September 10th BIOS E108 Mechanisms of cell death in neurodegenerative diseases. Part II -Apoptosis -Ubiquitin Proteasome pathway UPP -Proteostasis: Chaperones Autophagy and Lysosomal protein degradation -Aberrant Cell Cycle re-entry

APOPTOSIS

Apoptosis: mechanism of programmed cell death Steps that lead to apoptosis: 1-Condensation of nuclear chromatin 2-Compaction of cytosolic organelles and blebbing on the cell surface 3-Decrease in cell volume (the cell shrinks) 4-Alteration of plasma membrane 5-Phagocytosis

Necrosis is characterized by membrane destruction and loss of electron density in the cytosol. Apoptotic features of nuclear fragmentation, but no substantial necrotic features, are observed in neurons treated with 25 pmol/cm2 ProTα Necrosis Apoptosis The Journal of Cell Biology, Vol. 176, No. 6, March 12, –862 Structural differences between necrosis and apoptosis

Apoptosis is of Greek origin meaning "falling off". The term is used in an analogy to the apparent suicide of leaves resulting in the very visible color changes associated with the Autumn/Fall and that eventually leads to the leaves falling from the trees. Similarly, cells go through a predetermined sequence of events resulting in death and removal from the body. APOPTOSIS

5-10. Scanning electron microscopy. Epithelial cells. Different stages. Flat cells (Fig. 5) undergo different forms of rounding, surface blebbing and cell retraction (6-9) preceding the typical apoptotic figure shown in (10).

These proteins initiate the process, and could be released from the mitochondria following mitochondrial injury or dysfunction CASPASES -Caspases 1-14, divided in 3 groups depending on their structure and substrate specificity. - Normally in the inactivated form of pro-caspases

Group I: Caspase 1, 4, 5, 13 Group II: Caspase 2, 3, 7 action of group II caspases on substrates determines: -no cell repair -no cell cycle progression -DNA fragmentation Group III: Caspase 6, 8, 9, 10 will activate caspases of group I and II, beginning a chain reaction that lead to programmed cell death

Mitochondrial apoptotic pathway. Death can be induced by the binding of ligands (such as FasL) to specific receptors (such as FAS) located at the cell surface. FAS contains a cytoplasmic death domain where FADD (Fas-associated death domain) can bind in presence of FasL, and recruit Pro-caspase 8 for subsequent activation in caspase 8. This induces caspase 3 activation. Caspase 3 cleaves I-CAD, the inhibitor of CAD (Caspase-activated DNase), which is released to enter the nucleus and cleaves DNA. In addition caspase 8 cleaves Bid protein, resulting in a truncated Bid (tBid) that, upon dimerisation of Bax or Bad, causes the release of cytochrome c from mitochondria. The mechanisms by which Bax leads to mitochondrial membrane permeabilisation and subsequent release of pro-apoptotic factors still remain unclear. It is proposed that Bax could interact with the permeability transition pore, or form channels by self-oligomerization. This leads to the mitochondrial release of cytochrome c and Smac/Diablo (Smac: second mitochondrial- derived activator of caspase; Diablo: direct IAP-binding protein with low pI), AIF (apoptosis inducing factor) and various procaspases. Bcl2 inhibits the release of cytochrome C and AIF in the cytoplasm and prevents the variation of the permeability transition pore. In the cytosol, cytochrome c binds to Apaf-1 (apoptosis- protease- activating factor). Both proteins form the apoptosome, which converts procaspase 9 in caspase 9. This results in activation of downstream effector caspases. Smac/DIABLO binds to IAP (Inhibitors of apoptosis) and prevent them from inhibition of the caspase 9 and caspase 3 activation. AIF has an indirect role in chromosome degradation as it activates endonuclease G, a DNase that moves from the mitochondria to the nucleus during apoptosis. Interestingly to note, the mtDNA is not fragmented during apoptosis. Apoptotic Pathways

Exogenous Pathways to Apoptosis These pathways work through the activation of cell surface death receptors, activated downstream of certain stimuli. Fas/CD95 TNF  R1 (tumor necrosis factor a receptor 1) The DD (Death Domain) of these receptor binds to adaptor proteins with death effector domain (FADD-Fas associated protein with death domain) forming a complex that contains also caspase 8. Caspase 8 derives following activation of pro-caspase 8 either by caspases or other proteins. This exogenous pathway is involved in Huntington’s diseases, stroke, Parkinson, but not in AD. However, caspase 8 is activated in cultured cell models, but in neurons this pathway is resistant to activation of death cell receptor. Thus it is still not clear how this pathway leads to neuronal death.

