Keywords
mucopolysaccharide storage disease - lysosome - endosomes - inflammation - autophagy
Introduction
Mucopolysaccharidoses (MPS) are a subgroup of the approximately 60 known lysosomal
disorders. There are 12 enzyme deficiencies known to cause the different types of
MPS, numbered from MPS I up to MPS IX. The first patients were described a hundred
years ago with distinct faces and cognitive deficits, hearing impairment, and multiple
skeletal changes, later defined as “dysostosis multiplex.”[1]
[2] In the middle of the last century, sugar-containing substances, the MPS, were identified
in the urine of patients, which were recognized as sugar chains with residues composed
of amino groups and acid sulfate- or acetic acid groups. With simple tests, such as
the Berry-spot test, their increased excretion in the urine of patients could be detected.[3]
[4]
[5] Correspondingly excessive storage in the lysosomes could be observed under the microscope
to further support the diagnosis of an MPS disease, for instance, with metachromatic
vacuoles in granulocytes of peripheral blood smears.[6] In the 1960s, Neufeld and Cantz recognized cell lines which corrected the pathological
storage in the lysosomes by transfer of unidentified substances in the culture media,
the so-called cross-correction by correcting factors, which they numbered systematically.[7] Soon after, they identified these factors as proteins, enzymes, degrading substances
in the lysosomes.[8]
[9]
[10] From that moment on it was clear that MPS were caused by deficient enzymes in the
lysosomes, giving rise to excessive storage of nondegraded material in the lysosomes,
which was thought to be a digesting cell organelle, a waste bag, the “cell stomach.”
Lysosomes and Their Discovery
Lysosomes and Their Discovery
Sixty years ago, the lysosomes were discovered by de Duve.[11] He and his colleagues worked on the liver enzyme glucose-6-phosphate, its action
on insulin, and its connection to the cell fraction he called the “microsomes,” consisting
of membranes found, for example, in the endoplasmic reticulum. In these experiments,
a nonspecific acid phosphatase was observed to have a very different behavior. It
was localized within sac-like particles surrounded by a membrane, which after centrifugation
was found not only in the mitochondrial fraction but also in the microsomal fraction.
He named the fraction “L” for light-mitochondrial particles gained by five-fraction
centrifugation protocol. In 1955, there were five more enzymes identified in this
fraction. Four of them were recognized as hydrolases, situated in these L-structures
with digestive function, whereas a fifth enzyme was confirmed as constituent of peroxisomes
(urate oxidase).[11]
de Duve proposed the name lysosomes for these cell organelles and recognized that
they were filled with enzymes, mainly acid hydrolases, which could perform specific
degradations in the acidic milieu of lysosomes, membrane-bound cell organelles, that
are found in human and animal cells.[11] In 1963, the first international symposium on lysosomes brought together many groups
working on the field of lysosomes. At that time, the possible involvement of lysosomes
in various pathological processes was suspected.
Lysosomal enzymes (many of them are hydrolases) can break down a wide variety of macromolecules
and cell deposits, which are delivered to the lysosomes via endocytosis, phagocytosis,
and autophagy. Their role is to digest substances into their smallest constituent
molecules to recycle them into newly created macromolecules or to excrete them via
exocytosis.[11]
Lysosomes constitute about 5% of the cell volume and can differ in shape and size.
Any disturbance of the degradation processes, irrespective of which macromolecules
are involved, leads to an increased size and number of these cell organelles, which
can be representative of the disease. This is seen in all MPS, as well as in related
disorders such as glycoproteinoses (i.e., fucosidoses, α-mannosidoses, aspartylglucosaminuria),
neurolipidoses (i.e., Sandhoff disease, Niemann–Pick disease, Tay–Sachs disease) or
in Gaucher disease, Fabry disease, and numerous other lysosomal storage disorders
(LSDs). Many of them exhibit distinct features of the deposited material, such as
foamy cells or sea-blue histiocytes in Niemann–Pick disease, Gaucher cells with striped
cytoplasma, large foamy histiocytes in Wolman's disease, Gasser cells with purplish,
metachromatic or Alder granules in MPS, periodic acid-Schiff (PAS) staining-positive
vacuoles in fucosidosis, or curvilinear bodies in Batten's disease.[6]
Lysosomes and Mucopolysaccharidoses
Lysosomes and Mucopolysaccharidoses
The group of MPS disorders is comprised of 12 different enzyme deficiencies, most
of them affecting hydrolases involved in the degradation process of glycosaminoglycan
(GAG) chains. All involved lysosomal enzymes are produced by the endoplasmic reticulum
and are subsequently glycosylated in the Golgi apparatus, where they are phosphorylated
with a mannose-6-phosphate (M6P) residue as recognition marker.[12] This marker enables the enzymes to be transported from the trans-Golgi network into
the lysosomes via a mannose-6-phosphate receptor (M6PR) situated in the lysosomal
membrane. Once there, hydrolases remove the M6P residue, which may be recycled to
the trans-Golgi network to bind newly synthesized enzymes. The lysosomal membrane
in total contains more than a hundred proteins responsible for the trafficking between
lysosomes and the cytoplasm of the cell. The membrane has many functions, such as
chaperone-mediated autophagy, transport of the hydrolases into the lysosome, and contains
transporters and channels for amino acids, sugars, and lipids.
