 can directly activate effector caspases (caspase-3) or initiate the mitochondrial pathway by cleaving Bid, a pro-apoptotic protein.
5. Integration with Intrinsic Pathway:
   * Caspase-8 cleaves Bid into tBid, which translocates to the mitochondria and promotes MOMP.
   * This connects the extrinsic and intrinsic pathways, amplifying the apoptotic signal.
6. Activation of Effector Caspases:
   * Caspase-8, along with the intrinsic pathway, activates effector caspases (such as caspase-3), leading to the execution of apoptosis.
7. DNA Fragmentation and Cell Death:
   * Similar to the intrinsic pathway, activated caspases induce DNA fragmentation, morphological changes, and the formation of apoptotic bodies.

Question 9

intrinsic pathway

Answer 9

1. Initiation:
   * Apoptosis is often initiated by various intracellular signals, such as DNA damage, cellular stress, or lack of survival signals.
   * These signals may activate pro-apoptotic proteins, such as Bax and Bak, while inhibiting anti-apoptotic proteins like Bcl-2.
2. Mitochondrial Outer Membrane Permeabilization (MOMP):
   * Pro-apoptotic proteins (Bax and Bak) promote the permeabilization of the outer mitochondrial membrane.
   * This allows the release of pro-apoptotic factors from the intermembrane space into the cytoplasm.
3. Release of Cytochrome c:
   * Cytochrome c, a protein normally residing in the mitochondrial intermembrane space, is released into the cytoplasm.
   * Cytochrome c plays a crucial role in the activation of downstream caspases.
4. Formation of Apoptosome:
   * In the cytoplasm, cytochrome c binds with Apaf-1 (apoptotic protease-activating factor 1) and procaspase-9, forming a structure known as the apoptosome.
5. Activation of Caspases:
   * The apoptosome activates procaspase-9, converting it into an active form, caspase-9.
   * Caspase-9 then activates effector caspases, such as caspase-3, which execute the process of apoptosis.
6. DNA Fragmentation and Cell Death:
   * Caspase-3 and other effector caspases cleave various cellular substrates, leading to DNA fragmentation and other morphological changes associated with apoptosis.
   * The cell undergoes controlled dismantling, forming apoptotic bodies that are engulfed and cleared by neighboring cells.

Question 10

2nd necrosis

Answer 10

• Description:
• Occurs when efferocytosis (phagocytic clearance of apoptotic cells) is impaired or overwhelmed.
• Process: Plasma membrane of apoptotic cells ruptures.
• Release of intracellular contents triggers inflammatory responses.
• Mechanisms:
• Caspase cleavage of the plasma membrane calcium pump, leading to calcium overload.
• DFNA5 forms necrosis-inducing pores post caspase-3 cleavage.

Question 11

oncosis

Answer 11

• Description:
• Term used for cell death accompanied by cellular and organelle swelling.
• Process:
• Often induced by ATP depletion and cell swelling, leading to plasma membrane rupture.
• Notes:
• Commonly seen in infarcts after tissue ischemia.

Question 12

necroptosis

Answer 12

• Characteristics: type of regulated necrosis
• Dependent on kinase activity of RIP1, RIP3, and expression of MLKL.
• Activation of the necrosome (RIP1-RIP3-MLKL complex).
• Leads to plasma membrane rupture and necrosis.
• Implications:
• Implicated in acute and chronic neurodegenerative disorders.

Question 13

ferroptosis

Answer 13

• Characteristics: regulated necrosis
• Iron-dependent form of regulated necrosis.
• Involves lipid peroxidation of polyunsaturated fatty acids.
• Inhibited by iron chelation and antioxidants (glutathione peroxidase 4) (lipid specific – ferrostatins, vit-E)
• Implications:
• Implicated in neurodegenerative disorders like Huntington’s and Parkinson’s disease.
• Dopaminergic neurons are especially vulnerable
• Large neuroinflammatory effect

Question 14

paraptosis

Answer 14

• Characterized by cytoplasmic vacuolation.
• Initiators:
• Originally observed during the overexpression of the insulin-like growth factor 1 receptor (IGF-1R).
• Neuronal Context:
• Overexpression of p44, a p53 isoform, leads to IGF-1R activation, memory defects, and neuronal death, possibly through paraptosis and autophagy.
• Cytoplasmic vacuolation, the hallmark of paraptosis, may occur through various mechanisms, including autophagy, lysosomal disruption, and mitochondrial permeability transition.
• Cytoplasmic vacuolation often linked to endoplasmic reticulum stress (ROS) and the unfolded protein response (UPR), typically associated with apoptotic cell death.
• Distinctive Features:
• Unlike most cell death forms, paraptosis may necessitate ongoing protein synthesis.
• Tauopathies Connection:
• Cytoplasmic vacuoles, resembling paraptosis, observed in degenerating neurons with hyperphosphorylated tau in Alzheimer’s disease and tauopathies

Question 15

glutaminergic excitotoxicity

Answer 15

Excitotoxicity Mechanisms:
1. NMDA Receptor Activation:
* Permits calcium influx into neurons.
* Overactivation leads to excessive intracellular calcium levels.
2. Calcium Overload:
* Activates various enzymes and pathways.
* Triggers mitochondrial dysfunction and reactive oxygen species (ROS) generation.
3. Mitochondrial Dysfunction:
* Impaired ATP production.
* Release of pro-apoptotic factors.
4. ROS Generation:
* Oxidative stress damages lipids, proteins, and DNA.
* Further exacerbates neuronal injury.

