Neuropharmacology 202 (2022) 108822

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Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm

New therapeutic approaches to Parkinson’s disease targeting GBA, LRRK2

and Parkin
Konstantin Senkevich a, b, c, Uladzislau Rudakou a, b, d, Ziv Gan-Or a, b, d, *
a
The Neuro (Montreal Neurological Institute-Hospital), McGill University, Montréal, QC, Canada
b
Department of Neurology and neurosurgery, McGill University, Montréal, QC, Canada
c
First Pavlov State Medical University of St. Petersburg, Saint-Petersburg, Russia
d
Department of Human Genetics, McGill University, Montréal, QC, Canada

A R T I C L E I N F O A B S T R A C T

Keywords: Parkinson’s disease (PD) is defined as a complex disorder with multifactorial pathogenesis, yet a more accurate
Parkinson’s disease definition could be that PD is not a single entity, but rather a mixture of different diseases with similar phe­
Genetic targets notypes. Attempts to classify subtypes of PD have been made based on clinical phenotypes or biomarkers.
Clinical trials
However, the most practical approach, at least for a portion of the patients, could be to classify patients based on
GBA
LRRK2
genes involved in PD. GBA and LRRK2 mutations are the most common genetic causes or risk factors of PD, and
PRKN PRKN is the most common cause of autosomal recessive form of PD. Patients carrying variants in GBA, LRRK2 or
PRKN differ in some of their clinical characteristics, pathology and biochemical parameters. Thus, these three
PD-associated genes are of special interest for drug development. Existing therapeutic approaches in PD are
strictly symptomatic, as numerous clinical trials aimed at modifying PD progression or providing neuro­
protection have failed over the last few decades. The lack of precision medicine approach in most of these trials
could be one of the reasons why they were not successful. In the current review we discuss novel therapeutic
approaches targeting GBA, LRRK2 and PRKN and discuss different aspects related to these genes and clinical
trials.

1. Introduction The most recent genome-wide association study (GWAS) calculated

Parkinson’s disease (PD) is a neurodegenerative disorder with
multifactorial pathogenesis and complex phenotypes, including various
motor and non-motor symptoms. Multiple lines of evidence suggest that
instead of viewing PD as a single disorder, it should be considered a
mixture of different diseases with similar phenotypes (Espay et al., 2020;
Sauerbier et al., 2016). Many attempts have been made to classify PD
into subtypes based on clinical presentation, neuroimaging and bio­
markers (Campbell et al., 2020; Fereshtehnejad et al., 2017; Lawton
et al., 2018; Qian and Huang, 2019; Thenganatt and Jankovic, 2014).
However, the most viable approach could be to classify patients based
on similar underlying disease mechanisms, potentially with similar
biomarkers, that lead to somewhat distinct phenotypes. One of the ways
this could be done is by using causative gene stratification, so that
treatment development could specifically target these genes (von Lin­
stow et al., 2020).
the heritability of PD as 22% (Nalls et al., 2019), yet only a small pro­
portion of PD patients carry pathogenic variants with Mendelian in­
heritance. LRRK2 and GBA mutations are amongst the most common
genetic causes or risk factors of PD (Gan-Or et al., 2015a; Healy et al.,
2008; Ross et al., 2011). The frequency of LRRK2 variants in PD is
1–40% and GBA variants are found in 5–20% of PD patients, depending
on the population (Gan-Or et al., 2015a; Healy et al., 2008; Ross et al.,
2011). Other mutations in familial genes are relatively rare and repre­
sent up to 1–2% of all PD cases. Bi-allelic variants in PRKN are the most
common autosomal recessive cause of PD, and can explain about 8–15%
of early-onset PD (with age at onset, AAO, below 50 years) (Alcalay
et al., 2010a; Bonifati, 2014; Kilarski et al., 2012; Klein and West­
enberger, 2012). In this review, we only refer to bi-allelic PRKN muta­
tion carriers, as the role of heterozygous PRKN mutations in PD is
controversial (Clark et al., 2006; Lubbe et al., 2021; Yu et al., 2021).
Patients carrying variants in LRRK2, GBA or PRKN may differ in some of

* Corresponding author. Department of Neurology and Neurosurgery McGill University 1033 Pine Avenue, West, Ludmer Pavilion, room 312, Montreal, QC, H3A
1A1, Canada.
E-mail address: ziv.gan-or@mcgill.ca (Z. Gan-Or).

https://doi.org/10.1016/j.neuropharm.2021.108822
Received 28 June 2021; Received in revised form 1 October 2021; Accepted 4 October 2021
Available online 7 October 2021
0028-3908/© 2021 Published by Elsevier Ltd.
K. Senkevich et al. Neuropharmacology 202 (2022) 108822

their clinical characteristics and pathology, compared to sporadic PD Table 1

(Fig. 1; Table 1) (Alcalay et al., 2010b, 2015b; Kalia et al., 2015;
Schneider and Alcalay, 2017; Thaler et al., 2018). Patients with LRRK2
mutations have a more benign course of PD, as they progress slower and
have less non-motor symptoms than sporadic PD, whereas GBA carriers
have more malignant course with faster progression and more
non-motor symptoms. Patients with biallelic PRKN mutations have an
early onset, very slowly progressive PD, which responds well to rela­
tively low doses of levodopa (Alcalay et al., 2014; Piredda et al., 2020).
Moreover, α-synuclein pathology is only present in about 50% of LRRK2
mutation carriers and rarely found in PRKN mutation carriers with PD
(Kalia et al., 2015; Schneider and Alcalay, 2017), in whom the neuro­
degenerative process is typically limited to the substantia nigra
(Schneider and Alcalay, 2017). Thus, these three relatively common
PD-associated genes lead to distinct PD subtypes and represent specific
interest for drug development.
Current therapeutic approaches in PD are symptomatic and do not
involve disease modification. Moreover, most current drugs, whether in
development or in clinical trials, target PD as a single clinical entity, and
do not consider stratification by subtypes. This lack of stratification may
be one of the reasons for our failure to develop disease modifying
therapies for PD. We suggest that our best chance to treat PD is by
identifying and treating specific subtypes of the disease. Out of all the
known genetic targets, GBA, LRRK2 and PRKN are likely the best can­
didates for drug development, although multiple challenges exist when
designing the clinical trials for patients with mutations in these genes.
2. GBA in PD – genetic, enzymatic, clinical and therapeutic
considerations
2.1. GBA and PD
Homozygous variants in GBA may cause a rare lysosomal storage
disorder, Gaucher disease (GD) (Stirnemann et al., 2017). Throughout
the 20th Century, scattered reports have been published on GD patients
and their relatives (carriers of GBA variants) who developed PD (Miller
et al., 1973; Neil et al., 1979; Soffer et al., 1980; Van Bogaert and
Froehlich, 1939). First genetic association studies of GBA mutations in
PD cohorts were reported in the early 2000s and were confirmed in a
large multicenter study (Aharon-Peretz et al., 2004; Lwin et al., 2004;
Sidransky et al., 2009), followed by numerous other studies. GBA
Differences of clinical and pathological features of GBA, LRRK2 and PRKN
associated PD.
Clinical/Pathological GBA associated LRRK2 PRKN associated
feature PD associated PD PD
Lewy body pathology + +/− –
Early age at onset + – +
Motor symptoms + – –
progression
Cognitive deficit + – –
Psychiatric symptoms + – +/−
Olfactory dysfunction + – –
+ more prevalent than in sporadic Parkinson’s disease; - less prevalent than in
sporadic Parkinson’s disease; +/− conflicting reports.
mutations are also associated with other synucleinopathies such as de­
mentia with Lewy bodies and REM sleep behavior disorder (RBD) (Chia
et al., 2021; Krohn et al., 2020b; Mata et al., 2008; Nalls et al., 2013), yet
it is still unclear whether GBA is associated with multiple system atro­
phy, a more severe form of synucleinopathy (Table 2) (Mitsui et al.,
2015; Sklerov et al., 2017; Wernick et al., 2020).
GBA encodes the lysosomal enzyme, glucocerebrosidase (GCase),
responsible for the degradation of several glycolipids such as gluco­
sylceramide (GlcCer) and glucosylsphyngosin (GlcSph). GCase is syn­
thesized in the endoplasmic reticulum (ER) (Do et al., 2019), and if
folded correctly, transported to the Golgi complex for processing and
then to the lysosome by LIMP2 (Reczek et al., 2007).
There are several different hypotheses regarding the pathogenesis of
GBA-associated PD, including loss of function, accumulation of GCase
substrates, alterations in the lysosomal membrane composition, ER
accumulation and ER stress due to misfolding, autophagy dysfunction,
mitochondrial dysfunction, mitophagy dysfunction and neuro­
inflammation (Gan-Or et al., 2015b; Senkevich and Gan-Or, 2020). Most
of these hypotheses are based on loss of function of GCase, which is
further supported by studies reporting reduced GCase activity in blood,
CSF and brains of patients without GBA mutations (Alcalay et al., 2015a;
Gegg et al., 2012; Parnetti et al., 2014). One hypothesis suggests that the
reduction of GCase activity may lead to the accumulation and oligo­
merization of α-synuclein due to its direct interaction with GlcCer
(Mazzulli et al., 2011). Accumulated α-synuclein may block the trans­
port of GCase from the ER to the lysosome, leading to further reduction
of GCase activity (Mazzulli et al., 2011). α-synuclein could also directly
interact with GCase in acid conditions further promoting its inhibition
(Yap et al., 2013). Other studies suggest that GlcCer metabolites such as
glucosylsphingosine (GlcSph) have higher potency to promote α-synu­
clein fibrils formation by direct interaction than GlcCer (Taguchi et al.,
2017). In human brains, there are conflicting evidence for accumulation
of sphingolipids (Gegg et al., 2015; Huebecker et al., 2019), which
question the suggested mechanisms involving GlcCer or GlcSph
accumulation.
Different GBA variants may have different effects on disease patho­
genesis. Some variants such as p.L444P and to a much lesser extent, p.