Enzyme Deficiency as Cause of Mucopolysaccharidoses
Enzyme Deficiency as Cause of Mucopolysaccharidoses
Each of the GAG degradation steps is performed by a specific lysosomal enzyme.[12] If one of the enzymes has reduced activity or is missing, the process of digestion
necessarily ends prematurely and none of the following steps will be performed. The
cause of lysosomal enzyme deficiencies relies on the presence of different types of
mutations in their respective genes. One possible outcome is a missense mutation,
which leads to the exchange of a single amino acid in the protein chain. This can
lower enzyme activity, limit half-life of the enzyme and thus its availability, make
it vulnerable to the acidic milieu in the lysosome, and cause it to be digested sooner.
In MPS, enzyme activity is typically lowered but residual activity remains. Other
types of mutations such as stop-mutations, deletions, or duplications are responsible
for a usually significant shortening of the protein chain, which cannot be processed
in the Golgi apparatus or transported to the lysosome and/or can be eliminated by
autophagy. In this case, no residual enzyme activity can be measured.
With the exception of the X-linked MPS II (Hunter's disease), all other MPS types
are inherited by an autosomal recessive trait. Patients with the recessive forms of
MPS have a high number of different gene mutations which affect in many combinations
both alleles and the amount of the produced enzyme proteins. Therefore, a wide range
of enzyme activity may be found, from zero to several percent of the normal activity,
which explains the wide spectrum of clinical severity one can observe from most severely
affected patients with first signs in utero to attenuated forms of MPS in adulthood.
GAGs are not only the main ingredient of the extracellular matrix of the connective
tissue but also necessary for the proteoglycan chains of the glycocalyx of cell surfaces.[12] Depending on their localization, their composition varies but is quite often distinctive
for a specific tissue or organ. GAGs are heparan sulfate (HS), dermatan sulfate (DS),
chondroitin sulfate (CS), keratan sulfate (KS), and hyaluronic acid (HA).[13] Sometimes, lysosomal enzymes can catalyze reactions in different types of GAG chains,
for instance, the deletion of the sugar iduronic acid in HS as well as DS, as in MPS
I, MPS II, or MPS VI.[14] Other lysosomal enzymes only involve the degradation process of HS (MPS III) or
KS (MPS IV).[15]
[16] Any enzyme deficiency leads to an accumulation and storage of fragments of the involved
GAG chain(s), an increase in lysosomes in size and number, as well as cell size. In
the long run, this damages cells and causes apoptosis; thus, all the accumulated fragments
will enter the bloodstream and be excreted into the urine of patients. These fragments
can help to identify the chemical properties of the excessively stored material, allow
to identify the type of MPS, and act as a biomarker.[15]
[16]
[17]
Lysosomes and Other Functions
Lysosomes and Other Functions
In 1983, de Duve redefined the lysosomes as polymorphic particles with differing size,
shape, and internal structure.[18] New discoveries regarding the cell surface were the observation of receptors/ligands
and the receptor-mediated fluid-phase nonselective endocytosis (pinocytosis, protocytosis)
or phagocytosis. In this process, material is first incorporated by temporary storage
in inactive vacuoles (first named as receptosomes or endosomes), which subsequently
fuse with lysosomes or regurgitate their contents out of the cell.[18] The sorting process, which determines what substances should be incorporated into
a cell and transported to/fused with the lysosome, is not only regulated by the cell
surface as well as endosomes, but also influenced in part by (1) the lysosomes, as
they are involved in membrane recycling, and (2) somewhat indirectly via the Golgi
apparatus. Membranes are necessary for all the vesicles involved in the digestion
processes and may be reused again. There is evidence that lysosomes are no longer
the place of no return for incorporated membrane material. However, the amount of
recycled membrane material is probably not very high. It was observed that heavily
vacuolated cells may be immobilized by high amounts of membrane material, which can
interfere with the recycling process.