Endogenous pathway It is based on the release of cytochrome c out of the mitochondrion and subsequent activation of caspases. The mitochondrion may disrupt following two alternative mechanisms: -osmotic disequilibrium that leads to expansion of the matrix space, swelling of intracellular structures and rupture of the outer membrane -opening of channels in the outer membrane Starts from mitochondria dysfunction!!!

-In any event, cytochrome c forms complexes with the apoptosis protease activating factor APAF1, and procaspase 9. -This leads to activation of procaspase 9 to caspase 9, that will ultimately lead to activation of caspase 3 -In this phase, ROS production increase and there is a massive release of proapoptotic stimuli from the mitochondria, like *AIF (Apoptotic Inducing Factor) *SMAC (Second Mitochondria Derived Activator of Caspases) *DIABLO (Direct Inhibitor of Apoptosis AIT-Binding protein with Low pI *also antiapoptotic proteins belonging to Bcl2 family are released. The antiapoptotic properties of Bcl2 are characterized by the capability of the protein to bind to APAF1 and to regulate the amount of cytochrome c released in the cytosol. AIF is released in the cytosol during the induction phase, translocates to the nucleus where it induces DNA fragmentation in large scale

The Ubiquitin Proteasomal Pathway UPP

Ubiquitin and Proteasonal degradation: the Ubiquitin Proteasomal Pathway UPP It is the cellular “system” to eliminate proteins, degrading them into small peptides, aminoacids. In this way, proteins tend NOT TO accumulate in the cell. After they have been synthesized and have exerted their activity in the cell, proteins have to be eliminated, since new proteins are soon newly synthesized. This pathway is to avoid accumulation of proteins. Protein accumulation is a phenomenon common to all the neurodegenerative diseases: failure of the UPP is one of the reason why proteins accumulate and then tend to form aggregates. MOST IMPORTANTLY, all the intracellular and extracellular lesions characteristic of neurodegenerative diseases DO CONTAIN ALSO UBIQUITIN.

-Ubiquitin is a small protein that occurs in most eukaryotic cells. Its main function is to mark other proteins for destruction, known as proteolysis. -Several ubiquitin molecules attach to the condemned protein (polyubiquitination), and it then moves to a proteasome, a barrel-shaped structure where the proteolysis occurs. Ubiquitin can also mark transmembrane proteins (for example, receptors) for removal from the membrane. -Ubiquitin consists of 76 amino acids. It is highly conserved among eukaryotic species: Human and yeast ubiquitin share 96% sequence identity.

The process of marking a protein with ubiquitin consists of a series of steps: -Activation of ubiquitin by binding to an ubiquitin-activating enzyme E1. -Transfer of ubiquitin from E1 to the ubiquitin-conjugating enzyme E2 via trans(thio)esterification. -Then, the final transfer of ubiquitin to the target protein can occur either: 1- directly from E2. This is primarily used when ubiquitin is transferred to another ubiquitin already in place, creating a branched ubiquitin chain, or 2-via an E3 enzyme, which binds specifically to both E2 and the target protein. The target protein is usually a damaged or non-functional protein that is recognized by a destruction-targeting sequence. Ubiquitins then bind to a lysine residue in the target protein, eventually forming a tail of ubiquitin molecules. This is the typical way to mark specific proteins for proteolysis. Finally, the marked protein is digested in the 26S-proteasome into small peptides, amino acids (usually 6-7 aa subunits). Although the ubiquitins also enter the proteasome, they are not degraded and may be used again.

Structure of a proteasome barrel Core Particle (CP) Responsible for protein degradation Contains protease active sites Regulatory Particle (RP) Responsible for substrate’s recognition Contains ATPase active sites

Proteostasis or Protein Homeostasis

Proteostasis: Protein Homeostasis

Proteostasis: regulation of the homeostasis of proteins during the life span of a cell Homeostasis: the property of a system that regulates its internal environment and tends to maintain a stable, constant condition. Proteostasis: “refers to controlling the concentration, conformation, binding interaction, and location of individual proteins, by re-adapting the innate biology of the cell, often through transcriptional and translational mechanisms”.