Table 2
Frequency of GBA variants in patients with synucleinopathies.
Synucleinopathy Frequency of all GBA References
variants

PD 5–20%* Gan-Or et al. (2015c)

DLB 8.1–32%* (Nalls et al., 2013; Shiner et al., 2021;
Tsuang et al., 2012)
MSA 10.8% Wernick et al. (2020)
RBD 9.5% Krohn et al. (2020b)

PD- Parkinson’s disease, DLB – dementia with Lewy bodies, MSA-multiple sys­
tem atrophy, RBD – REM sleep behaviour disorder. * high frequency; 20% in PD
Fig. 1. Parkinson’s disease (PD) genetic subtypes under PD as an umbrella term and 32% in DLB are due to inclusion of studies with Ashkenazi Jewish
on the example of PRKN, LRRK2 and GBA carriers. population.

2
K. Senkevich et al.
N370S, may lead to ER retention (Fernandes et al., 2016; Ron and
Horowitz, 2005; Thomas et al., 2021). This retention can lead to loss of
function since GCase does not reach the lysosome, or it can lead to gain
of toxic function if retention leads to ER stress and cell death (Fernandes
et al., 2016; Thomas et al., 2021). Truncating GBA mutations such as
c.84GG and IVS2+1 result in absolute absence of the protein (Beutler,
2006), which argues for a loss of function mechanism. Other variants
may affect the function of the enzyme by changing the conformation of
the active site (Toffoli et al., 2020). Given these distinctive effects of
different GBA variants, drug development for GBA-associated PD should
take these differences into account, which may lead to mutation-specific
treatments in the future.
2.2. GBA-PD biomarkers – current status
Homozygous and compound heterozygous GBA variants causing GD
lead to a significant reduction of GCase activity, making it an excellent
biomarker for early GD detection (Ho et al., 1972). Similarly, the
accumulation of GCase substrate GlcCer and its deacetylated form,
GlcSph, leads to the formation of enlarged macrophages, called Gaucher
cells, which are deposited in organs and tissues and cause GD symptoms
(Stirnemann et al., 2017). Thus, the level of GlcSph can serve as a
biomarker of GD progression and response to treatment (Dekker et al.,
2011; Rolfs et al., 2013).
In PD, however, neither reduced GCase activity nor accumulation of
GlcCer or GlcSph can be used as reliable biomarkers. While the average
GCase activity has been repeatedly demonstrated to be reduced in blood,
cerebrospinal fluid (CSF), fibroblasts and brains of GBA variants carriers
(Alcalay et al., 2015a; Gegg et al., 2012; Lerche et al.; Pchelina et al.,
2017), there is a big overlap between GCase activity levels in PD patients
and controls with and without GBA variants (Alcalay et al., 2015a; Gegg
et al., 2012; Gündner et al., 2019; Moors et al., 2019). Similarly, con­
flicting evidence on GlcCer and GlcSph levels do not support these
substrates as potential biomarkers in GBA-PD (Gegg et al., 2015; Guedes
et al., 2017; Huebecker et al., 2019; Pchelina et al., 2018). Reduced
GCase activity was also reported in blood and brains of a subgroup of
sporadic PD patients with age-dependent effect (Alcalay et al., 2015a;
Huebecker et al., 2019; Muñoz et al., 2020; Rocha et al., 2015). While
this subgroup of patients may benefit from future GBA-targeted therapy,
neither reduced GCase activity nor substrate levels are likely to be good
biomarkers.
Previous studies have suggested that some immune markers may be
associated with GBA-PD (Chahine et al., 2013; Miliukhina et al., 2020),
whereas other studies showed no difference in immune biomarkers be­
tween GBA-PD, sporadic PD and other subgroups of the disease (Galper
et al.; Thaler et al., 2021). As there are no reliable biomarkers for
GBA-PD and its progression, additional studies are needed to identify
such biomarkers.
2.3. Clinical features of GBA-PD
On the individual level, it is difficult to identify a GBA-PD patient
based on their clinical presentation, as their symptoms are overall
typical. However, as a group, the disease course of GBA-PD patients is
more severe: they have earlier average AAO, higher frequencies of non-
motor symptoms, and faster motor and non-motor progression
compared to sporadic PD (Alcalay et al., 2012; Cilia et al., 2016; Creese
et al., 2018; Liu et al., 2016; Maple-Grødem et al., 2021; Zhang et al.,
2018). Multiple studies have demonstrated faster cognitive decline and
higher frequency of psychiatric symptoms in GBA-PD (Alcalay et al.,
2012; Cilia et al., 2016; Creese et al., 2018; Iwaki et al., 2019; Liu et al.,
2016). GBA-PD patients also have higher incidence of anxiety, depres­
sion and hallucinations as compared to non GBA carriers (Creese et al.,
2018; Swan et al., 2016). A meta-analysis reported a higher occurrence
of wearing-off and faster motor progression compared to non-carriers
(Zhang et al., 2018). Moreover, recent studies suggested increased rate
3
Neuropharmacology 202 (2022) 108822
of cognitive decline in GBA-PD patients after deep brain stimulation
(Lythe et al., 2017; Mangone et al., 2020).
Classification of GBA variants can be taken from GD, where severe
variants (e.g. p.L444P, c.84GG, p.V394L) lead to the neuronopathic
forms of GD (types 2 and 3) and mild variants (e.g. p.N370S, p.R496H)
lead to the non-neuronopathic form of GD (type 1). Several other GBA
variants are associated with increased PD risk (p.T369M, p.E326K) but
do not cause clinical GD (Berge-Seidl et al., 2017; Mallett et al., 2016). In
PD, severe GBA variants lead to a more severe PD phenotype compared
to mild variants (Cilia et al., 2016; Gan-Or et al., 2008; Thaler et al.,
2018). Moreover, the phenotype of GBA-PD has a dose-dependent effect,
as patients with homozygous or compound heterozygous variants may
have earlier AAO and more severe motor impairment and faster cogni­
tive decline (Thaler et al., 2017). It is therefore possible that the levels of
GCase activity are associated with PD severity, yet to further examine
this hypothesis, reliable lysosomal GCase activity measurement methods
need to be established.
2.4. GBA-targeted therapy
2.4.1. Substrate-reduction therapy
One of the suggested strategies for treatment of GBA-PD is substrate
reduction therapy, assuming that GlcCer and/or GlcSph may accumulate
and be involved in the pathogenic process in GBA-PD (Sardi et al., 2017;
Taguchi et al., 2017). Substrate reduction therapy, targeting excessive
accumulation of GlcCer by glucosylceramide synthase inhibition, is
already being used in non-neuronopathic GD (eliglustat and miglustat)
(Gary et al., 2018). Previous studies showed that miglustat may pene­
trate the blood-brain barrier, yet it had no clinical effect on neuro­
nopathic GD (Schiffmann et al., 2008; Treiber et al., 2007). Based on the
findings suggesting that sphingolipids may induce and promote the
accumulation of α-synuclein, it was hypothesized that glucosylceramide
synthase inhibition could alleviate α-synuclein burden and PD symp­
toms (Sardi et al., 2017; Taguchi et al., 2017). Preclinical experiments
were performed on murine models with transgenic α-synuclein p.A53T
overexpression and Gaucher model with homozygous p.D409V (Sardi
et al., 2017). This GBA variant has not been previously associated with
PD and was reported only in a single GD patient. In these models, the
glucosylceramide synthase inhibitor GZ667161 demonstrated target
engagement, reduction of α-synuclein accumulation and improvement
of cognitive symptoms (Sardi et al., 2017).
These findings prompted the researchers to continue the clinical
development of the glucosylceramide synthase inhibitor GZ667161
analog venglustat (GZ/SAR402671) in humans (Peterschmitt et al.,
2021) (Table 3). Recently, three phase 1 studies on healthy volunteers
reported on the safety and tolerability of venglustat (Peterschmitt et al.,
2021). The safety profile allowed to start a phase 2 placebo-controlled
study, MOVES-PD, including 221 GBA-PD patients (NCT02906020).
The study was divided into two stages. Safety, tolerability and target
engagement were demonstrated in GBA-PD patients during the first part
of the study, reported in 2019 (Fischer et al., 2019). The results of the
second part of MOVES-PD study were recently reported in the
AD/PD-2021 and MDS 2021 conferences (2021; J. Peterschmitt et al.,
2021). Target engagement was clearly demonstrated by a significant
decrease (~75%) of GlcCer levels in the CSF as well as in plasma of
patients receiving venglustat. The primary and secondary endpoints
were changes in MDS-UPDRS parts I, II and III and PD cognitive rating
scale (PD-CRS) at 52 weeks. The primary motor endpoint, sum of
MDS-UPDRS parts II and III, was increased in the treatment group (7.29
± 1.36) compared to the placebo group (4.71 ± 1.27; p = 0.17),
meaning that those who received the treatment deteriorated faster.
PD-CRS total score was decreased in the venglustat treated group
(− 0.73 ± 1.36), demonstrating cognitive deterioration. Moreover, a
higher rate of psychiatric disorders was reported in treatment group
compared to placebo group. The progression of symptoms was reported
starting week 13 of treatment. These negative effect on motor symptoms
K. Senkevich et al.
Table 3
Therapeutic approaches for GBA.
Type of Mechanism Drug name
treatment
Substrate Glucosylceramide synthase inhibition Venglustat (GZ/
reduction SAR402671)
therapy
Chaperone Inhibitory chaperone that facilitates Ambroxol
transportation of GCase to the lysosome
Ambroxol
Activator GCase activator BIA 28–6156/LTI-
291
Gene therapy Replacement of mutated GBA with WT PR001
copy of the gene
and cognition led to discontinuation of the development of venglustat
for GBA-PD.
Despite sparse clinical data and the lack of relevant preclinical
models, venglustat demonstrated target engagement and significantly
decreased concentration of GlcCer in clinical trials. Nevertheless, dete­
rioration of PD symptoms was reported rather than improvement, sug­
gesting that substrate reduction therapy may not be a useful approach
for GBA-PD. There are many possible reasons for clinical trial failures,
and in this case, we should also consider that either the hypothesis was
wrong and there is no GlcCer accumulation, or that even if GlcCer
accumulation exists, it does not contribute to PD progression.
Why did patients deteriorate in the venglustat trial?
Sphingolipids are very important components of cellular membranes
(Posse de Chaves and Sipione, 2010), and GlcCer has a central role in
sphingolipid homeostasis, as it is an intermediate metabolite and a
precursor of the vast majority of sphingolipids (Gault et al., 2010).
GlcCer can be further converted to GlcSph through deacetylation and to
LacCer by the addition of galactose (Gault et al., 2010; Olsen and Fær­
geman, 2017). It is possible that by decreasing the levels of GlcCer in
neural tissue below normal values, sphingolipids metabolism is dis­
rupted, and neuronal membranes get damaged. In support of this hy­
pothesis, peripheral neuropathy was reported as an adverse effect of
substrate reduction therapy in GD (Ficicioglu, 2008; Giraldo et al.,
2018). In addition, hand tremor is one of the known common adverse
