Prodromal sensory neuropathy in Pink1−/−SNCAA53T double mutant Parkinson mice

Parkinson's disease (PD) is frequently associated with a prodromal sensory neuropathy manifesting with sensory loss and chronic pain. We have recently shown that PD‐associated sensory neuropathy in patients is associated with high levels of glucosylceramides. Here, we assessed the underlying pathology and mechanisms in Pink1−/−SNCAA53T double mutant mice.


INTRODUC TI ON
Parkinson's disease (PD) is a complex neurodegenerative disease that primarily affects motor systems of the basal ganglia and manifests with muscle rigidity, tremor, slowness of movement and difficulty walking. [1] PD also involves multiple extranigral brain regions and the peripheral nervous system [2] leading to non-motor symptoms, particularly olfactory dysfunction, rapid eye movement (REM) sleep disorder, dysautonomia, restless leg disorder [3][4][5][6][7] and chronic pain that presents as musculoskeletal, neuropathic, visceral and dystonic pain and is highly prevalent in PD patients. [8][9][10][11][12][13] PD-associated pain is frequently associated with small or mixed fibre sensory neuropathies involving somatosensory and autonomic nerves. [14][15][16][17][18] Patients often complain about constipation, visceral discomfort and pain, gut and bladder dysfunction and deregulation of exocrine glands. A loss of somatosensation may remain unnoticed, unless it is associated with burning pain in the hands and feet. [19] The progressive loss of sensory function parallels the progression of the disease, but may precede motor symptoms for more than 10 years, [19,20] suggesting that sensory neurons are particularly vulnerable and likely represent a source of the propagation of prionlike alpha-synuclein. [21][22][23] The predominant morphological features of PD are intraneuronal alpha-synuclein-rich deposits, the major components of Lewy bodies, [2,24,25] but the pathophysiology is only partly understood.
Mitochondrial damage and dysfunction of protein degradation via the proteasome and via autophagolysosomes contribute to the progressive loss of dopaminergic and other neurons. Defective mitophagy can lead to leakage of mitochondrial DNA to the cytosol that triggers innate immune system responses. [26] It has been recognized in recent years that metabolic deregulation of bioactive lipids including ceramides and their metabolites increases the toxicity of alpha-synuclein. [27][28][29] Patients who are heterozygous carriers of glucocerebrosidase (GBA1) mutations tend to develop a rapidly progressive disease, [30] and such mutations propagate alpha-synuclein deposition in PD model organisms. [31][32][33][34] GBA1 mutations constitute the highest independent genetic risk factor for sporadic PD. [35] GBA1 is a lysosomal enzyme that catalyses the degradation of ceramides to glucosylceramides (GlcCer) and the subsequent generation of lactosylceramides (LacCer) and gangliosides. Low GBA1 activity and accumulation of GlcCer even occur in PD patients without known mutations of GBA1 [8,36,37] and point at a link between PD and Gaucher disease, a lysosomal storage disorder that is caused by homozygous loss-of-function mutations of GBA1. [38] We have recently shown in patients with sporadic PD that high concentrations of plasma GlcCer are associated with pain ratings and with sensory loss as assessed by quantitative sensory testing, [8] suggesting that GlcCer may contribute to the progression of the disease from peripheral sensory neurons to the brain and to clinical PD-associated pain. [8] GlcCer are components of membrane barriers and maintain organelle curvature, but overloading interferes with the lysosomal membrane charge and leads to leakage. [39] It also accumulates in mitochondrial membranes. [40] Hence, GlcCer constitutes a link between lysosomal and mitochondrial pathology in PD.
There are no remedies that specifically alleviate PD-associated pain or prevent sensory loss, [41,42] in part owing to limited knowledge of PD-associated somatosensory pathology in model organisms. Motor function and the morphological characteristics of PD have been studied in several rodent, primate and fly disease models, but sensory function and the underlying pathological features of sensory neurons have been rarely addressed, [43] and the few studies of non-motor manifestations such as olfaction, anxiety-like behaviour and gastrointestinal function are fragmented and, in part, inconclusive.
In the present study, we used a previously described Mice had free access to food and water, and they were maintained in climate-controlled rooms with a 12 h light-dark cycle.
Behavioural experiments were performed between 10 am and 3 pm.

