Results
Persistently low biopterin and high neopterin plasma levels
in multiple sclerosis patients
Neopterin is used as activity and prognostic marker in inflammatory
diseases, cancer, some infections and rheumatoid arthritis (Altindag et
al., 1998; Husain et al., 2013; Prat et al., 2008; Sucher et al., 2010).
Neopterin is produced in excess if the activity and expression of GCH1
exceeds the capacity of the two downstream enzymes (PTPS and SPR) to
convert the GCH1 product, neopterin-3-phosphate (neopterin-3P) into BH4.
Excess neopterin in serum or plasma mainly originates from activated
immune cells. Overall, MS patients had increased neopterin levels (Fig.
1A), but there was no obvious association with the clinical disease
activity (Fig. 1B, 1C). In parallel, biopterin levels were persistently
reduced (Fig. 1A-C), but there was no inverse direct association of high
neopterin with low biopterin, except in few patients (Fig. 1C). High
neopterin but low biopterin suggested excessive consumption of BH4
presumably in activated immune cells. In support, biopterin levels
dropped over time in human whole blood assays stimulated with LPS (Fig.
1D), and microarrays of the spinal cord of EAE mice showed increased
GCH1 but reduced RNA levels of the recycling enzyme, quinoid
dihydropteridine reductase (QDPR) (Suppl. Fig. 1).
The pterin data of MS patients suggested two alternative therapeutic
interpretations assuming that the biopterin/neopterin balance regulates
immune functions, which is strongly suggested by previous publications
(Cronin et al., 2018; Huber et al., 1984; Schoedon et al., 1986). Either
the supplementation with BH4 may beneficially restore normal
antioxidative capacity of BH4 and iron homeostasis, or supplementation
may further stimulate immune cell proliferation and activity and
increase the autoimmune attack. To answer this question we used two
models of autoimmune encephalomyelitis in mice (EAE), with relapsing
remitting (RR-EAE; SJL mice) or primary progressive (PP-EAE; C57BL6
mice) course of the disease.
Sapropterin (BH4) treatment aggravates EAE in
mice
Mice were treated with sapropterin (BH4) or DAHP (GCH1-inhibitor) or
vehicle perorally starting at the day of immunization. Sapropterin
treated mice reached higher maximum scores in SJL RR-EAE mice (Fig. 2A)
and in C57BL6 PP-EAE mice (Fig. 2B) and increased the frequency of mice
with high scores. DAHP oppositely reduced scores during the first
relapse (Fig. 2A). Both drugs had no effects on body weights. The C57BL6
experiment was done with vehicle and sapropterin only.
BH4 treatment mildly increases infiltration of lymphocytes
in the spinal cord in EAE
mice
The observed increase of EAE scores in sapropterin-treated mice was
associated with a higher number of T-cells infiltrating the lumbar
spinal cord white matter, which was revealed by FACS analyses (Fig. 3A,
B) and immunofluorescence analyses (Fig. 4). Both CD4+ and CD8+ T-cells
were increased. FACS results for individual myeloid cell populations
were not significant, but immunofluorescence studies suggested more
hotspots of cellular invasion, in particular with staining of CD11b
(activity marker) and F4/80 (Fig. 4). Overall proliferation of T-cells
and myeloid cells in the spleen was however not affected by sapropterin
or DAHP treatment as compared to vehicle treated EAE mice (Suppl. Fig.
2), suggesting that oral BH4 medication did not reinforce T-cell
proliferation but was permissive for CNS infiltration. We therefore
asked how BH4 medication could affect BBB integrity.
We have two major hypotheses (i) high levels of circulating BH4 activate
endothelial NOS (NOS3) at the BBB, facilitating BBB breakdown (Wu et
al., 2009) or (ii) BH4 leads to changes of lipid homeostasis thereby
affecting barrier functions. The second hypothesis is based on its
coenzyme function for AGMO (Watschinger et al., 2015) and our previous
studies in colitis mice where BH4/DAHP altered LPAs and ceramides
(Zschiebsch et al., 2016), both clinically relevant for MS (Kurz et al.,
2018; Schiffmann et al., 2012; Schmitz et al., 2017). Because NO likely
has dual effects in EAE (Giovannoni et al., 1998; Wu et al., 2009) we
opted for the putative lipid-paths. It is of note that eNOS is localized
at the BBB in endothelial cells (Thiel et al., 2001) whereas AGMO is
strongly expressed in choroidal cells at the brain-to-CSF barrier
(Suppl. Fig. 4).
Sapropterin (BH4) treatment leads to increased long-chain
ceramides in EAE
mice
We reanalyzed our previous microarray data of EAE versus naïve mice
(Schmitz et al., 2014) (GEO dataset
GSE60847) for search of lipid metabolizing genes. The RNA studies of the
lumbar spinal cord revealed previously not recognized deficits in EAE of
fatty acid desaturases (FADS and stearoyl CoA desaturases SCD1, SCD2)
and fatty acid elongases (ELOVL) (Fig. 5A), but increased
glucocerebrosidase (GBA) that degrades glucosylceramides (Fig. 5A). GCH1
was increased as expected as well NOS2 and NOS3 (Suppl. Fig. 1). The
loss of desaturases, in particular FADS1, which is involved in FA18:2
and FA18:3 generation (linoleic and linolenic acid), is remarkable in
light of the protective preserving effects of these omega-lipids in EAE
and/or human MS (Bittner et al., 2013; Bjornevik et al., 2019).
A lipidomic screen of plasma and lymph nodes of EAE mice treated with
sapropterin or vehicle revealed lipid alterations in plasma but not in
the lymph nodes in sapropterin versus vehicle treated EAE mice (Suppl.
Fig. 3). In plasma, polyunsaturated fatty acids, in particular linolenic
acid (FA18:3), were reduced in sapropterin treated mice (Fig. 5B, 5C).
It is of note, that feeding of mice with linolenic acid preserves the
BBB in EAE mice because it restores gating properties of TASK1 two-pore
potassium channels (Bittner et al., 2009).
Targeted LC-MS/MS lipidomic analyses were used to assess signaling
lipids in more detail (Fig. 5D-F; Suppl. Fig. 3C). Using lipid species
of five classes (28 different species) as input, canonical discriminant
analysis clearly separated groups and allowed a >95%
correct prediction of group membership based on the first two CanDisc
scores (Fig. 5D). The clear separation was based mainly on ceramides of
different chain lengths. Ceramides were increased in sapropterin treated
EAE mice and oppositely regulated in DAHP-treated mice as compared to
vehicle (Fig. 5E). Sapropterin also increased the pro-inflammatory
endocannabinoid, 2-AG (Fig. 5E right), whereas DAHP reduced ethanolamide
endocannabinoids (AEA and OEA). Polar plots give an overview of multiple
bioactive lipid species (Fig. 5F) and reveal an increase of ceramides
and unsaturated LPAs in sapropterin-treated mice, whereas lipids were
mostly normal in DAHP treated mice. Scatter plots of the concentrations
of individual mice are shown in Suppl. Fig. 3C. Ceramide homeostasis is
crucial for the maintenance of plasma membrane integrity (Silva et al.,
2007) and increased levels suggest leaky membranes and dysfunctions of
lysosomal breakdown (Rao et al., 2007). Because the alterations were
evident in plasma, it is a systemic effect reaching the BBB and likely
affecting membrane attachment and invasion of immune cells.