effects of miglustat (Giraldo et al., 2018). If similar adverse effects occur
with venglustat, it could have increased the MDS-UPDRS score results in
the treatment group.
2.4.2. GCase activators in development
Almost 20 years ago, a novel strategy to increase GCase activity was
demonstrated in fibroblasts from GD patients with homozygous GBA p.
N370S mutations (Sawkar et al., 2002). Chaperones are small molecules
that can cross the blood-brain barrier, bind to GCase in the ER and
facilitate its folding and transport to the lysosome. Different groups have
shown that such chaperones can potentially increase GCase activity in
the lysosome (Fernandes et al., 2016; Thomas et al., 2021). In recent
years, multiple strategies to increase GCase activity have been proposed
(Han et al., 2020; Jung et al., 2016). At least 14 small molecules have
been assessed for efficacy for GCase activation in GD, and preclinical
results on five molecules were reported in GBA-PD models (isofagomine,
S-181, ambroxol, NCGC607 and LTI-291) (Aflaki et al., 2016; Burbulla
et al., 2019; Han et al., 2020; Mullin et al., 2020).
Ambroxol is a pH-dependent competitive GCase inhibitory chap­
erone. Various studies suggest a different effect of ambroxol depending
on the mutation, with reduced effect of ambroxol in p.L444P carriers
(Ivanova et al., 2018; Khanna et al., 2010; Luan et al., 2013; Maegawa
et al., 2009; Sanchez-Martinez et al., 2016). Yet, molecular docking
studies showed similar binding effect of ambroxol to p.N370S and p.
L444P (Thirumal Kumar et al., 2019). Currently, there is no definite
explanation for the different effects of ambroxol in different experiments
and on different genotypes (Ivanova et al., 2018, 2021). Several studies
Neuropharmacology 202 (2022) 108822
Registered clinical trial Cohort Phase Current status
(clinicaltrials.gov)
NCT02906020 GBA-PD 2 Halted due to negative effect on
clinical phenotype
NCT02941822 GBA-PD and 2 Positive outcome in terms of safety
sporadic PD and target engagement
NCT02914366 PD-dementia 2 The study in process
– GBA-PD 2 The study in process
NCT04127578 GBA-PD 1/2a Preliminary report. Recovery of
GCase activity. Possible severe
immune response
suggested a dual role of ambroxol as a competitive and non-competitive
inhibitor in the GBA p.N370S model (Kopytova et al., 2021; Maegawa
et al., 2009). It is also possible that variants leading to conformational
changes of the GCase active site could decrease ambroxol binding. This
might mean that ambroxol (or other chaperones) could be beneficial
only for specific GBA variants and not for others. This possibility should
be further explored and considered in the future design of clinical trials.
Isofagomine is a potent competitive inhibitor of GCase. Preclinical
studies on isofagomine on PD and GD models in vitro and in vivo
demonstrated efficacy for GCase activation (Khanna et al., 2010; Korn­
haber et al., 2008; Richter et al., 2014; Sanchez-Martinez et al., 2016;
Sun et al., 2012). Despite showing some evidence for efficacy in pre­
clinical trials for GD, a phase 2 clinical trial in GD patients was halted
due to lack of efficacy (NCT00446550). Isofagomine has not been tested
in PD.
Recently, a positive effect of the heat shock protein amplifier, ari­
moclomol, on GCase activity has been demonstrated in primary fibro­
blasts and neuronal-like cells from GD patients (Fog et al., 2018).
Arimoclomol is a well-known molecule and demonstrated some positive
biochemical and clinical effects in phase 2/3 open-label clinical trials for
Niemann-Pick Disease Type C and Amyotrophic Lateral Sclerosis (NC
T00706147; NCT02612129). This molecule is currently considered for
PD clinical trials. Other new molecular chaperones are currently being
developed (Aflaki et al., 2016; Burbulla et al., 2019).
2.4.3. Ambroxol in clinical trials
Recently, an open-label, non-randomized clinical trial of ambroxol
has been completed (Mullin et al., 2020) (Table 3). This study included
eight patients with and ten patients without GBA mutations. Dose
constituted 1260 mg orally daily for 180 days with follow-up assessment
at 270 days mark. Ambroxol concentration increased in CSF and was at a
level of 11% of its blood level, demonstrating partial blood-brain barrier
permeability. Target engagement was demonstrated by increased GCase
activity in blood, although in the CSF it was decreased. The authors
suggested that in the CSF, ambroxol may inhibit GCase but cannot
dissolve from it due to the absence of cellular structures and lysosomes.
This hypothesis was supported by in vitro experiments in acellular con­
ditions (Mullin et al., 2020). A moderate increase of α-synuclein levels in
the CSF has also been reported, yet the nature of this increase and its
relevancy to PD is unclear. Furthermore, most of these changes occurred
in the group of non-carriers of GBA variants. Some positive changes in
motor and cognitive performance have also been reported in the study.
However, due to the lack of placebo group or blind randomization, these
results are difficult to interpret and require replication in additional
trials. A phase 2 clinical trial (NCT02914366) testing ambroxol for
treatment of PD with dementia, regardless of GBA status, is currently
ongoing (Silveira et al., 2019). While this study examines a more specific
population (PD with dementia), it is not limited exclusively to carriers of
GBA variants. Therefore, there is a reduced likelihood of success,
because not all PD patients with dementia necessarily have reduced
GCase activity. Overall, the current results support the continuation of
4
K. Senkevich et al.
clinical trials testing ambroxol and similar molecules in GBA-associated
PD, yet with careful consideration of the participant’s genotype and the
trial design.
2.4.4. LTI-291 as a GCase activator
Preclinical studies and clinical trials on other GCase activators are
currently ongoing. Two phase 1 trials (single and multiple ascending
doses) on allosteric GCase modulator LTI-291 (currently BIA 28–6156/
LTI-291) have been completed, demonstrating safety and partial blood-
brain permeability of the drug (den Heijer et al.). The authors report that
preclinical experiments demonstrated the efficacy of LTI-291 by
increasing GCase activity and decreasing GlcCer accumulation for GBA
p.E326K and p.N370S in vitro and in vivo. The single ascending dose arm
of the study included 40 healthy volunteers and the multiple ascending
dose arm included 39 middle-aged or elderly healthy subjects. In total,
five carriers of GBA variants were included in the study including four p.
T369M carriers and one p.E326K. No serious adverse events were re­
ported in both studies. The authors did not identify significant changes
in glycosphingolipid level in response to LTI-291 and GCase activity was
not measured (den Heijer et al.).
2.5. Gene therapy targeting GBA
There are several possible approaches for gene therapy in GBA-PD,
the most obvious being gene therapy using a viral vector containing a
normal copy of the GBA gene. In mouse models, direct administration of
the GBA adenoviral vector into several brain regions led to a decrease in
α-synuclein accumulation in the substantia nigra and striatum, and to an
increase in GCase activity (Rocha et al., 2015). Direct brain adminis­
tration of adenoviral vectors involves a number of risks and limitations,
and could be practically difficult in patients, who are very different from
one another, unlike the inbred mice. An alternative approach is the
systemic administration of a viral vector with GBA. In a mouse model, a
similar effect was shown with an increase in GCase activity and a
decrease in the concentration of α-synuclein in the brain (Morabito
et al., 2017). Systemic administration of the vector in a mouse model for
the neuropathic form of GD also demonstrated a decrease in neuro­
degeneration and an increase in lifespan (Massaro et al., 2020). In
humans, systemic injection of a viral vector with a gene may lead to
off-target effects, meaning that the effect on other genes could be un­
expected (Zhang et al., 2015).
Despite these potential limitations, recently the first PD patients have
received gene therapy with the GBA gene, in a Phase 1/2a clinical trial of
PRV-PD101 (PROPEL, NCT04127578) (Table 3). The preclinical data for
this gene therapy, an in vitro study using rodent neuronal cell lines and
an in vivo study using rodent models, demonstrated increased GCase
activity, decreased glycolipid substrate accumulation and improved
motor symptoms. The safety of the drug was also examined by injection
to non-human primates (Sheehan et al., 2020). In the PROPEL study, the
adenoviral vector (adeno-associated virus 9) containing GBA (PR001A)
is administrated intracisternally. The PROPEL study design is a multi­
center, randomized, double-blind, escalating dose controlled sham
procedure. The study is conducted in patients with GBA-PD bearing
pathogenic GBA mutations, although the definition of pathogenicity is
not detailed in the protocol. In a recent company report, positive effect
of PR001 administration in two patients with GBA variants (Type 2 GD
patient and GD patient with GBA-PD) was described. According to the
report, both patients demonstrated increased level of GCase in CSF from
undetectable to normal, four and three months after the treatment
respectively. The GBA-PD patient developed severe adverse event
related to immune-mediated response to the AAV9 viral vector three
months after the treatment. This led to the protocol amendment to
modify the immunosuppressive treatment (globenewswire.com, 2021).
5
Neuropharmacology 202 (2022) 108822
2.6. Strategies and future directions
To design a trial for GBA-PD in which the true effects of the treatment
will be visible, several important factors should be considered:
1) Targeting mutations that fit the specific properties of the treatment –
as different mutations have different effects on GCase structure and
function, these properties need to be considered when deciding
which mutation carriers should be enrolled into the trial. For
example, a chaperone may be beneficial for mutations that lead to ER
retention, but it may not be useful for mutations in which GCase is
not synthesized at all, such as the c.84GG mutation. GCase activators
that work in the lysosome may be beneficial for mutations that allow
GCase to be transported to the lysosome, but may not be beneficial
for mutations that are fully retained in the ER. Therefore, in each
trial, the researchers should consider the known properties of their
drug and of the specific mutation carriers that they enroll into the
trial, to make sure that the treatment fits the known mechanism of
the mutation.
2) Balancing the trial arms with similar GBA mutations – Several studies
have suggested that the rates of motor and non-motor progression in
carriers of severe GBA mutations are faster than among mild GBA
mutations or the GBA risk factors p.E326K and p.T369M. If the
treatment and placebo arms of a trial will not be balanced in terms of
