Transcript Document

The Lysosome and Lysosomal
Storage Disorders (LSD)
Part I: Historical, Physiological and
Pathological Considerations
Serge Melançon, MD
January 2010
Synopsis (Part 1A)
• Historical notes and scientific
partners
• The endosomal-lysosomal system
• The ‘synthetic’ pathway
• The ‘endocytotic’ pathway
• Retrograde transport
Synopsis (Part 1B)
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Cofactors for lysosomal enzymes
Secretion-recapture pathway
Plasma membranes and lipid rafts
Molecular genetics
Microglia
Blood-brain barrier (BBB)
Causes of lysosomal storage
Residual enzymatic activity
Effects of lysosomal storage
The discovery
of lysosomes
Why?
In 1955, Belgian scientist Christian de Duve
observed that the cells released an enzyme
called acid phosphatase in much larger
amounts when they were repeatedly frozen
and thawed before centrifugation
de Duve C (1975) Exploring cells with a centrifuge. Science 189, 186-194
To explain this phenomenon, de Duve
suggested that the digestive enzyme must
have been encased in some sort of
membrane-bound organelle within the
cell.
After estimating the probable size of the
lysosomes, he was able to identify the
organelle in images produced with an
electron microscope
Lysosomes are spherical organelles contained by a single layer
membrane, though their size and shape vary to some extent.
Using brightfield or phase contrast
microscopy, the size of these cellular
organelles range from 0.25 to 0.5 mm
Several hundred lysosomal structures can be seen in the above phase micrograph
• "Lysosome" was the name given
because of these enzymes' ability to
"lyse" the cell
• Initially referred to as "suicide bags"
since one of the functions of lysosomes
is to rupture when the cell dies,
• Rupturing releases hydrolytic enzymes
that digest all parts of the cell, including
proteins, DNA, RNA, carbohydrates,
lipids and cellulose.
CELL GEOGRAPHY
101
micro.magnet.fsu.edu/.../ images/animalcell.jpg
Rough ER
The RER is where hydrolytic
enzymes are manufactured
before being transported to
the Golgi apparatus (complex),
where they undergo additional processing and are
transformed from an inactive to an active state
fig.cox.miami.edu/ ~cmallery/150/cells/ER.jpg
Golgi complex
• The Golgi is the distribution
and shipping department for
the cell's chemical products
• It plays three important roles
1 Modification of complex molecules (such as proteins) by the
addition of sugars (glycosylation)
2 Proteolysis of peptide molecules which makes them become
active
3 Sorting of molecules for either, transport out of the cell,
incorporation in the cell membrane, or transport to another
part of the cell
The Golgi is divided into three
functionally separate areas.
• The trans-Golgi network
(TGN) (closest to the cell
membrane) which performs
proteolysis and sorts molecules
for their destination
• The medial Golgi which adds
sugars to both lipids (fats) and
peptides (proteins)
• The cis face (closest to the
nucleus and the ER) receives
transport vesicles from the
smooth ER
Mitochondria
io.uwinnipeg.ca/~simmons/ 1115/cm1503/Image110.gi
The mitochondria are essential in the
production of energy (via adenosine
triphosphase; ATP) and lipid biosynthesis
Peroxisomes contain enzymes
That convert hydrogen
peroxide to water and
render potentially toxic
substances safe for the
cell
And also initiate the production of
phospholipids, which are typically used to
build up cell membranes.
The endosomal–lysosomal system
• The lysosome is one component of a series of
unconnected (?) intracellular organelles,
collectively known as the endosomal–lysosomal
system or vacuolar apparatus (De Duve and
Wattiaux,1966)
• The various components of the system were
described over 30 years ago by Novikoff (1973)
micro.magnet.fsu.edu/ cells/celldigestion/imag.
The endosomal–lysosomal system
• The main components are
– the early endosome, situated at the cell periphery,
– the late endosome, which is perinuclear, and
– the lysosome.
• They form a chain that is responsible for
– the trafficking and digestion of endocytosed molecules
– and participate actively in sorting and recycling
The lysosome (De Duve 1955)
‘terminal compartment of the system’
• Characteristics
• a membrane,
• a low internal pH,
• vesicles containing hydrolytic enzymes.
• The membrane contains
• transport systems to carry particles between
lumen and cites,
• an electrogenic proton pump (V-type H+ATPase, Arai et al, 1993).
• several membrane proteins of uncertain
function (Eskelinen et al, 2003).
The lysosome
micro.magnet.fsu.edu
/.../ lysosomesfigure1.jpg
Role of the lysosome
• Substrate breakdown using endocytosis
• Secretion of its content after fusion with
the plasma membrane (Luzio et al, 2000).
• Phagocytosis of bacteria and cellular
debris to form phagolysosomes.
• Calcium-regulated exocytosis for
membrane repair (Reddy et al, 2001)
Lysosomes’ partners and hangouts
Synthesis and trafficking of lysosomal
enzymes (the 'synthetic' pathway)
• Glycoproteins synthesized in the rough
endoplasmic reticulum (RER)
(At this early stage they are inactive)
• Translocate through the ER membrane with
the help of N-terminal signal sequences
• Once in the lumen of the ER, undergo Nglycosylation and lose the signal sequence.
