1. Figure 1: GBA activity was assessed using the 4-MUG whole-cell assay in both TS207 parent and
GBA knockout neuroglioma cell lines. TS207 GBA knockout cells have consistently low to no amounts
of activity compared to TS207 GBA parental cells. Activity decreases with CBE treatment, a potent,
selective, irreversible inhibitor of GBA, in a dose-dependent manner at 30 minutes (A). This effect is
already exacerbated at 24 hours (B). A dose response of isofagomine or ambroxol has little effect
after 30 minutes of incubation (A); however, with extended treatment of the drug after 24 hours, a
dose responsive increase with ambroxol is seen (B). This also provided information that ISF may
need a longer incubation time in these cells in order for an effect to be visualized in GBA activity.
Profiling Glucocerebrosidase (GBA) Levels and Activity in Mutant B Lymphocytes, Mutant
Fibroblasts, and A53T Transgenic Mouse Cortical Neurons
Zack Brittingham1, Paula Loos1, Peter Buckett1, Warren Hirst1
1Neurodegeneration & Neurological Disease, Neuroscience Research Unit, Pfizer, 610 Main Street, Cambridge MA
Fig. 2: GBA mutant B lymphocytes in Table 1 were tested for endogenous levels of GBA by western
blot (A) and the results were quantified (B). TS207 GBA knockout and parent cells acted as positive
and negative controls. GBA mutations L444P and N370S overall have less GBA, however there are
exceptions which is an example of donor-to-donor variability.
ABSTRACT
Parkinson’s disease (PD) is a neurodegenerative disorder characterized phenotypically by
loss of balance, muscle rigidity, and body tremors, and biochemically by the presence of
Lewy bodies, or fibrillar aggregates of α-synuclein protein in the brain.1 Glucocerebrosidase
(GBA) is a lysosomal hydrolase involved in breaking down the glycolipid glucocerebroside
into ceramide and glucose, and mutations in this enzyme are the most common genetic risk
factor of PD, as there has been a recent association with decreased GBA activity and
increased α-synuclein levels.2 Chaperones are proteins that assist in the folding or unfolding
of select proteins, seen more prevalently in disease models or in structures that require
protein-folding assistance. Chaperones are also involved in the prevention of aggregation of
protein subunits, ensuring they maintain their functionality.3 Therapeutically, chaperone
therapy has been discussed as a potential treatment for Gaucher disease.4 The protein GBA1
has had known associations with Gaucher disease, where build-up of the glucocerebroside
substrate can result in this rare autosomal recessive lysosomal storage disorder, and
Gaucher disease and Parkinson’s disease have many biochemical and morphological
similarities.2 Specifically, these chaperones could be used to select for and bind to mutant
GBA, thereby stabilizing it and ensuring proper folding and trafficking from the ER to the
lysosome.5 Isofagomine (ISF) has been shown to both stabilize and promote lysosomal
trafficking of GBA, even though it has poor selectivity as an iminosugar with the possibility to
fluctuate between reversible inhibition of enzymatic activity and improving protein
translocation.5,7 Additionally, ambroxol (ABX) has been shown to increase GCase activity in
both mutant fibroblasts and healthy controls by binding and stabilizing the protein.8,9 It has
been used preliminarily in enzyme enhancement therapy for Gaucher, and has been shown
to interact with both the active and non-active site residues of GBA.9 Our research has been
targeting activators or chaperones of GBA in order to increase GBA levels and/or activity in
the lysosome in-vitro, thus reducing the amount of glucocerebrosidase substrate and the
amount of α-synuclein aggregates while restoring lysosomal function.
Table 1: B Lymphocyte Lines, Demographic, & Genetic Information
from Coriell Institute
4-MUG WHOLE CELL ASSAY
Glucocerebrosidase activity was measured using the non-native substrate 4-
methylumbelliferyl-β-glucuronide (4-MUG) that cleaves when it binds to the active site of
GBA. The cleaved form of 4-MUG is 4-MU, or 4-methylumbelliferone, which fluoresces.