the ratio of severe to mild mutations, it may lead to one of the arms of
the trial to progress faster than the other, unrelated to the treatment.
Since motor and non-motor symptoms are typically the end-points of
trials, this imbalance may lead to false positive or false negative
results, depending on which arm harbors more severe mutation
carriers.
3) Potential GBA modifiers may bias the trial – The penetrance and AAO
of GBA-PD, as well as the activity of GCase, may be affected by ge­
netic variants in genes such as CTSB, TMEM175 and LRRK2 (Alcalay
et al., 2015a; Krohn et al., 2020a; Sosero et al., 2021). Whether these
and other variants affect the rate of progression of GBA-PD has not
been studied, yet it is a reasonable possibility. Hence, to avoid po­
tential effects of these genetic variants on the rate of progression and
subsequently on trial results, these potential modifiers should also be
taken into account during the randomization process, to ensure that
they do not bias the results. Finally, it was shown that PD polygenic
risk score (PRS) may affect GBA-PD penetrance and age at onset, and
that imbalance in PRS between trial arms may lead to false positive
or false negative results in clinical trials (Leonard et al., 2020). We
therefore recommend that prior to randomization, these genetic data
are collected and considered in the randomization to trial arms. At
minimum, and much less preferable, post-hoc analyses using these
data should be performed.
4) Combination of drugs – It is possible that to fully address the effects
of specific mutations, combinations of drugs such as chaperone +
activator, will be required. One potential trial design for this com­
bination could include four arms: chaperone only, activator only,
chaperone + activator and placebo. Such trial will be cost-effective
and could answer multiple questions in a single trial.
5) Overshooting the desired therapeutic effect – Since the lysosomal
sphingolipid metabolism pathway is crucial for cell maintenance, it
is possible that increasing GCase activity to levels that are too high
may lead to undesired results. This may occur in gene therapy
approach, in GCase activators which may not only activate the
mutated GCase, but also the wildtype, and in other enzymatic
modulators.
One of the main challenges for the researchers designing trials will be
the identification and recruitment of GBA-PD patients. While GBA var­
iants are common in PD, they progress faster, which may be both an
advantage and a limitation. The main advantage is that in a disease that
progresses faster, it would be easier to see an effect in a shorter time
K. Senkevich et al.
frame or with a smaller trial. The main limitation is that when designing
a trial focused on PD patients at early stages and/or drug-naïve PD, in­
dividuals with GBA-PD will not likely stay at these stages for a long time,
and will therefore be more difficult to recruit. The high frequency and
faster progression to dementia may also hamper recruitment, if these are
the exclusion criteria. GBA genotyping is a potential obstacle on its own,
as the presence of the pseudogene GBAP1 makes the sequencing of GBA
challenging. Methods that properly distinguish between the gene and
the pseudogene are therefore necessary for correct identification of all
GBA-PD patients. All these challenges should be carefully considered
when designing a GBA-targeting trial. To facilitate these trials, we
recommend that clinicians routinely screen for GBA variants in all their
patients, whether through research initiatives or through clinical ini­
tiatives such as PD GENEration (parkinson.org/PDGENEration) and
others. Since GBA mutations are common in RBD patients (Krohn et al.,
2020b), and since they may be associated with faster conversion, tar­
geting RBD patients with GBA mutations may be an attractive oppor­
tunity, as they are at a much earlier stage of the disease.
3. Therapy targeting LRRK2
The importance of LRRK2 in PD emerged from linkage studies in
families with autosomal-dominant, late-onset PD (Paisán-Ruı́;z et al.,
2004; Zimprich et al., 2004). Seven mutations have been reported to
cause PD: p.N1437H, p.R1441C/G/H, p.Y1699C, p.G2019S and p.
I2020T (Ross et al., 2011), while additional variants such as p.R1628P
(Zhang et al., 2017), p.G2385R (Xie et al., 2014) and p.M1646T (Sosero
et al., 2021) may increase the risk of PD. The most common pathogenic
LRRK2 variant p.G2019S was reported in ~4% of familial PD cases and
~1% in sporadic PD (Lesage et al., 2007), but it has a much lower or
higher prevalence in some ethnicities. Due to founder effects, in
Ashkenazi Jews and North African Arabs p.G2019S can be found in
10–15% and ~39% respectively in sporadic PD patients and up to 26%
and 40% respectively in familial PD cases (Hassin-Baer et al., 2009; van
Dijkman et al., 2018). The penetrance of LRRK2 variants is incomplete
and estimated to be from 25% to 43% in older adults (Lee et al., 2017).
3.1. Mechanism and pathology of LRRK2-PD
LRRK2 encodes a 2527 amino acid protein, leucine-rich repeat kinase
2, containing six independent domains. The enzymatic part of the pro­
tein consists of the Ras-like GTPase (ROC), C-terminal domain of ROC
(COR) and a kinase domain. Other domains, an N-terminal armadillo
repeat region (ARM), an ankyrin repeat (ANK) domain, a leucine-rich
repeat (LRR) domain and a C-terminal WD40 domain, play a role in
protein-protein binding (Bae and Lee, 2015). All the clearly pathogenic
LRRK2 variants are located in the catalytic domains and may induce
hyperactivation of the kinase domain (Bryant et al., 2021). Rab10
GTPase is one of the key substrates phosphorylated by LRRK2 and may
be used as a biomarker of the kinase activity (Rideout et al., 2020).
Studies using p.R1441C knock-in mutation in mice showed hyper­
phosphorylation of Rab10, which was suppressed by LRRK2 inhibition
(Dhekne et al., 2018). This suggests that Rab10 is a physiological sub­
strate of LRRK2 and that pathogenic variants do lead to hyper­
phosphorylation in these models. Rab GTPase family regulates many
important aspects of cell biology through their role in intracellular
trafficking (Pfeffer, 2018). LRRK2 phosphorylates 14 different Rab
proteins (out of 50 that were tested), which are possibly physiological
substrates of LRRK2 (Steger et al., 2017). Various cellular pathways
involving these Rab GTPases were proposed as potential mechanisms
underlying PD pathology (thoroughly reviewed in (Bae and Lee, 2020)):
disrupted ciliogenesis, reduced neuroprotective signaling, impaired
maintenance of lysosomal and mitochondrial homeostasis, disrupted
vesicle trafficking, promotion of α-synuclein propagation, inflammatory
response – just to name a few.
Pathological findings in patients with different LRRK2 variants may
6
Neuropharmacology 202 (2022) 108822
vary. While the autopsies of the majority of p.G2019S carriers (65%)
showed Lewy bodies (LB) containing neurons, most carriers of other
LRRK2 variants (70%) did not have that PD hallmark pathology (Kalia
et al., 2015). This may suggest that LRRK2-PD represents a distinct
neuropathological condition, as PD is pathologically characterized by
α-synuclein accumulation.
3.2. LRRK2-PD clinical features
The clinical features of LRRK2-PD are often reported as indistin­
guishable from idiopathic PD (Obergasteiger et al., 2020; Zhao and
Dzamko, 2019), but accumulating evidence suggest that LRRK2-PD
patients have a milder disease phenotype. A recent longitudinal
assessment of pooled prospective data compared 155 LRRK2-PD patients
with 889 sporadic PD patients (Ortega et al., 2021). This study reported
possible slower rate of cognitive decline based on MoCA scores and
slower motor disease progression based on MDS-UPDRS III scores, albeit
the results were not statistically significant (Ortega et al., 2021).
Another study used the longitudinal data from PPMI for 158 LRRK2-PD
and 598 sporadic PD patients and reported 60% lower rate of non-motor
symptoms progression in carriers of LRRK2 mutations (Ahamadi et al.,
2021). Furthermore, LRRK2-PD was linked to lower likelihood of
non-motor symptoms like olfactory disturbance (Marras et al., 2011;
Saunders-Pullman et al., 2011), depression (Piredda et al., 2020) and
RBD (Bencheikh et al., 2018; Saunders-Pullman et al., 2015). It is
interesting to note that unlike sporadic PD, LRRK2-PD seems to have no
sex effect and prevalence in women is roughly similar to men (Gan-Or
et al., 2015d; Kestenbaum and Alcalay, 2017). Also, homozygous car­
riers of p.G2019S were reported to be comparable to heterozygous
carriers when it came to AAO, cognitive performance, depression status
and other non-motor symptoms (Kestenbaum and Alcalay, 2017).
As mentioned before, LRRK2-PD patients have less abundant α-syn­
uclein pathology (Kalia et al., 2015). GBA-PD and PD associated with
SCNA multiplications are characterized by abundant α-synuclein pa­
thology and more malign PD course (Cilia et al., 2016; Konno et al.,
2016; Schneider and Alcalay, 2017). It is therefore possible that the
lower rates of α-synuclein pathology in LRRK2-PD may be the reason (or
just an indicator) for the more benign clinical course observed in these
patients. It is also possible that other, α-synuclein independent mecha­
nisms, are driving PD development in carriers of LRRK2 variants.
3.3. LRRK2 inhibitors in development
3.3.1. Kinase inhibitors
The gain-of-function effect of LRRK2 mutations on the kinase activity
became an appealing target for therapeutic development because it is
usually easier to inhibit rather than to activate a protein. Additionally,
various kinase inhibitors have already been developed for use in
oncology (Kannaiyan and Mahadevan, 2018). Therefore, the approach
of kinase inhibition is not new, although it needs to be further improved
in order to increase selectivity and penetrance of blood-brain barrier.
There are several non-selective LRRK2 kinase inhibitor compounds
that have been so far only tested in preclinical models of PD, and which
were recently discussed in detail, covering their efficacy and safety as­
pects (Wojewska and Kortholt, 2021). The safety concerns emerge from
the fact that LRRK2 is expressed in lung and kidneys where LRRK2 in­
hibitors might have detrimental effects (Baptista et al., 2013; Herzig
et al., 2011; Tong et al., 2012). These concerns were supported by the
studies in rodents that showed that LRRK2 knockout animals or those
treated with LRRK2-inhibitors display pathological changes in kidneys
or lungs (Andersen et al., 2018; Baptista et al., 2013; Hinkle et al., 2012).
However, the analysis of 1455 carriers of LRRK2 loss-of-function vari­
ants showed that there is no significant association between suppressed
levels of LRRK2 and kidney or lung pathology (Whiffin et al., 2020).
Another study in nonhuman primates reported that the LRRK2 inhibitors
induced only minor changes in lung tissue which did not impair lung
K. Senkevich et al. Neuropharmacology 202 (2022) 108822