Analysis of nociception and somatosensory function
Nociceptive tests were performed at 2, 4, 10 and 12 months of age with 8 mice per group. Mice were habituated to the test room and the test chambers for three consecutive days before the baseline measurements.
The latency of paw withdrawal on point mechanical stimulation was assessed using a Dynamic Plantar Aesthesiometer (Ugo Basile).
The steel rod was pushed against the plantar paw with ascending force (0-5 g, over 10 s, 0.2 g/s) and then maintained at 5 g until the paw was withdrawn. The paw withdrawal latency was the mean of three consecutive trials with at least 30 s intervals.
The sensitivity to painful heat stimuli was assessed by recording the paw withdrawal latency with a Hot Plate (52°C or 30-55°C surface) or with the Hargreaves test (IITC Life Science). In the latter, an infrared lamp was placed with a mirror system underneath the respective hind paw. By pressing the start button the lamp starts to emit a heat-beam until the paw is withdrawn, which stops the lamp.
The mean of three replicate tests was used for statistical analysis.
The Orofacial Pain Assessment Device (OPAD, Stoelting) allows for evaluation of facial thermal nociception by using a rewardconflict paradigm. The OPAD cage consists of a plexiglass chamber with metal grid floor and an adjustable slit giving access to the nipple of the reward bottle. The slit is flanked with PC-controlled thermal peltier elements. To receive the reward (diluted milk in water), the mouse has to touch the thermodes with its cheeks. Mice were fasted overnight to increase their appetite. Mice were trained three times a week for 2 weeks at innocuous temperatures (36.5-38°C) to get a stable baseline of at least 600 licks in 18 min. During test periods, temperature circles starting from neutral (37°C, 3 min) to aversive cold (15°C, 10°C each 3 min) or aversive heat (45°C, 54°C each 3 min) were applied using a ramping protocol. Circles were repeated up to 20 min. The ANY-maze software (Version 4.99, Stoelting) registered licks and contacts with the thermodes. The average and total numbers of lickings and contacts at the defined temperatures were used for analysis. Ramping times were excluded.

Thermal gradient ring
A thermal gradient ring (Ugo Basile) was used to assess the temperature preferences and exploration of the ring platform. [45] The TGR provides a circular running track for the mouse to move freely.

Olfactory and motor functions
Behavioural studies of olfactory sensitivity and discrimination, motor functions and Phenomaster analyses of drinking, feeding and voluntary wheel running are explained in the Data S1.

Culture and staining of primary DRG neurons
Primary adult dissociated DRG neuron-enriched cultures were prepared by dissecting mouse dorsal root ganglia (DRGs) into 1x PBS (Phosphate Buffered Saline, Gibco), followed by digestion with 5 mg/ml collagenase A and 1 mg/ml dispase II (Roche Diagnostics).
Triturated cells were centrifuged through a 15% BSA (bovine serum albumin) solution and plated on poly-L-lysine and laminin coated cover slips in Neurobasal medium (Gibco) containing 2% (vol/vol) B27 supplement (Gibco), 50 μg/ml Pen-Strep, 100 ng/ml NGF and 200 mM L-glutamine. After incubation for 2 h, 2 ml Neurobasal medium was added, and neurons were cultured for up to 48 h depending on the experimental requirements. Cells were kept at 37°C, 5% CO 2 , 95% humidity. Primary DRG neurons were used for calcium imaging and immunohistochemical staining.
To assess the effects of GlcCer24:1 (Avanti Polar Lipids, #860549P) on outgrowth, morphology and excitability neurons were cultured in the presence of 1-10 µM GlcCer24:1 or vehicle, starting at plating of the neurons. The vehicle was a 2:1 mixture of chloroform and methanol, the final dilution of the vehicle was 1:10 000 or 1:1000, respectively. For assessment of culture longevity, tiled images of live cultures were captured daily to assess the whole coverslip. Culture density was assessed by analysing the percentage area covered with neurons and dendrites using FIJI ImageJ. After background subtraction, thresholds were set using Li's algorithm, and binarized images were analysed using the particle counter. For immunofluorescence studies, cultures were washed and subsequently fixed with 4% paraformaldehyde in 1x phosphate buffered saline (PBS) at 24 or 48 h. Immunostaining was performed for neuronal marker (NF200, TUJ1), alpha synuclein, the growth cone marker, phalloidin-Alexa594 and nuclear DAPI to assess outgrowth, dendritic trees, morphology and viability. Arborization was assessed using Scholl-analysis implemented in ImageJ.

Calcium imaging
Calcium influx in sensory neurons upon stimulation with capsaicin is a biological correlate of nociceptive sensitivity. [46][47][48] Capsaicin is a VALEK Et AL.