Quality Control The competition between cellular protein folding and degradation. It has a crucial role maintaining a correct proteostasis. Crucial role of quality control during aging

Mechanisms of protein degradation The Ubiquitin Proteasome Pathway The Lysosomal Pathway Autophagy

The lysosome CD63 BSA

Protein ubiquitination and the UPP and lysosomal pathways

Autophagy : a method to control the degradation of proteins, organelles, structure and aggregates through the lysosomal pathway

Mechanisms of Autophagy Macroautophagy: a system to degrade entire regions of the cytosol, including organelles. First organelles are included into de novo formed double layers membranes, which are then sequestered and targeted to the lysosomes. Very important in neurodegeneration as it destroys dysfunctional organelles such as mitochondria. It also target for degradation large aggregates that might disrupt the functionality of the cell mTOR kinase involved in the regulation of this autophagic mechanism. CMA or Chaperone Mediated Authophagy: a type of selective autophagy aimed at removing from the system selected, soluble proteins. Relation to this kind of autophagy and degradation of misfolded proteins. Microautophagy: so far poorly characterized in mammals.

Different types of Autophagy

Role of Autophagy in human disease

Autophagy and aging

Consequences of Macroautophagy failure during aging

A simplified model of Autophagy regulation

Role of the component of nutrient signaling in the regulation of Autophagy

Role of chaperone proteins in the maintenance of a correct proteostasis -Inhibiting formation of larger aggregates -Favoring protein degradation/Autophagy -Regulating the Insulin Signaling Pathway

Chaperones “An older person who is responsible for young people on social occasions” “To go somewhere with someone as his/her chaperone” Proteins that help control protein homeostasis in any given time of the life of the cell Stress Environmental Chemical Pathophysiological states Chaperones expression

1-To recognize nascent polypeptides 2-During translation they contribute to a) maturation of folding intermediates b) assembly into multimeric complexes c) transport to organelles Chaperones’ Functions

Cell Stress Response in physiological and pathological conditions

Chaperones involved in neurodegeneration

Modified from Chaperones that influence protein misfolding

Chaperones regulate the formation of aggregates in a model of “HD” worm: age and client protein factors

Influence of environmental stress (temperature) on chaperones’ activity and aggregates formation Current Opinion in Structural Biology 2010, 20:23–32 Htt and paramyosin aggregates Htt aggregates

Signaling pathways controlling aging and longevity: role of chaperones

Age-associated decline correlates with reduced control of proteostasis

Does proteostasis occur at an extracellular level? Are there Extracellular Chaperones? In neurodegenerative diseases protein aggregates make it to the extracellular compartment

Proteins identified as extracellular chaperones

Extracellular proteostasis

Extracellular chaperones to protect from Inflammation Membrane disruption and to favor Endocytosis and Degradation

Aberrant cell cycle re-entry

The Cell Cycle

G0= resting or quiescent phase; G1=first gap phase; S= DNA replication phase; G2= second gap phase; M=mitosis. Neuronal aberrant cell cycle re-entry The cell cycle in cells and mature neurons

Evidences that link Cell Cycle Events (CCE) to neuronal death -KO models -Brain analysis -NGF deprivation: death with increased cell cycle markers -Drugs that block cell cycle prevents NGF-deprivation related cell death -Expression of cell cycle proteins increase *in brain of neurodegenerative diseases (AD) *in vulnerable neurons

Cell cycle re-entry: a permissive event to neurons vulnerability The two hit hypothesis

Cell cycle proteins involved in neuronal development

The developing cerebral cortex

Cell cycle proteins and neuronal development

Cell cycle proteins control neurons differentiation and development

Cell cycle initiation and cell death in mature or immature neurons

Hypothesis on how cycling neurons can be toxic

Expert Rev Mol Med Jun 29;12:e19. Review. The two hit hypothesis: aberrant cell-cycle re-entry renders neuron vulnerable to a second toxic insult

Evidences of accumulation of cell cycle related proteins in AD PCNA Cyclin B1 Cyclin D cdk4nonAD

Cortex Hippocampus WT APPTgR1.40 DNA synthesis and centromere duplication in models of AD