function (Baptista et al., 2020). However, the short duration of the study
(2 weeks) raises questions on the generalizability of the safety conclu­
sions and their meaning for human patients who would be taking the
drug for extended periods of time. Preclinical results regarding the
safety effects associated with lungs and kidneys LRRK2 inhibition are
conflicting and may not be relevant for humans. Of note, the two
non-selective LRRK2 kinase inhibitor compounds that reached clinical
trials (discussed later), did so without reporting preclinical studies.
Targeted inhibition of specific LRRK2 variants (i.e., p.G2019S) in
heterozygous patients might be a safer choice to avoid the potential side
effects of systemic inhibition discussed earlier. A few preclinical studies
suggested efficacy of compounds EB-42486 and EB-42168 as potent
inhibitors of LRRK2 p.G2019S (Bright et al., 2021; Garofalo et al., 2020).
However, EB-42486 was not able to cross the blood-brain-barrier and
the ability of EB-42168 to cross it was not tested. Overall, targeted in­
hibition of specific LRRK2 variants is a viable approach for precision
medicine, although we still do not have enough preclinical data to start
clinical trials pursuing this target.
As of today, there are two LRRK2 kinase inhibitor compounds that
reached clinical trials, DNL151 and DNL201 (Table 4). In May 2021, it
was announced that DNL151 was selected to be advanced to late-stage
studies, testing it further in PD patients with LRRK2 gain-of-function
mutations and in patients with sporadic PD (denalitherapeutics.com,
2021). The DNL201 compound will be kept as a backup. DNL151 and
DNL201 are orally available small molecules that can penetrate the
blood-brain barrier. Both were tested in phase 1/1b randomized,
double-blind, placebo-controlled trials, as described below. The pre­
clinical data for DNL151 or DNL201 was not published by the company,
but according to the reports from phases 1/1b of the clinical trial, the
drug was well tolerated and considered safe. Moreover, DNL151
demonstrated a favorable safety and tolerability profile, robust target
engagement, as well as significant change in biomarkers that indicate
lysosomal function. Reduction of phosphorylated (pS935) LRRK2 and
pT73 Rab10 levels imply possible normalization of kinase activity
(denalitherapeutics.com, 2021). DNL151 was tested in 36 PD patients,
with either LRRK2-PD or sporadic PD (NCT04056689) and in 184
healthy volunteers (NCT04557800). DNL201 met the objectives of
safety, tolerability, CSF exposure levels, target engagement, kinase in­
hibition and lysosomal biomarkers in 122 healthy volunteers
(NCT04551534) and in 29 PD patients with and without LRRK2 muta­
tions (NCT03710707) (denalitherapeutics.com, 2018, 2020).
3.3.2. Antisense oligonucleotides
Antisense oligonucleotides (ASOs) are single-stranded DNA mole­
cules that are designed to specifically bind to their target mRNA via base
pairing and inhibit or alter gene expression by causing steric hindrance,
inducing alternative splicing or triggering target degradation (Chery,
2016). ASOs are an alternative approach for inhibiting LRRK2 activity
by decreasing the levels of translated protein or inducing splicing al­
terations which will affect the catalytic core of the protein. Preclinical
studies of ASOs targeting LRRK2 reported some promising results
(Table 4). Decreased loss of dopaminergic neurons and improved grip
strength was reported in murine models, along with decreased α-synu­
clein aggregation in primary neurons of mice with and without the p.
G2019S variant after intracerebral ventricular injections of ASOs (Zhao
et al., 2017). It was noted that the treatment did not affect the gene
expression in kidneys and lungs. Whether or not these findings are
relevant in humans needs to be examined. Some in vitro studies also
reported a protective effect of ASOs that induce skipping of exon 41,
containing the p.G2019S variant. These protective effects include
endoplasmic reticulum Ca2+ homeostasis, which was normalized in
neurons with p.G2019S variant derived from induced pluripotent stem
cells (Korecka et al., 2019a) and mitophagy rate, presumably altered by
p.G2019S, which was restored in PD-patient-derived fibroblasts car­
rying the p.G2019S variant (Korecka et al., 2019b). Additionally, there
was a decrease of Rab10 phosphorylation and normalization of auto­
phagosome maturation after the transgenic mice expressing either
human wildtype LRRK2 or p.G2019S were treated with ASO blocking
the 5’ splice site of exon 41 (Korecka et al., 2020). There is currently a
Phase 1 randomized placebo-controlled clinical trial aiming to evaluate
the safety and tolerability of LRRK2 ASO BIIB094 in 82 PD patients with
and without genetic variants (NCT03976349). The BIIB094 molecule,
delivered to the CNS via an intrathecal injection, is thought to lower
LRRK2 protein levels by binding mRNA and mediating its degradation
(ionispharma.com).
3.3.3. GTPase modulators
The kinase domain is not the only target from the catalytic core of
LRRK2, as the ROC GTPase domain have also been suggested to play a
role in PD pathogenesis. It was pointed out that all pathogenic variants
in the ROC domain increase the proportion of GTP-bound LRRK2, while
the protective variant p.R1398H decreases it (Nixon-Abell et al., 2016).
It is not yet clear if the observed relationship is due to changes in affinity
of the protein for GTP, decreased GTPase activity or due to indirect