RNA analyses
Quantitative RT-PCR (qRT-PCR) were done according to standard procedures (Data S1) using Primers described in Table S1a. RNA sequencing and data analysis is described in Data S1 and was essentially as described in Ref. [49].
For analysis of neurite outgrowth and morphology, primary neuron cultures were washed in PBS, fixed in 4% PFA and immunostained with primary antibodies (Table S1). Tiled images were captured on an inverted Axio Imager Z1 fluorescence microscope (Zeiss). For quantification, RGB images were converted to binary images using threshold setting implemented in ImageJ, and the particle counter was used to assess the area covered with neuronal immunoreactive structures.

Transmission electron microscopy
Mice were terminally anaesthetized with carbon dioxide and perfused transcardially with cold 0.9% sodium chloride (NaCl) followed Sections were analysed using a Zeiss electron microscope (Zeiss EM900) and imaged with a slow-scan CCD-Camera. The area, width and brightness of white mitochondria was measured with the respective tools in FIJI ImageJ.

Oxygraph analysis of mitochondrial OXPHOS activity
Brain sections (prefrontal cortex, hippocampus), DRGs, and trigeminal nerve and ganglia were immediately transferred into ice-cold Respiration medium MiR06Cr containing 280 U/ml catalase. Tissue was homogenized using a motor-driven tightly fitting glass/Teflon Potter-Elvehjem homogenizer. Mitochondrial respiration was measured using high-resolution respirometry (Oxygraph-2k, Oroboros Instruments) with DatLab software 6.1.0.7 (Oroboros Instruments). A "substrate-uncoupler-inhibitor titration" protocol was used essentially as described in Ref. [46]. LEAK-respiration was induced by the addition of complex I -linked substrates pyruvate (5 mM), malate (0.5 mM) and glutamate (10 mM). Complex I -linked respiration was measured after adding ADP (2.5 mM) in a saturating concentration. To measure complex II-linked respiration, rotenone (0.5 µM) was added to block complex I followed by the addition of succinate (10 mM). Maximum uncoupled respiration (ETS, electron transfer system capacity) was PRODROMAL SENSORY LOSS IN PD MICE | 1063 measured after stepwise titration of FCCP (carbonyl cyanide-p-trifluo rmethoxyphenylhydrazone). Residual oxygen consumption (ROX) was determined after sequential inhibition of complex III with Antimycin A and complex IV with azide. Absolute respiration rates were corrected for ROX and normalized for the protein content. Citrate synthase activity was measured to confirm comparable numbers of mitochondria.

Lipid analyses
Lipid analyses were done as described previously [50] and are briefly explained in Data S1.
RNA sequencing and data analysis is described in Data S1 and was essentially as described in Ref. [50].

Statistics
Group data are presented as mean ± SD or mean ± SEM, the latter for behavioural time course data, specified in the respective figure legends. Data were analysed with SPSS 24 and Graphpad Prism 8.0 and Origin Pro 2020. Data were mostly normally distributed, or lognormally distributed. For testing the null-hypothesis that groups were identical, the means of two groups were compared with 2sided, unpaired Student's t tests. The Mann-Whitney U test was used as a non-parametric alternative in case of violations of t test requirements. Time course data or multifactorial data were submitted to two-way analysis of variance (ANOVA) using e.g., the factors 'time' and 'genotype'. For respirometry, the within subject factor "stimulus" and between subject factor "group" were used. In case of significant differences, groups were mutually compared at individual Principal component analysis was used to define the lipid species, which accounted most for the variance between genotypes.
Further multivariate analyses included canonical discriminant analysis to assess the predictability of group membership and separation of genotypes and tissues. A53T mice had normal paw withdrawal latencies upon mechanical stimulation but showed a strong loss of thermal heat sensitivity in Hargreaves and Hotplate tests ( Figure 1A), and they preferred cooler temperatures in a Thermal Gradient Ring (TGR) test, in which mice can freely choose their temperature of well-being ( Figure 1B). The TGR behaviour suggested paradoxical heat sensation and loss of cold sensation, which was confirmed for trigeminal hot/cold stimulation in the Orofacial Pain Assessment Device (OPAD), in which mice have to touch heated or cooled metal bars to get access to a rewarding milk bottle. Pink1 −/− SNCA A53T showed a preference for unpleasant cold temperatures when compared to WT mice ( Figure 1C).

