Alessandra d’Azzo, PhD
Department of Genetics
St. Jude Children’s Research Hospital, Memphis TN USA
GALACTOSIALIDOSIS
Galactosialidosis (GS) is a neurosomatic lysosomal storage disease belonging to thegroup of the glycoproteinoses. The disease is inherited as an autosomal recessive trait
and is caused by genetic lesions in the CTSA gene encoding the lysosomal Protective
Protein/Cathepsin A or PPCA. The cathepsin A enzyme’s catalytic activity is distinct
from its protective function towards β-galactosidase (β-GAL) and neuraminidase 1(NEU1), with which PPCA forms a complex. In this configuration the two glycosidases
acquire their full activity and stability in lysosomes. Deficiency of PPCA results incombined NEU1/β-GAL deficiency.
GS is a prototypical lysosomal storage disease (LSD) of glycoprotein catabolism and it is
one of the few LSDs caused by a primary defect in one of the lysosomal cathepsins.
However, the secondary severe loss of NEU1 activity probably accounts for most of theovert clinical manifestations seen in patients and for the disease pathogenesis. As for
sialidosis, caused by primary deficiency of NEU1, GS primarily affects cells of thereticuloendothelial system.
Based on the age of onset and the severity of the symptoms, patients with GS areusually classified into three clinical types.
Early Infantile GS
The early infantile type of GS presents with signs of the disease between birth and 3months of age; these include non-immune hydrops fetalis, edema, coarse facies,
proteinuria and telangectasias. In addition, these patients develop visceromegaly,
skeletal dysplasia, renal and cardiac failure and variable neurological involvement.
Ocular abnormalities, including corneal clouding and cherry-red spot, and heart
involvement with cardiomegaly and thickening of the septum have been seen in a
number of patients. These very severe patients die within the first year of life likely
because of heart and kidney failure. Importantly, early infantile type of GS may be
associated with fetal loss.
Late Infantile GS
The late infantile type of GS comprises a distinct group of patients characterized bymild or absent cognitive impairment. Patients usually present with phenotypicalterations and symptoms within the first 2 years of life and they slowly progress intoadulthood. These include coarse facies, hepatosplenomegaly, dysostosis multiplex,
especially of the spine, and growth retardation associated with muscular atrophy. Heartinvolvement with thickening of the mitral and aortic valves, and hearing loss arerecurrent features. Cherry-red spots and corneal clouding can occur in some patients,
while seizures and overt neurologic signs are very rare. Many of the patients diagnosed
with the late infantile phenotype survive into adulthood and are still alive. Therefore,
they may develop additional kidney and pulmonary complications.

Alessandra d’Azzo, PhD
Department of Genetics
St. Jude Children’s Research Hospital, Memphis TN USAJuvenile/AdultThe majority of GS patients belong to the juvenile/adult type. The reason for the dual
designation is that their age of onset and clinical course vary greatly and patients
present with a broad and continuous spectrum of severity of their symptoms . They aremostly of Japanese origin. In contrast to the late infantile types, patients with thejuvenile/adult form of GS have a more severe clinical presentation. Besides
characteristic features described above, patients develop severe neurologic
manifestations, including myoclonus, cerebellar ataxia, generalized seizures, progressive
cognitive impairment and mental retardation.
As it is the case for sialidosis , a few examples of atypical patients with confirmed
diagnosis of GS but no oligosacchariduria have been reported. These findings highlightthe fact that GS should be taken into consideration for some of the undiagnosed cases,
even in the absence of this diagnostic marker of the disease.
A total of 27 mutations in the CTSA gene have been reported, including smalldeletions/insertions, missense mutations, splicing variants and only one nonsense
mutation.