Table 4
Therapeutic approaches for LRRK2.
Mechanism Drug Registered clinical trial Cohort Phase Current status
name (clinicaltrials.gov) o
r reference

LRRK2 kinase inhibitor
p.G2019S selective LRRK2
kinase inhibitor
Antisense oligonucleotide
(inhibit translation)
Antisense oligonucleotide
(Induces skipping of exon
41)
GTPase modulator
DNL151 NCT04557800 Healthy 1/1b Completed with positive outcome in terms of safety and target
NCT04056689 volunteers/PD engagement.
Selected to be advanced to late stage studies.
DNL201 NCT04551534 Healthy 1/1b Completed with positive outcome in terms of safety and target
NCT03710707 volunteers/PD engagement.
Will be kept as backup for DNL151.
PFE-360 Baptista et al. (2020) – Preclinical Reported ~50% reduction in LRRK2 activity in the nonhuman primate
brain with minor or reversable changes in lungs.
GNE- Baptista et al. (2020) – Preclinical Reported that low doses were sufficient to completely inhibit brain LRRK2
7915 activity in macaque and to avoid major impairment of lung function.
MLi-2 Baptista et al. (2020) – Preclinical
EB- Bright et al. (2021) – Preclinical Ex vivo study reported selective p.G2019S LRRK2 inhibition measuring
42168 Ser935 and The73 Rab10 biomarkers.
BIIB094 NCT03976349 PD 1 Recruiting PD patients with and without genetic variants.
ASO 41- Korecka et al. (2020) – Preclinical Reported to decrease Rab10 phosphorylation and normalize
1 autophagosome maturation in transgenic mice; normalize mitophagy rate
and endoplasmic reticulum Ca2+ homeostasis in vitro.
FX2149 Li et al. (2015) – Preclinical Reported to decrease LRRK2 autophosphorylation, reduce
neuroinflammation, and decrease neuronal degeneration in vitro and in
murine model. More potent and brain-penetrant than earlier analogues.