Loss of thermal nociception in middle-aged
Nociceptive tests were repeated in younger mice to assess the onset of thermal sensory loss. There were no differences at 2 months of age between mutant and WT mice. The first significant protraction of paw withdrawal upon heat stimulation occurred at 4 months of age ( Figure 1D), hence at least 11 months before the earliest occurrence of spontaneous motor deficits.
There was no sensory loss in single mutant mice at 9-10 months of age but rather nociceptive hypersensitivity in Pink1 −/− mice

Accumulation of glucosylceramides in DRGs and loss of sphingolipids in the sciatic nerve
We have previously shown in PD patients, that sensory loss and pain owing to PD-associated polyneuropathy were linearly associated with high plasma GlcCer. [8] Hence, we hypothesized that PD associated polyneuropathy is caused, at least in part, by GlcCer accumula-  Figure S2B). The accumulation of ceramides also manifested as an increase of ceramide immunofluorescence in cultured DRG neurons obtained from 12 months old mice ( Figure 2D).
In the DRGs, the accumulation of ceramides predominated whereas loss of sphingosines and sphinganines predominated in the sciatic nerve ( Figure 3). Hence, GlcCer accumulates in somata, likely with a gain of toxic function, and downstream SPH metabolites are missing in axons, likely resulting is a loss of function. It is of note that the alteration of the signalling lipids was not associated with a disruption of the citrate cycle, which requires metabolic lipids, as revealed by normal citrate synthase levels in the DRGs ( Figure   S2D). Normal citrate synthase levels suggested normal mitochondrial numbers. However, mitochondrial damage is a predominant feature of PD, and GlcCer accumulation likely disrupts organelle membranes. Therefore, we studied mitochondrial morphology and respiratory function to assess a link between GlcCer and mitochondrial pathology.

Mitochondrial pathology in distinct DRG neurons with reduced mitochondrial respiration
Based on the lipid patterns, we expected that GlcCer overload would be associated with pathological alteration of organelle morphology and function. [39,52] We analysed the ultrastructural morphology of DRGs of 15-17 months old mice to address this hypothesis (Figure 4).
There were no overt differences in terms of cellularity, neuron number or shape, myelin sheaths or vascular cells between genotypes ( Figure 4). DRG neurons of both genotypes showed a high The box shows the interquartile range, the whiskers show minimum to maximum. Groups were compared with unpaired, two-sided Student's t tests or ANOVA according to the number of groups and data structure. OPAD data were compared with ANOVA for repeated measurements followed by post hoc comparison with correction of alpha according to Šidák. In E, data of single mutant mice were compared with two-way ANOVA followed by post hoc Šidák. Asterisks show adjusted p values with significant difference at *p < 0.05, **p < 0.001, ***p < 0.0001 VALEK Et AL. | number of autophagolysosomes, similar to the abundance of autophagolysosomes in DRGs of younger mice. [53] There was also no difference in the ultrastructural morphology of dark neuronal mitochondria (crista type) between Pink1 −/− SNCA A53T and wild-type control mice, but some neurons of Pink1 −/− SNCA A53T mice were highly packed with very prominent white mitochondria (Figure 4), which do not normally occur in the DRGs of younger mice. [53] The frequency of neurons with such mitochondria was 0%-3.5% in sections of wild-type DRGs and 6%-15% in sections of Pink1 −/− SNCA A53T DRGs. These white mitochondria were bloated (higher width Figure 4C) and had fewer or disorganized cristae, reflected by higher brightness (i.e., no structure inside) ( Figure 4C). Similar changes of mitochondrial morphology have been previously described in neurons of the cortex and substantia nigra of Parkin-SNCA double mutant mice, [54] or mitochondria of neuronal cells exposed to beta-amyloid. [55] Mitochondrial lipidome studies of Parkin knockout mice showed elevated levels of some ceramide species. [56] Hence, elevated GlcCer likely contribute to these morphological indices of mitochondrial damage. In agreement with this conclusion, accumulation of GlcCer owing to GBA1 mutations are supposed to interfere with mitochondrial respiration [52] and with the autophagolysosomal removal of damaged mitochondria via mitophagy. [57,58] To assess if pathological mitochondrial morphology was associated with respiratory dysfunction, we measured cellular oxygen consumption and OXPHOS activity with a 'substrate-uncoupler-inhibitor' titration protocol in freshly prepared tissue of the prefrontal cortex, There were no differences between genotypes at the two sites of the brain, but oxygen consumption, Complex-I and C-II respiration and maximum uncoupled respiration were significantly reduced in DRGs of Pink1 −/− SNCA A53T mice. The difference was not caused by lower numbers of mitochondria as revealed by normal citrate synthase levels ( Figure S2D). The data suggest that sensory neurons of Pink1 −/− SNCA A53T mice may suffer from energy deficits and mitochondrial dependent redox stress.