Lysosomes help to degrade endocytosed extracellular material as well as intracellular
substances sequestered by autophagy. In the latter case, lysosomes fuse and form autolysosomes
and phagolysosomes.[19] Many proteins are involved in this machinery of trafficking, interacting, and maintaining
the charge difference of ions between the cellular cytoplasm and lysosomes. Lysosomes
can fuse with the plasma membranes of the cells to repair them or defend against parasites
too big to be tackled by single cells. The integrity of the lysosomal membrane is
also protecting the cell from autolytic processes. But lysosomal overloading with
stored material can induce lytic properties against cellular structures and result
in apoptosis, necrosis, and cell death.
Lysosomes and Cell Signaling
Lysosomes and Cell Signaling
The cytosolic site of the lysosomal membrane is involved in signaling complexes, especially
the mammalian target of rapamycin complex 1 (mTORC1) and small GTP-ases of the RAG
family. The mTORC1 complex regulates the cellular metabolism arising from nutrients,
energy, and growth factors. As part of this complex, mTOR can influence the transcription
factor EB (TFEB) via phosphorylation, which in turn influences the transcription of
genes encoding lysosomal proteins leading to an upregulation. TFEB plays an important
role in the exocytosis pathway of lysosomes, as they move closer to and fuse with
the plasma membrane to excrete their contents, removing stored material from the cell
amounting to a rescue from lysosomal overloading.[20] For this process, TFEB induces an increase in intracellular calcium levels through
the activation of the lysosomal calcium-channel Mucolipin 1. This physiological cell-saving
and clearing function can reduce accumulation of pathogenic stored proteins as could
be observed in experiments for Huntington's disease or Parkinson's disease.[21] Protein aggregates in these two diseases as well as Alzheimer's disease can cause
severe neurodegenerative disorders and it appears to be a promising therapeutic approach
to clear the cell via lysosomal autophagy, which will then degrade and excrete the
aggregates, rescuing the cell from any excessively stored material.[22]
Autophagy
Autophagy is an essential and physiological process for homeostasis in each cell and
depends on lysosomes. Autophagy declines with age but also in disease, for instance,
LSDs.[23] Excessive storage of material in the lysosomes impairs the process of autophagy
in each cell, disturbs the elimination of damaged cell components, aggregated proteins,
and other components; hampers any recycling processes; and diminishes energy metabolism
and homeostasis in each cell.[24] Selective autophagy, such as aggrephagy, is responsible for clearing cells of misfolded
proteins, whereas mitophagy and ribophagy take care of dysfunctional mitochondria
or defective ribosomes. Lipophagy removes lipid droplets, with reduced clearance observed
in atherosclerosis or hepatic steatosis. Glycophagy degrades accumulated glycogen
and xenophagy prevents cell damage by removing pathogens such as viruses, bacteria,
and parasites. Mitophagy also reduces DNA damage as it suppresses the oxidative stress
by reducing mitochondrial reactive oxygen species .[25] All these cellular autophagy steps are promoting cell survival and should be understood
as a process for better cell survival.
Although in LSDs the underlying gene defects, the impaired metabolic pathways, the
stored material, and the speed of accumulation may vary, the result looks the same:
the cell clearance and autophagy are reduced, energy and homeostasis are impaired,
and survival of cells is endangered. Several late-onset neurodegenerative disorders
and neuronopathic LSDs, such as neuronal ceroid lipofuscinoses, sphingolipidoses,
or glycogenosis type II (Pompe's disease) have shown to be very susceptible to effects
on lysosomal storage and defective autophagy.[23] This might be explained by the high metabolic requirements of neurons, their shape
and polarization, the protein-clearing processes in these long-lived cells, or the
disturbed function in myocytes due to vacuoles filled with stored material.
Lysosomes and Their Role in Drug Uptake
Lysosomes and Their Role in Drug Uptake
The accumulation of stored material in lysosomes can result not only from defective
degrading enzymes but also from disturbed transport function of transporter proteins
or channels in the lysosomal membrane. Independent of the underlying cause, the function
of lysosomes, and sooner or later of other cell organelles as well, is impaired, leading
to reduced autophagy, induced inflammation, and altered calcium homeostasis. Therefore,
an enhanced clearance by TFEB overexpression could restore the defective cell metabolism.
The enhancement of hydrolases synthesis and maturation might help to slow down or
even reverse the storing process.