 Assay buffer: 50% PBS (pH 7.4), 50% NaOAc (pH 4.0), 1.25mM 4-MUG
 Cells cultured in 96-well plates; media aspirated before adding 50uL assay buffer, 1 hour
incubation at 37oC, add 150uL glycine stop buffer (pH 10.8)
 Bottom-read fluorescence: ex 355nm, em 450nm
REFERENCES
1. Smith, Wanli W. et. al. “Endoplasmic reticulum stress...” Hum. Molec. Genet. 2005, 14(24), 3801-3811.
2. Siebert, Marina et. al. “Glucocerebrosidase is shaking...” Brain: Jour. of Neurosci. 2014, 137, 1304-1322.
3. Berg. Biochemistry 7th ed. 2006.
4. Ellis, RJ. et. al. “Molecular Chaperones” Annu. Rev. Biochem. 1991, 60, 321-347.
5. Khanna, Richie et. al. “The Pharmacological Chaperone Iso…” FEBS Jour. 2010, 277(7), 1618-1638.
6. Sun, Ying et. al. “Ex Vivo and In Vivo Effects of Isofagomine...” Jour. of Biol. Chem. 2012, 287, 4275-4287.
7. Patnaik, Samarjit et. al. “Discovery, Structure-Activity...” Jour. of Med. Chem. 2012, 55, 5734-5748.
8. McNeil, Alisdair et. al. “Ambroxol improves lysosomal...” Brain: Jour. of Neurosci. 2014, 137, 1481-1495.
9. Maegawa, GH. et. al. “Identification and characterization...” J. Biol. Chem. 2009, 284(35), 23502-23516.
ACKNOWLEDGEMENTS
I would like to take the opportunity to thank my advisor Paula Loos for the instruction she provided and
dedication she took to assist in my learning. Additionally, I would like to thank everyone on the GBA Team and
otherwise at Pfizer for providing me an invaluable educational opportunity.
Cell Line Disease Model GBA Sex Age
GM10870 GD, Type I N370S / N370S F, Caucasian 63
GM10871 GD, Type I N370S / N370S F, Caucasian 71
GM10873 GD, Type I N370S / N370S M, Caucasian 79
GM08752 GD, Type II L444P/L444P M, Mexican 1
GM08753 No GD L444P/WT F, Mexican 25
ND05256 Healthy Wild-type F, Caucasian 66
ND00700 Healthy Wild-type F, Caucasian 74
AG10097A Healthy Wild-type M, Caucasian 52
Cell Line Disease Model GBA Sex Age
Detroit 551 Healthy Wild-type F Fetal
ND37180 PD N370S / WT M, Caucasian 80
GM08760 GD, Type II L444P/L444P M, Mexican 1
AG06858A Healthy Wild-type M, Black 47
GM00288B Healthy Wild-type M, Caucasian 64
Table 2: Fibroblast Lines, Demographic, & Genetic Information from
the Coriell Institute
Fig. 6: LAMP1 levels of
GM08760 (L44P h0),
GM00288B (WT), and
AG06858A (WT) fibroblasts
with varying concentrations of
ISF for 6 days, including some
where ISF was washed out for
the last 24 hours. LAMP1 levels
are compared to Actin and the
DMSO vehicle. It is shown that
LAMP1 levels among diseased
and wild-type fibroblasts
remain fairly constant with ISF
treatment. LAMP1 increases
can be associated with
lysosomal biogenesis, that of
which we do not see here.
Fig. 4: Mutant fibroblasts were tested for endogenous GBA levels (DMSO vehicle) and treatment
with ISF using western blot (A) and the results were quantified (B). Healthy donor Detroit 551
fibroblasts have more GBA than both PD (ND37180) and GD (GM08760) diseased fibroblasts. 6 Day
treatment with ISF increases levels of GBA in a dose-dependent manner. CBE, a GBA inhibitor, has
little to no effect on endogenous levels of GBA.