7
K. Senkevich et al.
inhibition of LRRK2 kinase activity. In vitro and in vivo studies testing
GTP-binding inhibitors reported some decrease in LRRK2 autophos­
phorylation, reduced neuroinflammation and suppression of
LRRK2-linked neuronal degeneration (Li et al., 2014, 2015). Addition­
ally, what makes GTP-binding inhibitors an interesting approach, is the
potential of great selectivity due to the fact that LRRK2 is one of only
four ROC GTPases in humans (Tomkins et al., 2018). We expect that
additional GTPase modulators will be developed and progress into
clinical trials in the future.
3.4. Can LRRK2 inhibitors be used for sporadic PD and GBA-PD?
Some genetic and experimental data suggest that LRRK2 may also be
relevant in sporadic PD. For example, LRRK2 is one of the top genes
implicated in PD genome wide association studies, even after removing
the effects of the p.G2019S mutation and other potentially pathogenic
variants (Lake et al., 2021). This association may be driven by regula­
tory, non-coding variants in LRRK2, potentially affecting its expression
or activity (Lake et al., 2021). Experimental data from different in vitro
and in vivo models suggest that LRRK2 is involved in mechanisms related
to sporadic PD, such as inflammation, α-synuclein accumulation and
others (Kluss et al., 2019). Therefore, it is possible that LRRK2-targeting
treatment will be relevant for some sporadic PD patients, who may be
genetically and biologically similar to LRRK2-PD. Identifying these
specific individuals, perhaps by a combination of genetics and bio­
markers such as LRRK2 activity, substrate phosphorylation and others,
could be crucial for treating sporadic PD patients with LRRK2 inhibitors.
Recently, experiments in cell models suggested that LRRK2 in­
hibitors may also be beneficial in GBA-PD. In these experiments, LRRK2
mutations led to reduced GCase activity, and treatment with LRRK2
inhibitors improved GCase activity (Ysselstein et al., 2019). However,
multiple lines of human data contradict these results and do not support
LRRK2 inhibitors as a viable treatment for GBA-PD. First, in a cohort of
PD patients with the LRRK2 p.G2019S mutation, plasma GCase activity
was increased rather than decreased (Alcalay et al., 2015a). In carriers of
the LRRK2 p.M1646T risk variants, plasma GCase was increased as well
(Sosero et al., 2021). Carriers of both GBA and LRRK2 mutations provide
additional compelling evidence that LRRK2 mutations are not likely to
decrease GCase activity. If they were, we would expect that individuals
with both GBA and LRRK2 mutations will have a more severe pheno­
type, as they supposedly carry two mutations that reduce GCase activity.
However, data from three independent cohorts suggest the opposite
(Omer et al., 2020; Ortega et al., 2021; Yahalom et al., 2019). In all three
cohorts (two cross-sectional and one longitudinal), PD patients with
both LRRK2 and GBA mutations had milder phenotypes, similar to those
who carry only LRRK2 mutations. Lastly, the major differences in pa­
thology and clinical course (Kalia et al., 2015; Schneider and Alcalay,
2017; Srivatsal et al., 2015), the fact that GBA mutations may also lead
to RBD or dementia with Lewy bodies while LRRK2 mutations do not
(Creese et al., 2018; Honeycutt et al., 2019; Krohn et al., 2020b; Nalls
et al., 2013; Ouled Amar Bencheikh et al., 2018; Pont-Sunyer et al.,
2015), together with the previous data, all support a separate mecha­
nisms for GBA-PD and LRRK2-PD, and may even suggest that LRRK2
mutations have a protective effect in GBA-PD. If this is true, LRRK2 in­
hibition in GBA-PD may even worsen their disease. We therefore
recommend, with the currently available data, not to pursue LRRK2
inhibition as a therapeutic strategy for GBA-PD.
3.5. Challenges, strategies and future directions in LRRK2-PD clinical
trials
Perhaps the main challenge in the development of LRRK2-targeting
treatment is related to the clinical course and rate of progression of
LRRK2-PD. Several studies have suggested that LRRK2-PD progresses
slower, and have less cognitive decline than sporadic PD or GBA-PD
(Ortega et al., 2021; Yahalom et al., 2019). Since it progresses slower,
8
Neuropharmacology 202 (2022) 108822
either larger trials or longer trials will be required to observe a drug
effect, unless the effect of the drug will be very large. However, a large
effect cannot be assumed in advance, even if in preclinical models it
seems this way. Therefore, trials for LRRK2 inhibitors should probably
be designed to be larger and longer. The reduced frequency and slower
progression to cognitive decline, make it a less favorable endpoint in
LRRK2-targeting trials, which will probably have to focus on motor
symptoms and other biomarkers. Such biomarkers may be proven
crucial for the development of a therapeutic LRRK2 inhibitor, if the
clinical signal will be relatively weak, but only if there is clear evidence
from human studies that changes in biomarkers are strongly correlated
with clinical changes. One of the questions that may arise is whether the
current biomarkers such as LRRK2 autophosphorylation, Rabs phos­
phorylation, urine BMP and others are reliable enough to move forward
to clinical trials (Rideout et al., 2020). The first trials on LRRK2 in­
hibitors may provide the data needed to answer this question.
Another challenge, similar to GBA but more acute, is the challenge of
recruitment. Since LRRK2 mutations are less common, recruitment
might prove difficult. Same as for GBA, we recommend that all clinicians
will genotype LRRK2 mutations in all their patients to make them trial-
ready. There are multiple research- or clinical-based initiatives aiming
to genotype LRRK2 in multiple cohorts worldwide. Having trial-ready
PD populations will be crucial for developing LRRK2 trials, especially
due to the size and trial duration issues mentioned above.
Less is known about LRRK2 modifiers, as opposed to GBA modifiers.
A family and population-based study suggested that DNM3 may be a
modifier of LRRK2-PD (Trinh et al., 2016), yet it was not replicated in a
follow-up study (Brown et al., 2021). More recently, a GWAS aimed to
identify LRRK2 modifiers nominated variants in the CORO1C region as
potential LRRK2 modifiers (Lai et al., 2020). Just like in GBA-PD,
polygenic risk score of PD also affected LRRK2 penetrance (Iwaki et al.,
2020). Although the effects of these variants on LRRK2-PD progression is
unknown, we suggest that in clinical trials targeting LRRK2, it will be
useful to match the treatment and placebo groups for CORO1C variants
and polygenic risk scores.
4. Therapy targeting PRKN
PRKN or Parkin is one of the autosomal recessive genes that was
identified through gene mapping in juvenile-onset PD patients (Kitada
et al., 1998). Homozygous and compound heterozygous mutations in
PRKN cause early-onset PD (EOPD) (Brüggemann and Klein, 1993). A
systematic review carried out to estimate the prevalence of biallelic
PRKN variants in EOPD showed 4.3% in sporadic EOPD cases, up to
15.5% in familial cases of EOPD (Kilarski et al., 2012).
While the role of biallelic PRKN mutations in PD is well established,
the association of rare heterozygous variants with the disease is
controversial, as different studies yielded contradicting results (Kay
et al., 2010; Lesage et al., 2008). A recent study tried to elucidate this
link by assessing the rare heterozygous single nucleotide variations
(SNVs) and copy number variations (CNVs) in a relatively large cohorts
of PD patients (Yu et al., 2021). The study reported no associations of
rare heterozygous PRKN variants with PD risk, nor with the AAO.
Another recent study suggested that the association of monoallelic PRKN
variants could be inflated by cryptic biallelic variants (Lubbe et al.,
2021). Therefore, we currently cannot consider heterozygous carriage of
PRKN mutations as a risk factor of PD, or such carriers as candidates for
Parkin-targeting trials.
4.1. Mechanism, pathology and clinical features of PRKN-PD
The parkin protein, encoded by PRKN, is an E3 ubiquitin protein
ligase that, which together with a kinase PINK1 plays a crucial role in
mitophagy – removal of dysfunctional mitochondria through auto­
phagy, hence supporting healthy mitochondrial function (Narendra and
Youle, 2011). One of the main triggers for mitophagy is the
K. Senkevich et al. Neuropharmacology 202 (2022) 108822