Synthesis and trafficking of the
lysosomal enzymes (LE)
the 'synthetic' pathway
• LE then move to the Golgi compartment to
acquire mannose 6 -phosphate (M6 - P)
ligand (marker)
• Process requires the sequential action of
two enzymes,
– a phosphotransferase (Reitman & Kornfeld,
1981; Waheed et al, 1981)
– a diesterase (Varki & Kornfeld, 1981; Waheed
et al, 1981).
Synthesis and trafficking of the
lysosomal enzymes
the 'synthetic' pathway
• M6-P marker separates glycoproteins
destined for the lysosome from
secretory glycoproteins
• Failure to acquire this marker results in
mistargeting of lysosomal enzymes;
they will not enter the lysosome and
substrate breakdown will not occur.
Formation of lysosomal recognition tag or marker, mannose-6-phosphate
the 'synthetic' pathway
• In the mucolipidoses II (I-cell disease) and III
(pseudo-Hurler polydystrophy, mistargetting is
precisely what happens
• These patients lack the first enzyme, i.e. the
phosphotransferase
• Since all enzymes requiring the M6-P marker fail to
enter the lysosome, they end up outside the cell
• Consequently, these patients have very high plasma
levels of all lysosomal enzymes
• This clinical finding led to the discovery of the M6-P
ligand and its receptor (Hickman & Neufeld, 1972).
the 'synthetic' pathway
• Not all lysosomal enzymes require the
M6-P ligand
• Glucocerebrosidase (Gaucher disease)
which is associated with the lysosomal
membrane, does not acquire the M6-P
residue, although it does undergo Nglycosylation and is targeted
• The precise mechanism by which this
occurs is unknown.
the 'synthetic' pathway
• The receptor–protein complex then moves to
the late endosome, where the low pH causes
it to dissociate (Gonzalez-Noriega et al, 1980)
• The hydrolase moves on into the lysosome
and the receptor is recycled either to the
Golgi to pick up another ligand, or to the
plasma membrane.
• The final steps in the maturation of the
lysosomal enzyme include proteolysis, folding
and aggregation.
Synthesis and trafficking of the
lysosomal enzymes
Transport of macromolecules to the
lysosome (the endocytotic pathway)
• The material to be broken down in lysosomes
may be extracellular or intracellular
• Extracellular materials enter the cell either by
endocytosis or phagocytosis, depending on
the nature of the molecule.
• Receptor-mediated endocytosis is the
process by which most biologically important
extracellular substances are internalized
The endocytotic pathway
the ‘endocytotic’ pathway
• This occurs by binding to specific cell
surface receptors (Goldstein et al, 1985)
• Ligands are first delivered to early
endosomes, and then transported to late
endosomes, probably by multivesicular
bodies
• They are then delivered to the lysosomes.
the ‘endocytotic’ pathway
• Phagocytosis is the route of entry into the
cell for microorganisms and cellular debris
• Such particles are incorporated into
phagosomes, which fuse with primary
lysosomes to form secondary lysosomes
• Finally, intracellular materials undergo
autophagy
The endocytotic pathway
the ‘endocytotic’ pathway
• Although a small amount of hydrolysis
takes place in endosomes, the bulk of it
takes place in the lysosome
• This is because it is only in the acid
milieu of the lysosome that hydrolases
are active
• The low pH of the lysosome is
maintained by the vacuolar proton pump
Retrograde transport from endosomes to
the trans-Golgi network
• Some intracellular transmembrane proteins, such
as acid-hydrolase receptors, processing peptidases
and SNAREs*, undergo retrograde transport from
endosomes to the trans-Golgi network (TGN) as
part of their normal trafficking
• This retrograde-transport pathway is exploited by a
subset of bacterial and plant protein toxins to
enable them to reach the endoplasmic reticulum
and eventually the cytosol.
*Soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptors
Retrograde transport
• Recent studies have begun to unravel the molecular
machinery that is involved in this retrograde
transport.
• Acid-hydrolase receptors such as S. cerevisiae
vacuolar sorting-10 protein (Vps10) and the
mammalian mannose 6-phosphate receptors
(MPRs) are selected for retrograde transport by a
five-subunit complex named 'retromer'
• In mammalian cells, this complex is mainly
associated with tubules that emanate from vacuolar,
early–late endosomal intermediates.
Retrograde transport
• Although MPRs seem to be capable of being
transported to the TGN from both early and late
endosomal compartments, protein toxins undergo
retrograde transport exclusively from early
endosomes.
• Not surprisingly then, protein toxins and MPRs
share some components of the retrogradetransport machinery that is associated with early
endosomal compartments
Retrograde transport
• The location of molecular devices that are involved
in the retrograde transport of MPRs and protein
toxins highlights the existence of an extensive
'tubular endosomal network' (TEN)
• This TEN sorts and recycles cargoes to various
destinations, including different domains of the
plasma membrane, the limiting membrane of
lysosomes and lysosome-related organelles, and
specialized storage vesicles, in addition to the
TGN
Components of the molecular machinery that mediates retrograde transport
from endosomes to the trans-Golgi network.
RETROMER COMPLEX
• Multisubunit complex that mediates the retrograde
transport of acid-hydrolase receptors between
endosomes and the trans-Golgi network (TGN)
• Recent studies have shown low level of retromer
in affected regions of the brain of patients with
Alzheimer disease.
PAUSE
Working model of Retromer