RNA sequencing reveals transcriptional changes in the DRG
To assess if and how mitochondrial pathology was associated with changes at transcriptional levels or reflected by changes of mitochondrial genes, we performed gene expression analyses by RNA sequencing of DRG tissue of prodromal (i.e., "healthy") middle-aged to old mice ( Figure 6, leading edge genes in Figure S3, pathways in Figure S4).
Mitochondrial genes or genes involved in mitochondrial biogenesis and transport were similar, but mice clustered perfectly according to genotype by using the 1000 top regulated genes (at 90% confidence level with 1.5-fold change) ( Figure 6A). Apart from the knockout of Pink1, XY scatter plots ( Figure 6A), Volcano ( Figure 6B) and MA plots ( Figure 6C) revealed a major loss of acidic fibroblast growth factor, Fgf1 in DRGs of Pink1 −/− SNCA A53T mice. FGF1 is known to restore the survival of dopaminergic neurons in PD models, [59,60] and is supportive of developing and postmitotic sensory neurons. [61][62][63][64][65] Fgf1 is a positive-control gene.

F I G U R E 3
Tissue specific analysis of sphingolipid species in DRGs, sciatic nerve and spinal cord. Box/scatter plots show the concentrations of the respective lipids (Y-axes labels) in pg/mg of tissue from spinal cord (SC), sciatic nerve (ScN) and DRGs in 12 months old Pink1 −/− SNCA A53T and wild-type control mice (n = 8 per group). Each scatter represents a mouse. The boxes show the interquartile range, the line is the median, the small open circle is the mean, the whiskers show minimum to maximum and the line shows the distribution according to a Gauss fit. Data were compared with two-way ANOVA for each lipid separately, followed by post hoc t tests using an adjustment of alpha according to Holm-Šidák. Lipids and sites with significant differences between genotypes are colour-coded. Red indicates a significant increase and blue a significant decrease VALEK Et AL. Gene ontology analyses and gene set enrichment analyses (GSEA) agreed with the top candidate pathways ( Figure S4). The major terms, which were enriched in downregulated genes (top 200 down) were "proteolysis", "cell differentiation", and "transmembrane" (mostly receptors). Major terms in upregulated genes (top 200 up) were "nuclease", "immune response", "lipid metabolism" and "ion channel/ion transport". Ion channels determine somatosensory neuron excitability and hence, they were of particular interest for the observed sensory losses. The gene regulations therefore led us to study thermosensitive TRP channels.

Loss of TRP-channels in DRGs and loss of TRPVcalcium signals in sensory neurons of the DRGs
GlcCer overload leads to membrane dysfunction [39] that likely impacts transmembrane ion channels and receptors. The mechanisms are, so far, unknown. Our behavioural studies suggested that thermo-sensory function is particularly vulnerable. Hence, we assessed expression and function of TRP calcium channels that mediate the sensation of heat and cold (Figure 7).
The RNA profile showed a reduction of TRPVs and TRPA1 and increase of TRPM6, whereas redox-sensitive TRPCs were mostly unaffected ( Figure 7A). The loss of TRPs was confirmed by RT-PCR ( Figure 7B) and immunofluorescence analysis of TRPV1 immunoreactive neurons in DRG sections ( Figure 7C counts, Figure 7D exemplary images). Further immunofluorescence studies of the DRGs at 3 and 9 months did not reveal a significant reduction of the numbers of TRPV1 or TRPA1 positive neurons in these younger mice, but TRPM8 was reduced ( Figure S5). Hence, the observed loss of thermal sensitivity at the behavioural level preceded the disappearance of the immunofluorescent signal of TRP channels.
There were no differences in gene expression of voltage-gated calcium or sodium channels, but a number of subunits of voltagegated potassium channels were increased ( Figure S4 . Complex-I-respiration was initiated by adding ADP in a saturating concentration. To measure C-II respiration, rotenone was added to block C-I, followed by adding succinate. Maximum uncoupled respiration was measured after stepwise titration with the uncoupler, FCCP. Residual oxygen consumption (ROX) was determined after sequential inhibition of complex III with antimycin A and complex IV with azide. Absolute respiration rates were corrected for ROX and normalized for the protein content, and citrate synthase activity was assessed to confirm equivalent numbers of mitochondria ( Figure S2D). Data were analysed per two-way ANOVA for repeated measurements (within subject factor "stimulus" versus between subject factor "genotype") and subsequent genotype comparisons for each period using t tests with an adjustment of alpha according to Holm-Šidák. Asterisks indicate significant differences between groups, *p < 0.05 VALEK Et AL. | data, where paw withdrawal upon mechanical stimulation was in the normal range.
To assess the functional implications of TRP channel changes, primary DRG neurons of 18 months old Pink1 −/− SNCA A53T and wild-type mice were stimulated with capsaicin to activate TRPV1 and formalin to activate TRPA1. It is of note that the immunofluorescent morphology of neurons, dendrites and growth cones was alike in Pink1 −/− SNCA A53T and wild-type DRG cultures ( Figure S6A).
Immunofluorescence studies also did not reveal overt differences of autophagy, protein aggregates, or membrane and ER markers ( Figure   S6B). AUCs were determined by integration from 175 to 520 s for formalin and 695-950 s for high K + . Peaks and AUCs were compared with two-sided, unpaired t tests, time courses by two-way ANOVA. *p < 0.05, **p < 0.001, ***p < 0.0001 level. It is conceivable that high levels of GlcCer directly interfere with the gating properties and membrane insertion of TRP channels.

