Lecture X Flashcards
What are lysosomes?
ubiquitous organelles located in the cytoplasm
What are lysosomes involved in?
autophagy
secretion of plasma membrane
repair of plasma membrane
energy metabolism control of the cell
What cells are really sensitive to lysosomal dysfunction?
neurons
What neurodegenerative diseases are caused by lysosomal dysfunction?
Alzheimer’s
Parkinson
Multiple Sclerosis
What happens when the lumen of the lysosomes is acidic?
the lysosomal enzymes work at an acidic pH
What does lysosomal location depend on? Where are they usually located?
depends on their stage of life
they can be perinuclear or far from the nucleus (not always random)
Describe how lysosomal enzyme are inserted into lysosomes:
lysosomal enzymes follow the normal protein synthesis of the cell
after synthesis, they are packed into vesicles in the golgi
they are modified and delivered to lysosomes thanks to their specific tag
How is lysosomal biogenesis regulated?
by many intracellular and extracellular signals that are integrated
*the state of the cell really impacts on the biogenesis of lysosomes
What is the membrane of the lysosome like?
phospholipid bilayer membrane that is very rich in carbohydrates
What is the lumen of a lysosome like?
it contains hydrolases and activators
What is the primary function of lysosomes?
degradation of substrates
Where does the degradation of substrates in lysosomes usually occur?
surface of intra-lysosomal vesicles and it is catalyzed by hydrolases (located in the lumen)
What happens if a lysosomal hydrolase is defective?
substrate is not degraded and accumulates
Describe the lysosome cycle:
material enters lysosomes either from the inside or outside of the cells
degradation of the materials results in building blocks being formed
blocks cn be recycled
Why is the lysosomal process tightly regulated?
in order to maintain the correct energy metabolism for the cell
How are hydrolases driven to the lysosomes?
they have specific tags
golgi are tagged with mannose 6 phosphate (M6P), which is recognized by M6P receptors
*after the glycosylation process, phosphate groups are attached to mannose residues
What is cross correction?
when hydrolases tagged with M6P and uptaken by neighboring cells that express M6P receptors
What is enzyme replacement therapy?
when a cell is enzyme deficient, a functional copy can be provided to be uptaken
*this is a way cells become cross-corrected
What is LYSET?
essential gene for lysosomal enzyme transport and for viral infections
*controls the enzymes that catalyze the addition of the phosphate groups on mannose residues
What happens if LYSET is missing?
enzymes cannot be tagged with M6P and cannot be targeted to the lysosomes
**leads to a reduction in lysosomal hydrolases so the cell has decreased capability of degrading substrates
What is the final outcome is LYSEt is missing?
lysosomal degradation disorder
What can the absence of LYSET result in, in terms of viruses?
viral infection resistance
What are some degradation pathways present in the cell?
ubiquitin proteasome system
autophagy (lysosomes are at the end of this process so there is a strict link between autophagy and lysosomal function)
How can lysosomes release their contents into the extracellular space?
lysosomes can fuse with the plasma membrane, which is regulated and induced at high Ca²⁺ concentrations
What physiological processes does lysosomal secretion mediate?
deregulation of immune cells
bone reabsorption by osteoclasts
defense mechanisms against parasites
platelet function
What is really important and regulates the cellular metabolism and growth?
nutrient sensing
What disorders is lysosomal dysfunction related to?
neurodegenerative diseases and lysosomal storage diseases
Why is lysosomal dysfunction so severe in the CNS?
neurons are very sensitive to the lysosomal dysfunction as they are metabolically active even though they are non-proliferating cells
What factors can contribute to lysosomal dysfunction?
genetic mutations:
loss of function mutations
gain of function mutations: lead to protein aggregation
physiological aging
Are lysosomes secondary players?
yes and the primary dysfunction is a genetic mutation
What are lysosomal storage diseases considered?
monogenic disorders due to mutations in genes that not only encode for lysosomal hydrolases but also for other proteins (ex: proteins associated with the membrane of the lysosome or proteins belonging to other cellular compartments (golgi and ER)
How many types of LSD are there?