Downstream Effects:
1. Neuronal Excitability Changes:
* Altered membrane potential and increased firing rates.
2. Cellular Swelling:
* Intracellular water influx leading to cell swelling.
3. Activation of Cell Death Pathways:
* Apoptosis, necrosis, and, in some cases, necroptosis.
4. Inflammation:
* Release of pro-inflammatory mediators.
* Microglial activation contributes to neuroinflammation

Question 16

autophagy

Answer 16

• Macroautophagy is the most well-studied form of autophagy. It involves the formation of a double-membrane structure called the autophagosome, which engulfs cellular components targeted for degradation.
• The autophagosome fuses with a lysosome, forming an autolysosome, where the contents are degraded by lysosomal enzymes.
  2. Initiation:
• Autophagy is initiated in response to various cellular stresses, such as nutrient deprivation, oxidative stress, or the presence of damaged organelles.
• During autophagy initiation, a structure known as the phagophore is formed, which expands to engulf cellular material.
  3. Degradation:
• The autophagosome, with its cargo, fuses with lysosomes, forming an autolysosome.
• Lysosomal enzymes then break down the contents of the autophagosome, releasing amino acids and other building blocks that can be used for cellular maintenance and repair.
  4. Role:
• Autophagy plays a crucial role in maintaining cellular homeostasis by eliminating damaged or unnecessary cellular components, promoting cell survival during periods of stress

Question 17

UPS

Answer 17

Proteasome:
1. Structure:
* The proteasome is a large protein complex found in the cytoplasm and nucleus of eukaryotic cells.
* It consists of a cylindrical structure made up of multiple protein subunits arranged in a barrel-like shape.
2. Function:
* The primary function of the proteasome is to degrade proteins that are tagged for destruction by ubiquitin, a small protein that is covalently attached to target proteins.
* Ubiquitin is added to proteins that need to be degraded in a process called ubiquitination.
* The proteasome recognizes these ubiquitinated proteins and unfolds them before translocating them into its catalytic core for degradation.
3. Degradation Process:
* The proteasome’s catalytic core contains proteolytic enzymes that cleave the peptide bonds in the target protein, breaking it down into small peptides.
* These peptides are then further degraded into amino acids by cellular peptidases.
4. Regulation:
* The ubiquitin-proteasome system is involved in the regulation of various cellular processes, including cell cycle progression, DNA repair, and the degradation of misfolded or damaged proteins

Question 18

protein folding

Answer 18

• Shaping of protein:
  1. Primary structure; polypeptide chain
  2. Secondary structure; beta pleated sheets & alfa helix
  3. Tertiary structure; covalent bonds (disulfide bridges between Cysteines)/ H-bridges / Salt bridges (ionised groups) / metal bridges / hydrophobic interactions
  4. Quaternary structure; multiple proteins to form one protein complex
• Posttranslational modification: (involves the folding/shaping)
• Protein folding in the ER by molecular chaperones
   Chaperones catalyse the self-assembling process by keeping the protein stable while folding
   Hsp70 is the most common version
   Hsp70 is used in refolding of proteins (heat-shock protein)

Question 19

protein aggregates in neurodegen

Answer 19

1. Protein Misfolding and Aggregation:
   * In neurodegenerative diseases, specific proteins undergo misfolding, adopting abnormal conformations that render them prone to aggregation.
   * The misfolded proteins aggregate to form insoluble structures, such as plaques or inclusion bodies.
2. Common Proteins Involved:
   * Different neurodegenerative diseases are associated with the aggregation of specific proteins. For example:
   * Alzheimer’s disease is characterized by the aggregation of beta-amyloid and tau proteins.
   * Parkinson’s disease involves the aggregation of alpha-synuclein.
   * Huntington’s disease is associated with the aggregation of mutant huntingtin protein.
3. Toxicity of Protein Aggregates:
   * The accumulation of protein aggregates can have toxic effects on neurons.
   * These aggregates may disrupt cellular processes, interfere with normal protein degradation mechanisms, and induce cellular stress.
4. Impaired Proteostasis:
   * Proteostasis refers to the maintenance of proper protein folding, trafficking, and degradation within cells.
   * Protein aggregates can overwhelm the cellular proteostasis machinery, leading to the accumulation of misfolded proteins and further exacerbating cellular dysfunction.
5. Neuronal Dysfunction and Death:
   * The presence of protein aggregates is often associated with synaptic dysfunction and impaired neuronal function.
   * Over time, the continued accumulation of aggregates can lead to neuronal death, contributing to the progressive nature of neurodegenerative diseases.
6. Spread of Aggregates:
   * In some neurodegenerative diseases, there is evidence that misfolded proteins can spread from cell to cell.
   * This spreading mechanism may contribute to the progressive nature of the diseases, as aggregates propagate through interconnected regions of the nervous system.
7. Inflammatory Response:
   * The presence of protein aggregates can trigger an inflammatory response in the brain.
   * Inflammation is thought to contribute to the progression of neurodegenerative diseases and may exacerbate neuronal damage.