Glucosylceramides lead to hyperexcitability of primary sensory neurons
To address the direct effects of GlcCer on neuronal structure and TRP channel function, DRG cultures were treated with GlcCer24:1.
Short-term treatment (24-48 h) had no effect on the morphology or outgrowth dynamics of the dendritic trees up to 10 µM GlcCer24:1 ( Figures S7 and S8). Viability in the first 2 days was not impaired, as assessed by the fraction of neurons responding to "high-K + " with a

Glucosylceramides reduce sensory neuron longevity
To assess the impact on culture longevity upon protracted GlcCer24:1 treatment, live cultures were captured daily and the coverage with neuronal bodies and neurites was analysed. The statistical analysis in Figure 8D revealed a premature decline of culture density in GlcCer24:1 treated cultures suggesting a negative impact of GlcCer24:1 on sensory neuron longevity, exemplified by a culture profile plot ( Figure 8E) that reveals the rarefication of neuronal somata at 96 h. Exemplary time courses of culture images are in Figure   S9A and quantification of neuronal numbers in Figure S9B.
Overall, the in vitro data shows that GlcCer24:1 causes hyperexcitability following short-term treatment of sensory neurons, presumably owing to membrane instability, which may ultimately result in calcium overload, mitochondrial damage, redox stress and premature damage of highly vulnerable thermosensitive neurons, which is reflected both in the mouse and the human phenotype of PD-associated sensory neuropathy.