Ambroxol Isofagomine
GBA ACTIVTY IN TS207 CELL LINE WITH GBA KNOCKOUT CONTROL
A) B)
GBA LEVELS IN MUTANT HUMAN B LYMPHOCYTES
GBA LEVELS IN MUTANT HUMAN FIBROBLASTS
GBA ACTIVITY IN MUTANT B LYMPHOCYTES
A)
B)
LAMP1 LEVELS ARE NOT AFFECTED BY ISOFAGOMINE
GBA ACTIVITY IN MUTANT HUMAN FIBROBLASTS
A53T TRANSGENIC MOUSE CORTICAL CULTURES HAVE DECREASED
LEVELS OF α-SYNUCLEIN AFTER CHAPERONE TREATMENT
Fig. 7: A53T mutant α-synuclein is overexpressed in the neurons of transgenic A53T mice. Cortical
cultures were prepared and doses of ABX & ISF were adminstered at day 11 for 6 days, after which
some treatments were washed out (WO) for an additional 3 days. α-Synuclein was specifically
detected by western blot (A, C) in A53T mice but not in wildtype (WT) neurons. Treatment with ABX
and its washout counterpart significantly decreased levels of A53T α-synuclein. ISF, which has a
higher binding affinity than ABX, is proposed to still be bound to GBA in the ISF-treated cells,
therefore not affecting levels of A53T α-synuclein since GBA is inactive. When ISF is washed out (ISF
WO), the GBA that was chaperoned to the lysosomes is no longer inhibited and is free to digest the
A53T mutant α-synuclein resulting in decreased levels. Caspase-3, which serves as an indicator for
apoptosis, was detected by western blot (B, D) in the same samples, and demonstrates a possible
increase in cell health compared to the vehicle with ABX treatment.
WESTERN BLOT
Glucocerebrosidase, LAMP1, and caspase-3 levels were measured using western blot &
quantification with normalization to Actin
 Cells were lysed with a triton lysis buffer (0.25% triton, 250mM sucrose, 10mM tris pH 7.5,
& 1mM EDTA) along with phosphate and protease inhibitors (PI, PH2, PH3, & PMSF)
 Samples were prepared with 25% NuPage LDS Sample Buffer (4x), 10% NuPage Reducing
Reagent (10x), and 65% sample a protein diluted with universal lysis buffer
 Samples were vortexed, heated for 10 mins. at 70oC, spun to eliminate condensation, & run
in 20-well 4-12% Bis-tris NuPage Novex gels in MES SDS running buffer (20x) at 200V
 Blots were transferred using iBlot nitrocellulose for 7 mins, rinsed in 0.1% Tween-20 in TBS
(TTBS), one hour RT in Rockland blocking buffer, overnight at 4oC in 1o Ab, rinsed in TTBS, 1
hour RT in 2o Ab, rinsed in TTBS, scanned using Licor Odyssey
Casp3
C)
B)
B)
A)
Fig. 5: Endogenous GBA activity measured with 4-
MUG substrate of Detroit 551 (WT), ND37180
(N370S/WT), and GM08760 (L444P/L444P) fibroblasts.
Healthy donor Detroit 551 fibroblasts have more GBA
activity than both PD (ND37180) and GD (GM08760)
diseased fibroblasts, with the L444P homozygous cell
line having a very small amount of relative GBA activity.
Fig. 8: α-Synuclein overexpression has been shown to inhibit translocation of GBA to the lysosome
from the ER, where aberrant GBA fails to fold correctly and is degraded. This results in decreased
proteasome activity and creates an influx of ROS, leading to ER and mitochondrial stress through
activation of PERK, an ER transmembrane kinase, & the unfolded protein response (UPR). Chaperone
synthesis is stimulated to assist with cell stress, ensuring misfolded proteins are removed.3 If ER stress
is too much for the cell, PERK leads to activation of caspase-3, a marker for cell apoptosis.1
Fig. 3: GBA mutant B lymphocytes,
the characteristics of which are
exemplified in Table 1, were tested for
endogenous GBA activity levels.
Although there is donor-to-donor
variability, the N370S homozygous
mutants demonstrate less GBA activity
in comparison to the wild-type cells.
A)
WT A53T
D)