accumulation of mitochondrial reactive oxygen species (ROS) and fail­ Table 5

ure of various antioxidant enzymes to adequately respond to the Therapeutic approaches for Parkin.
oxidative stress (Schofield and Schafer, 2021). Hence, disruption of this Mechanism Drug Registered Cohort Phase Current
important self degradation mechanism could lead to cytotoxic levels of name clinical trial status
ROS detrimental to dopaminergic neurons (Fujita et al., 2014; Kolodkin (clinicalt
rials.gov) o
et al., 2020). It was also suggested that by regulating mitophagy, Par­
r reference
kin/PINK1 have a role in the innate immunity modulation, and dis­
ruptions can lead to loss of dopaminergic neurons due to Parkin WO- Miller and - Preclinical Data
activators; 2018/ Muqit limited
neuroinflammatory response (Sliter et al., 2018). autoinhibitory 023029 (2019) to in vitro
Neuropathological studies, albeit only a few, showed that PD pa­ mechanism WO- assays.
tients carrying biallelic PRKN variants have an atypical neuropathology, disruptors 2010/
with mostly absent α-synuclein accumulations and LB (Schneider and 011839
Selective MF-094 Kluge et al. - Preclinical Data
Alcalay, 2017), as well as localized neurodegeneration limited only to
inhibitors of MF-095 (2018) limited
the substantia nigra and locus coeruleus (Dawson and Dawson, 2010). In ubiquitin- to in vitro
addition to early onset of disease, other clinical features of PRKN carriers specific assays
clearly differ from sporadic PD. The disease progression is much slower protease 30
(Alcalay et al., 2014), cognition is relatively preserved in advanced
disease and dementia is absent in most patients (Alcalay et al., 2014;
targeting drugs will become much larger. However, the current evidence
Piredda et al., 2020), and olfaction is mostly preserved (Wang et al.,
are conflicting, and recent large-scale analyses suggest that the previous
2017). Milder disease features observed in PRKN carriers could be
reported association between heterozygous PRKN variants and PD may
related to mostly absent α-synuclein pathology and pathology confined
be due to cryptic second mutations that have not been identified (Lubbe
to specific brain regions.
et al., 2021). Another issue is that even if the association was true, the
reported odds ratio is small, similar to those of top SNPs in PD GWAS.
4.2. PRKN targeting drugs in development
This would mean that for many of the PRKN heterozygous carriers,
having PD is likely not related to their PRKN genetic status, and they
At this point, no drug that specifically and directly targeting PRKN is
might not benefit from Parkin-targeting drugs. Including these patients
in clinical trials. However, there are multiple small molecules that are
in trials may therefore dilute the signal and in a case of a drug that could
going through preclinical development, mostly aimed at activation of
work, lead to false negative results. We therefore recommend at this
mitophagy. One of the evaluated approaches is the direct activation of
stage to focus only on biallelic PRKN-PD patients for clinical trials,
Parkin by disrupting its autoinhibitory mechanisms. Two small mole­
despite the aforementioned limitations of such trials.
cules (WO-2018/023029 and WO-2010/011839) showed this effect in
vitro but in vivo evidence are yet to be published (Miller and Muqit,
5. Conclusions
2019). Another proposed drug target is the ubiquitin-specific protease
30 (USP30), which removes ubiquitin from substrates of Parkin
Genetic studies have provided new and important insights into the
(Table 5) (Komander et al., 2009). Inhibiting this enzyme could poten­
pathogenesis of PD, and now allow for stratifying patients based on their
tially compensate for reduced Parkin activity. Two compounds (MF-094
genetic background towards clinical trials targeting specific genetic
and MF-095) showed effective and selective inhibition of USP30 in vitro
variants. Despite some major limitations discussed in this review, the
(Kluge et al., 2018). Additionally, there are multiple studies aimed at
most promising targets thus far are GBA, LRRK2 and PRKN, for which
supporting mitochondrial function by targeting the electron transport
specific treatments are in different stages of clinical development. Many
chain or by providing antioxidative effects but since these mechanisms
challenges still exist, including recruitment, biomarkers identification,
are not specific to PRKN-PD, they will not be discussed in this review.
trial design and others. Nevertheless, through international collabora­
tions and data sharing, we can enable large and properly designed
4.3. Challenges, strategies and future directions
clinical trials for PD patients who carry mutations in these specific
genes, towards future development of efficient treatments.
Some of the challenges we will face when designing a clinical trial
targeting Parkin are similar to those of LRRK2, but amplified. As biallelic
Financial disclosures
PRKN mutations carriers with PD are much rarer, recruiting cohorts that
will be large enough for meaningful clinical trials will be difficult and
ZGO received consultancy fees from Lysosomal Therapeutics Inc.
likely require years of preparations in multiple sites. As PRKN-PD can be
(LTI), Idorsia, Prevail Therapeutics, Inceptions Sciences (now Ventus),
considered as an independent, rare disease, smaller trials might be
Ono Therapeutics, Denali, Handl Therapeutics, Neuron23, Bial Biotech,
performed, with the hope of a large enough effect size of the drug that
Guidepoint, Lighthouse and Deerfield. Other authors have nothing to
will be detected in a smaller group during the trial. The rate of pro­
disclose.
gression of PRKN-PD and the overall conserved cognitive function also
pose major hurdles, since it will be difficult to obtain a significant signal
Acknowledgement
in both motor and cognitive domains as outcomes of the trials. For this
reason, identifying specific and reliable biomarkers for PRKN-PD pro­
ZGO is supported by the Fonds de recherche du Québec - Santé
gression may be crucial for future trials. While the slow progression of
(FRQS) Chercheurs-boursiers award, in collaboration with Parkinson
PRKN-PD is generally a disadvantage towards trial development, it may
Quebec, and is a William Dawson Scholar. KS is supported by a post-
also be conceived as an advantage, as more patients might be eligible to
doctoral fellowship from the Canada First Research Excellence Fund
enroll to the trial, as many are still defined at early stages of the disease.
(CFREF), awarded to McGill University for the Healthy Brains for
The development of international collaborations and registries of
Healthy Lives initiative (HBHL). Fig. 1 was created with BioRender.com.
monogenic form of PD will advance our ability to perform clinical trials
of PRKN-PD even with the above limitations (Klein et al., 2018). How­
ever, these trials should still be designed to be longer than typical trials.
An important issue to solve is the role of heterozygous PRKN variants
as risk factors in PD. If they are, then the population eligible for Parkin-

9
K. Senkevich et al. Neuropharmacology 202 (2022) 108822

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