DISCUSS ION
Somatosensory neuropathies are a frequent manifestation of PD, and the onset is often years before the occurrence of motor dysfunction. [16] The high vulnerability of sensory neurons in PD is not well-understood. In contrast with olfactory sensory neurons, which are also affected early in the course of PD, only a few neurons of the dorsal root ganglia are dopaminergic. [71] We show in the present study that Pink1 −/− SNCA A53T double mutant mice phenocopy the human prodromal sensory neuropathy.
These mice give new insight into the pathogenic events that culminate in an early PD-associated sensory neuropathy. They show a progressive loss of thermal sensation starting far earlier than the earliest occurrence of clinical motor dysfunction. The sensory phenotype is reminiscent of the QST phenotype of PD patients that is characterized by a predominant loss of thermal sensation with/ without mechanical hypersensitivity. [18,72] Thermal sensation relies on TRPV positive DRG neurons [73,74] and non-myelinated C-fibres, [75] whereas mechanosensation is mediated primarily through myelinated neurons with A-delta fibres. [76] The phenotype suggests distinct vulnerabilities of these subsets of sensory neurons.
At the transmission microscopy level, we did not detect overt morphological signs of neuronal death or damage in DRGs of Pink1 −/− SNCA A53T mice. However, studies in PD patients show that the density of sensory fibre terminals in the skin is reduced, [15,72] reminiscent of small fibre polyneuropathies in metabolic diseases [77] suggesting that metabolic deregulation may contribute to the sensory decline in PD, possibly involving lipids. Indeed, in PD patients, sensory loss and pain intensity ratings are associated with high levels of plasma glucosylceramides. [8] In agreement with the human data, we show now that GlcCer tional consequences of GBA1 deletion assumed that the observed pathology was caused by glucosylceramides without directly measuring these lipids. It was reported that GBA1 deletion or mutation increases alpha-synuclein aggregates, [28,29] leads to mitochondrial calcium overload, [58] ER stress [78] and disruption of autophagolysosomal pathways [79] (Graphical illustration in Figure 9). It is of note that Gaucher disease may manifest with a sensory neuropathy in some patients [80] but is not a predominant feature, possibly because of the younger age as compared to sporadic PD and the polygenetic nature of PD. Disease manifestations in Pink1 −/− SNCA A53T are likely a function of "genes x GlcCer x age".
In Gaucher disease, a metabolite of GlcCer, glycosylsphingosine (GlcSph), rather than GlcCer itself, was postulated to be a biomarker for disease progression. [81,82] Both, GlcCer and their metabolites, glucosyl-and galactosyl-sphingosines (psychosines) were shown to disrupt mitochondrial membranes and oxidative phosphorylation. [83,84] In line with these mechanistic reports, our ultrastructural and OXPHOS studies show that subsets of neurons have high numbers and maxima of capsaicin evoked calcium influx and of high K + evoked calcium influx. The areas were calculated from 180 to 500 s for capsaicin and 550-780 s for high K + . The box is the interquartile range, the line is the median, the open circle is the mean, whiskers show minimum to maximum. The scatters are neurons. Areas and maxima were compared with independent, two-sided t tests for each stimulus. The analysis included n = 159 and n = 131 neurons in control and GlcCer24:1 cultures. The cultures were from five mice per treatment. The asterisks indicate significant differences between groups, **p < 0.001, ***p < 0.0001. (D) For long-term exposure, cultures were treated with 1 µM GlcCer daily. Half of the medium was replaced with fresh medium containing 1 µM. Hence, concentrations in the cultures increased by 50% each day. Images of live cultures were captured daily (exemplary images are shown in Figure S7). Tiled images were stitched to observe all neurons. The coverage of the cover slips with neuronal bodies and dendrites was assessed in binarized images in FIJI ImageJ. The density of the cultures (box/scatter plots) declined in GlcCer24:1 conditions at 72 and 96 h; 2-way ANOVA for "time" X "treatment" and subsequent t test for "treatment" with adjustment of alpha according to Šidák. *adjusted p < 0.05. (E) Exemplary culture 3D surface plots of 96 h cultures treated with GlcCer24:1 versus vehicle. The intensity ranges from blue (cover slip bottom without growth) to high intensity (red) of neuronal bodies (rainbow colours). Red peaks represent the neuronal somata. The low number of the red crests in the GlcCer24:1 culture shows the thinning of the neuronal culture VALEK Et AL. | of white mitochondria that do not normally occur at younger age and were associated with respiratory dysfunction.
The mitochondria had a bloated appearance with partially disrupted cristae, and these morphological abnormalities bear some resemblance to those described in human PD patients' brains. [85][86][87] Mitochondria are susceptible to morphological EM artefacts but we did not observe swollen mitochondria in DRGs in younger mice in a previous technically identical study, [53] and swollen mitochondria were consistently more abundant in Pink1 −/− SNCA A53T mice than in controls and were only evident in specific neurons. Owing to the genetic defect of Pink1, we assume that damaged mitochondria were not efficiently removed by mitophagy. [88][89][90] Although there was no ultrastructural pathology of autophagolysosomes, lysosomal pathology likely contributed to the pathology because glycosphingolipids act as detergents on lysosomal membranes and disrupt the function of lysosomal enzymes. The functioning of GBA1 depends on folding and proper transfer from the ER to the lysosomes, a process that requires transport via the lysosomal integral membrane