more than 7-
What is the primary pathogenic event is LSDs?
accumulation of substrates that are either not degraded ot not completely targeted
What substrate is involved in Matachromatic leukodystrophy (MLD)?
sulfatide (major compnent of myelin)
What is the enzyme responsible for the degradation of sulfatide in MLD?
arylsulfatase A (ARSA), which works with SAP-B as a cofactor
What happens if there is a defect in ARSA or SAP-B?
there is an accumulation of sulfatide that is not degraded to galactosylceramide and leads to MLD
What leads to Krabbe disease?
accumulation of GalCer, which happens if the β-galactosylceramidase enzyme does not remove the galactosyl group from GalCer
What else is the enzyme β-galactosylceramidase involved in?
catabolism of galactosylsfingosin (or psychosine) , which is converted to sphingosine and lactosylceramide (LacCer)
What are sulfatide, GalCer and psychosine all present in?
myelin
What is the outcome of the MLD and Krabbe disease?
there is a dysfunction of myelinating cells, which in the end, die
What happens if there is a dysfunction of myelinating ceels that die?
astrogliosis is triggered, which leads to microglia activation and neuronal inflammation
demyelination occurs → secondary neurodegeneration
What is primary neurodegeneration?
accumulation of substates causes primary damage in neurons before demyelination occurs
Can patients have both primary and secondary neurodegeneration?
yes
they can experience not only severe CNS damage, but also dysfunctions in the PNS due to schwann cells being affected
What do LSDs lead to?
severe diseases where patients lose the neuronal transmission and their ability to talk, to walk, to swallow, etc. due to primary and secondary neuronal degeneration
What presents the most severe phenotypes?
absence of protein because the progression of the disease is very rapid and the patient dies early
What is a problem when addressing LDLs?
timing since it is based on genetics, the accumulation of substrates begins early in life and progress rapidly
they also have multiorgan pathologies
What are some therapeutic approaches?
we can prevent the storage burden by reducing or blocking the synthesis of the molecules that cannot be degraded (not efficient)
we can increase residual enzymatic activity if the mutation has not abrogated the enzyme’s function completely
we can also provide a source of the functional enzyme from the outside, which is called “enzyme replacement therapy” (most proven approach): we can deliver the enzyme systemically, or the=rough vehicles such as viral vectors (gene therapy) or a cell (cell therapy) or a combination of the 2
How can the functional enzyme be delivered to the CNS?
via hematopoietic stem cell (HSC) transplant
What is alogeneic transplant?
patient receives healthy cells from a donor to replace their own stem cells
What is an autologous ex-vivo gene therapy?
HPSC are isolated from the patient and correct and then re-introduced to the patient
What are some advantages to autologous ex-vivo HSC gene therapy compared to allogenic HSC transplant?
timing: autologous is faster
more enzyme is available using gene therapy
immunosuppression is not needed since the cells are derived from the patient
What can the Libmeldy drug be used to treat?
mucopolysaccharidosis type I (an LSD in which there is an accumulation of glycosaminoglycans)
*therapy is successful if patient is treated before the onset of symptoms since LSDs progress very rapidly
Do the treatments described before provide a cure?
no, they only slow down the progression of the disease
How can we treat the disease?
the idea is to maximize the expression and bioavailability of the functional enzyme, particularly in the CNS
What are potential ways we can maximize expression and bioavailability of the functional enzyme in the CNS?
we can work on vector design: generating a viral vector that is efficient in transducing directly in the brain or HSC that are retransplanted in the patient
increasing the levels of available enzyme to improve cross-correction
improving engraftment
What is a hypothesis that has been predicted to maximize expression and bioavailability of the functional enzyme?
Using a signal peptide, such as ApoEII, that binds to LDL receptors
the presence of this tag could improve the uptake by the endothelial cells and hopefully transocytosis into the brain parenchyma (allows for the BBB crossing of the enzyme)
What can iPSCs be used for?
disease modeling and cell therapeutic approaches since they are able to differentiate
What should be used for drug screenings?
2D cultures not organoids because organoids suffer from heterogeneity and variability