Often in LSDs, the intralysosomal ion concentration is altered and the lysosomal pH-value
is aberrant. This has additional effects within the lysosomes, such as altered activity
of all enzymes as they depend on the acidic milieu, as well as reduced intracellular
autophagy and impaired cellular trafficking. To modulate the cellular and lysosomal
calcium levels might be an efficient strategy to influence degradation and clearance
of accumulated macromolecules.[26]
Substances bound to the cell surface are taken up by endocytosis and delivered to
the lysosomes for digestion in most cases. Packing and conformation of these substances
may play a role in their effective degradation by the lysosomal milieu. There is clear
evidence that under physiological conditions, lysosomal enzymes are synthesized in
the endoplasmic reticulum and marked with M6P. There are two different M6P receptors
identifying the M6P residue, designated MPR46 and MPR300. Usually, M6P containing
enzymes are transported via clathrin-coated vesicles to the endosomes, where the acidic
milieu induces the dissociation of the M6P–ligand complex which is then recycled to
the trans-Golgi network or to the cell surface. But approximately 5 to 20% of the
newly synthesized enzymes escape this cell sorting mechanism and are secreted from
the cell into the extracellular space from where they may be recaptured again by M6PR
on the cell surface and transported to the lysosomes.[27] This effect was the basis of the experiments of Neufeld and coworkers: The escape
of enzymes from cells of one MPS type and their recapture by the M6P receptors on
the cell surface of cells with a different MPS type, and therefore a different enzyme
defect, resulted in the reciprocal cross-correction of excessive storage in the combined
cell culture of fibroblasts derived from different MPS patients.[7]
[8] The same basic principle is used in enzyme replacement therapy (ERT). Patients get
periodical enzyme-rich infusions of recombinant, synthesized enzymes which can be
captured by receptors on cell surfaces and incorporated into cells and lysosomes,
where they alleviate enzymatic deficiencies and help diminish stored material.
Besides these well-known pathways for lysosomal trafficking of hydrolytic enzymes,
there is clear evidence that there are alternate, M6P-independent pathways for lysosomal
enzyme sorting. Markmann et al showed that non-phosphorylated enzymes (without M6P)
were secreted from cells and could be recaptured via low-density lipoprotein (LDL)
receptor and LDL receptor-related protein 1, which might impact on improving efficiency
of ERT in different LSDs. Furthermore, other sugar receptors on cell surfaces, such
as asialoglycoprotein, mannose, or fucose receptors, might play a role in the uptake
of lysosomal enzymes.[27]
Lysosomal Storage Disorders as Inducers of Inflammation
Lysosomal Storage Disorders as Inducers of Inflammation
Immune system abnormalities and irregularities are observed in LSDs. There are various
abnormalities, such as increased production of autoantibodies (Gaucher disease, Fabry
disease), increased immune-mediated reactions in MPS, or an inappropriate activation
of microglia in neurodegenerative foci of Niemann–Pick disease.[28] The impaired cell metabolism in LSDs leads to disturbances in autophagy, impaired
autophagosome–lysosome fusion, accumulation of undegraded macromolecules in the cell,
dysfunction of other cell organelles such as mitochondria, and manifests with increased
expression of chemokines/cytokines, increased signs of inflammation and neovascularization.
Especially in Gaucher disease, the consistently increased levels of proinflammatory
cytokines and chemokines as well as the chronic stimulation of the immune system may
be responsible for the production of monoclonal and polyclonal immunoglobulins, which
result in the development of multiple myeloma in these patients.[28]
The accumulation of GAGs in MPS starts a cascade of intracellular responses, such
as metabolic, inflammatory, and immunological effects in several organs. Especially
the immune system and the skeleton share some of the same regulatory molecules, such
as classical cytokines, receptor proteins, adaptor proteins, transcription factors,
and signal transducers influencing the interplay between bone-forming osteoblasts
and bone resorbing osteoclasts, which can be observed in severe changes of bone formation
and rheumatoid-like chronic arthritis of many joints with increasing stiffness and
contractures. The mechanism of the aberrant activation of neuroinflammatory responses
in those MPS types with predominant HS storage is not yet fully understood, but in
animal models with MPS type IIIB the administration of oral corticosteroids as immunosuppressive
therapy was noticed to be beneficial.[28]
Conclusions
This review only partially highlights some of the currently held views of LSDs and
their multisystemic consequences, but should give a glimpse of the complex function
of lysosomes in the cell. It is no longer considered just a “bag for the digestion”
of undegraded and therefore stored material, but has been shown to be a crucial component
in many processes such as autophagy, immune response, cell energy metabolism, oxidative
stress, and many more. There appears to be great potential to develop therapies and
administer drugs on multilevel targets to improve the clinical signs and symptoms
of patients with MPS. Successful stem cell transplantation or the continuous administration
of ERT might improve some of the clinical symptoms, but the future in therapy will
be as multisystemic and complex as the MPS themselves are.