protein-2, LIMP2, [91,92] lysosomal acidic pH, negative membrane charge and negative calcium gradients [93] (Graphical illustration Figure 9). Intralysosomal calcium is kept low via outward calcium transport through TRP channels, [94,95] which is impaired by accumulating lipids. [96] It is of note that GBA1 dysfunction increases neuronal sensitivity towards calcium overload. [58] GlcCer accumulation, membrane instability and calcium F I G U R E 9 Putative signalling paths of GlcCer in PD-associated pathology. Pink1 deficiency and mutant SNCA cause genetic PD, and mutations of GBA1 are associated with sporadic PD. The enzyme glucocerebrosidase, GBA1 is needed for degradation of glucosylceramides (GlcCer). The graph illustrates how elevated GlcCer may initiate or further aggravate PD-associated pathology of mitochondria, lysosomes and membranes. Ceramides and GlcCer are essential components of membranes, required for membrane fluidity, the composition of lipid rafts and functioning of transmembrane ion channels like TRP channels. Membrane ceramides are generated in membranes via neutral sphingomyelinase (nSMase) on demand, and GlcCer are inserted into the outer leaflet of the plasma membrane. The compartmentalized homeostasis of membrane ceramides and GlcCer is disrupted upon overload with these lipids, which may lead to hyperexcitability or loss of TRP channels. GlcCer are degraded via GBA1, an acidic lysosomal enzyme that is transferred from the ER to the lysosome via LIMP2. Malfunction of GBA1 can arise from mal-transport, mutation, high pH or calcium overload. The resulting accumulation of glycosphingolipids further disrupts membrane charge, acidification, enzyme functions, lysosomal membrane integrity and autophagolysosomal flux. Autophagolysosomal degradation is the major pathway for removal of SNCA aggregates and of defective mitochondria via mitophagy. Mitophagy depends on Pink1-Parkin mediated ubiquitin labelling of the mitochondrial membrane. GlcCer overload promotes the aggregation of SNCA and may aggravate the failure of mitophagy, leading to a release of Bax and oxidative species (ROS). ROS further compromise lysosomal ATPase and TRP channels, leading to further accumulation of sphingolipids and calcium overload. Δψm, depolarization of mitochondrial transmembrane potential; ASM, acidic sphingomyelinase; ASAH, acid ceramidase; Cer, ceramides; dhCer, dihydro-ceramides; ER, endoplasmic reticulum; GBA1, GBA2 glucocerebrosidase alpha and beta; GlcCer, glucosylceramides; GlcSph, glucosylsphingosines; LIMP2, lysosomal integral membrane protein; Pink1, PTEN induced kinase; SM, sphingomyelin; SMase, sphingomyelinase; SNCA, alpha synuclein; Sph, Sphingosine; TRP channel, transient receptor potential channel; Ub, ubiquitin PRODROMAL SENSORY LOSS IN PD MICE | 1075 overload likely coincide or converge on axonal damage and loss of nerve terminals. Considering that heterozygous GBA1 mutations are the strongest independent risk factor for sporadic PD, we assume that GlcCer accumulation occurs early in the course of the disease and leads to mitochondrial damage and channel dysfunction.
Heat-evoked calcium influx in somatosensory neurons is mediated through TRPV channels. They are regulated by phosphorylation, redox modification, calmodulin and clathrin-mediated endocytosis. [69,[97][98][99][100][101] TRP channels are also sensitive to lipid-mediated modification, including phosphoinositides [68,69,102] and oxidized linoleic acid metabolites. [103,104] It is conceivable that GlcCer directly change the gating properties or interfere with the desensitization of TRP channels. We observed an exaggerated calcium influx in GlcCertreated cultures, which was not specific for TRPV1 stimulation, but also occurred upon depolarization pointing to membrane instability in agreement with an in vitro Gaucher model, where sphingolipid accumulation made plasma membranes more pliable. [105] To stop progression of neurodegeneration, one would like to know the causal chain of pathophysiological events. From the genetics of Pink1 −/− SNCA A53T mice, we expected SNCA aggregates and defective mitophagy. Indeed, we observed bloated dysfunctional mitochondria. GlcCer accumulation likely aggravates the failure of mitophagy because of the disruption of lysosomal functions.
Incomplete removal of damaged mitochondria results in oxidative stress and activation of innate immune responses, [26,106,107] which occurs concurrently with ceramide accumulation in the aging Pink1 −/− SNCA A53T brain. [66] Based on the Pink1 −/− SNCA A53T double mutant phenotype and the combined GlcCer and mitochondrial pathology, we propose that GlcCer accumulation results in membrane instability and mitochondrial membrane disruption, and hyperexcitability leads to calcium overload with subsequent secondary damage, ultimately converging on a loss of sensory nerve terminals. We have evaluated multiple mechanistic aspects, which contribute to the prodromal sensory loss in Pink1 −/− SNCA A53T mice. There is no single gene or pathway acting as single disease operator, but pathological events coincide and converge on small fibre neuropathy. The combined deregulation of sphingolipid metabolism, TRP channel dysfunction and loss and mitochondrial damage most convincingly explained the behavioural phenomena of early prodromal somatosensory loss and PD-associated pain. Because of the very early onset, QST for heat sensitivity may allow for early diagnosis, and pharmacological manipulation of GlcCer metabolic pathways may offer novel therapeutic options.

ACK N OWLED G EM ENTS
We thank Anke Biczysko for excellent technical assistance in ultrastructural analyses.

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflicts of interest. The funding institution had no role in data acquisition, analysis or decision to publish the results.

PEER R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1111/nan.12734.