(http://www.hgmd.cf.ac.uk/ac/index.php; http://www.ncbi.nlm.nih.gov/clinvar;
Coutinho MF et al., 2011 https://www.ncbi.nlm.nih.gov/pubmed/21214877
Caciotti et al 2013 https://www.ncbi.nlm.nih.gov/pubmed/23915561;
Annunziata and d’Azzo 2017 http://www.ncbi.nlm.nih.gov/pubmed/28603679)
Aside from its cathepsin A activity at acidic pH, PPCA also functions as a deamidase
and esterase at neutral pH. In vitro, the enzyme can deamidate selectedneuropeptides, such as substance P and neurokinin, and can function as
carboxypeptidase on oxytocin-free acid, bradykinin, and endothelin I (ET-1). All
enzymatic activities of PPCA were shown to be drastically reduced in lymphoblastoid
cells and fibroblasts from numerous GS patients. In addition, it was also demonstrated
that an enzyme hydrolyzing the C- terminus of ET-1 was deficient in tissues from a GS
patient, a finding that implicates cathepsin A activity in the degradation of endothelin I(ET-1) in human tissues. Interestingly, increased levels of cathepsin A in heart tissue in anengineered mouse model induced the upregulation of several other cathepsins. Incontrast, inhibition of cathepsin A activity has protective properties and the use of thisapproach has been proposed for the treatment of heart failure after myocardial
infarction. More recent studies have also showed that the activities of cathepsin A andthe cathepsin A homolog, serine carboxypeptidase Scpep1, are both needed toregulate ET-1 levels and to control vasoconstriction, hyper-proliferative corneal
dystrophy and abnormal skin thickening. It is therefore becoming increasingly clear thatlysosomal enzymes like cathepsin A can directly or indirectly been implicated in multiple
biological processes that may shed light on the complexity of phenotypic alterations inpatients.
(Kase et al 1990 https://www.ncbi.nlm.nih.gov/pubmed/2244901
Jackman et al 1992 https://www.ncbi.nlm.nih.gov/pubmed/1694176
Seyearantepe et al., 2008 https://www.ncbi.nlm.nih.gov/pubmed/18391110
Pan X et al., 2014 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3937211

Alessandra d’Azzo, PhD
Department of GeneticsSt. Jude Children’s Research Hospital, Memphis TN USA
Pan X etal., 2017 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5325571
Annunziata and d’Azzo 2017 http://www.ncbi.nlm.nih.gov/pubmed/28603679)
PPCA and its relationship with NEU1
Extensive biochemical studies have been done on complex formation and mode ofassembly of PPCA and NEU1; although a full understanding of the way these proteinsinteract awaits the resolution of the 3 dimensional structure of the complex. Detailed
information about these studies can be found in the following papers:(Bonten et al 2009 http://www.ncbi.nlm.nih.gov/pubmed/1966647;
Bonten et al 2014 http://www.ncbi.nlm.nih.gov/pubmed/24337808)
Mouse model of GS
A mouse homozygous for a null mutation at the Ppca (Ctsa) locus was one of the first
genetically engineered animal models for an LSD. These mice have a clinical andpathological presentation that closely recapitulates the early onset forms of GS. Tissues
and cells isolated from mutant mice have no cathepsin A activity and severe
secondary deficiency of Neu1, while β-Gal is partially reduced only in some cell types.
Extensive morphological changes are recognizable already in the first weeks of life, withsevere vacuolation and lysosomal expansion in some cells of most systemic organs andthe central and peripheral nervous system. As the disease primarily affects thereticuloendothelial system, pathologic changes are first detected in tissues and organs
of epithelial origin. Similarly to patients with the severe form of the disease, mutant micepresent with severe early-onset nephropathy, associated with edema and proteinuria,
in addition to oligosacchariduria. Time-dependent splenomegaly and heartinvolvement are also characteristic features of the disease. Homozygous knockouts areinfertile, because of structural changes in the blood-epididymal barrier, resulting inaltered sperm motility, and have a reduced lifespan of ~6–9 months, although genderdifferences have been documented. The complete loss of Neu1 activity explains why
many phenotypic abnormalities in Ppca KO mice are similar to those seen in the Neu1
KO model, although on close examination features that are unique for one or the otherdisease model have been identified. The most overt difference is seen in thecerebellum; early in life Ppca KO mice acquire acute and progressive ataxia that is
associated with regional loss of cerebellar Purkinje cells and impaired motor
coordination. This phenotype is not observed in the Neu1 KO mice at least not until theend of their lifespan (5–7 months). Because the expression levels of PPCA in Purkinje cells
are greater than those of Neu1, it is possible that these neurons are more sensitive to theloss of cathepsin A activity than of Neu1 activity, but more rigorous testing is needed tocorroborate this hypothesis.
(Zhou et al 1995 http://www.ncbi.nlm.nih.gov/pubmed/7590240;
de Geest et al 2002 http://www.ncbi.nlm.nih.gov/pubmed/12023988;
Annunziata and d’Azzo 2017 http://www.ncbi.nlm.nih.gov/pubmed/28603679)
PPCA regulates chaperone-mediated autophagy
A serendipitous finding gave the first indication of an in vivo physiological role of thecathepsin A activity of PPCA. The enzyme was found to co-purify with the lysosomal
associated membrane protein 2a (LAMP2a) from lysosomal preparations of rat liver.

Alessandra d’Azzo, PhD
Department of Genetics
St. Jude Children’s Research Hospital, Memphis TN USA
LAMP2a is one of three isoforms of LAMP2 that are generated through alternative
splicing of its mRNA. They are highly homologous, differing only in the composition of
their transmembrane domain and short carboxy-terminal cytoplasmic tail; their heavily
glycosylated, luminal domains are identical. The LAMP2a is the only isoform that serves
as a receptor for chaperone-mediated autophagy (CMA). CMA is activated inresponse to cellular stress and promotes lysosomal internalization and degradation ofcytosolic protein substrates. Human and mouse GS fibroblasts showed increased levelsof CMA. Thus, Ppca-deficient cells are actively undergoing CMA.
(Cuervo et al., 2003 http://www.ncbi.nlm.nih.gov/pubmed/12505983)
Therapy for galactosialidosis
The availability of a faithful animal model for GS has been instrumental for testing
investigative therapies; they include bone marrow transplantation, enzyme
replacement therapy (ERT), bone marrow-mediated ex vivo and in vivo gene therapy.
All these experimental approaches have been overall very successful in correcting theclinical manifestations characteristic of the disease, including partial reversal of thebrain pathology. These promising results have spearheaded the development byUltragenyx Pharmaceutical Inc. of an ERT therapy using a recombinant human PPCA
(rhPPCA). The rhPPCA was s taken up by deficient human fibroblasts via the mannose-6phosphate receptor pathway and rescued NEU1 and β-GAL activities. An in vivo proof
of concept study in PPCA KO was conducted to evaluate the efficacy of rhPPCA via
biweekly intravenous administration for 8 weeks. The study had as goals to evaluate thetissue distribution of rhPPCA and normalizitation of the NEU1 and β-GAL activities; todemonstrate reduction of lysosomal storage in affected tissues and to demonstrate
reduction in accumulation of sialylated glycoconjugates in urine. Dose-dependent
increase in cathepsin A activity in affected tissues such as liver, spleen, kidney andheart was observed; interestingly, increased cathepsin A activity was also measured in
the brain, although to a smaller extent. Overall, rhPPCA treatment corrected NEU1activity and normalized β-Gal activity in multiple tissues. In the group of mice thatreceived the highest amount of protein (20 mg/kg rhPPCA), the vacuolization
characteristic of GS was no longer evident. Finally, the treatment resulted in decreased
total sialic acid in urine.
These findings should encourage the development of an ERT-mediated clinical trial forthe treatment of the mild late infantile group of GS patients with no neuropathic signs.
These findings were also presented at several conferences during the last years
including the ISMRD family conferences in July 2015 and November 2017.
(Zhou et al 1995 http://www.ncbi.nlm.nih.gov/pubmed/7590240;
Hahn et al 1998 https://www.ncbi.nlm.nih.gov/pubmed/9843984;
Leimig et al 2002 https://www.ncbi.nlm.nih.gov/pubmed/11964280
Bonten et al 2004 https://www.ncbi.nlm.nih.gov/pubmed/15084520
Hu et al 2012 http://www.ncbi.nlm.nih.gov/pubmed/22008912;
Annunziata and d’Azzo 2017 http://www.ncbi.nlm.nih.gov/pubmed/28603679)

Alessandra d’Azzo, PhD
Department of Genetics
St. Jude Children’s Research Hospital, Memphis TN USAIn conclusion, despite the rarity and complexity of galactosialidosis, remarkableprogress has been made over the last decade towards a clinical trial for the patients. If
the bench work will continue at this pace, I am inclined to predict that additional newfunctions for PPCA will be uncovered that might open new possibilities for alternative
therapies. In this respect I believe that the most helpful hints on the roads to suchdiscoveries often come from the patients themselves and their families. Their insightfuland courageous drive is a daily encouragement for us scientists to move ahead andkeep looking forward.