Elovl5 Expression in the Central Nervous System of the Adult Mouse

ELOVL5 (Elongase of Very-Long Fatty Acid 5) gene encodes for an enzyme that elongates long chain fatty acids, with a marked preference for polyunsaturated molecules. In particular, it plays an essential role in the elongation of omega-3 and omega-6 fatty acids, precursors for long-chain polyunsaturated fatty acids (PUFAs). Mutations of ELOVL5 cause the spino-cerebellar ataxia type 38 (SCA38), a rare autosomal neurological disease characterized by gait abnormality, dysarthria, dysphagia, hyposmia and peripheral neuropathy, conditions well represented by a mouse model with a targeted deletion of this gene (Elovl5–/– mice). However, the expression pattern of this enzyme in neuronal and glial cells of the central nervous system (CNS) is still uninvestigated. This work is aimed at filling this gap of knowledge by taking advantage of an Elovl5-reporter mouse line and immunofluorescence analyses on adult mouse CNS sections and glial cell primary cultures. Notably, Elovl5 appears expressed in a region- and cell type-specific manner. Abundant Elovl5-positive cells were found in the cerebellum, brainstem, and primary and accessory olfactory regions, where mitral cells show the most prominent expression. Hippocampal pyramidal cells of CA2/CA3 where also moderately labeled, while in the rest of the telencephalon Elovl5 expression was high in regions related to motor control. Analysis of primary glial cell cultures revealed Elovl5 expression in oligodendroglial cells at various maturation steps and in microglia, while astrocytes showed a heterogeneous in vivo expression of Elovl5. The elucidation of Elovl5 CNS distribution provides relevant information to understand the physiological functions of this enzyme and its PUFA products, whose unbalance is known to be involved in many pathological conditions.


INTRODUCTION
Polyunsaturated fatty acids (PUFAs) are fatty acids containing two or more double bonds on their carbon chain, and are classified, depending on the position of the first double bond from the methyl terminal end, in ω-6 PUFAs and ω-3 PUFAs. In addition to a structural role in membranes, various PUFA metabolites also possess signaling functions, including roles in neurogenesis, neuronal survival, synaptic activity and regulation of brain inflammation (Guillou et al., 2010;Bazinet and Layé, 2014;Serhan et al., 2014). These multiple actions are the basis of the positive outcomes of PUFA-targeting treatments in various CNS pathological conditions, such as Parkinson's disease, Alzheimer's disease and Multiple sclerosis (Bousquet et al., 2011;Yanai, 2017;Penesová et al., 2018;Hernando et al., 2019). In mammalian organisms, essential fatty acids have to be introduced through the diet as precursors and then metabolized into more complex derivatives, by elongation and desaturation reactions performed by different enzymes located in the endoplasmic reticulum (ER) (Moon et al., 2009). A key and rate-controlling role is played by ELOVLs (ELOngation of Very Long-chain fatty acids), which are elongase enzymes responsible for and initial condensation reaction necessary to elongate fatty acids (Leonard et al., 2004;Jakobsson et al., 2006;Sassa and Kihara, 2014).
Two ELOVL5 missense mutations [(c.214C > G (p.Leu72Val) and c.689G > T (p.Gly230Val)] have been identified as the genetic cause of the Spinocerebellar Ataxia 38 (SCA38) (Di Gregorio et al., 2014). SCA38 is a rare form of autosomic dominant neurological disorder characterized by gait ataxia, nystagmus, peripheral neuropathy, hyposmia and pure cerebellar atrophy (Borroni et al., 2016). Such pathological features were recapitulated in a mouse model with a targeted deletion of the Elovl5 gene (Elovl5 −/− ) showing motor coordination and balance defects, loss of the sense of smell and a selective cerebellar atrophy (Hoxha et al., 2017).
Such a variety of neurologic alterations associated with mutations of different ELOVLs, might be explained by their tissue-specific expression and tissue-specific role. While ELOVL2, ELOVL7, and ELOVL1 have a restricted CNS distribution (Tvrdik et al., 2000;Lein et al., 2007), Elovl4 expression is abundant and well characterized in both adult and perinatal mouse brain (Sherry et al., 2017). In contrast, the Elovl5 expression pattern remains largely uninvestigated so far. This work is aimed at mapping Elovl5 expression in the adult mouse CNS with the final aim to provide relevant insights to understand its role in specific CNS areas and cell types.

Animals
Elovl5 −/− mice have been kindly provided by Dr. Moon and Dr. Horton of the UT Southwestern Medical Center (Moon et al., 2009). Brains analyzed to study the expression of Elovl5 were collected from adult (6 to 12 months old) Elovl5 −/− and Elovl5 +/− (C57/BL6J background) and wild type mice (C57/BL6J) of both sexes bred in our Animal Facility at NICO (Orbassano, Italy). All experimental procedures on adult mice and on pups have been approved by the Ethical Committee of the University of Torino and authorized by the Italian Ministry of Health (authorization number: 161/2016-PR and 510/2020-PR).

Tissue Preparation
To assess Elovl5 expression in brain slices mice were anesthetized using a cocktail of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight) via intraperitoneal injection. The mice were intracardially perfused initially with a physiological solution (NaCl 0.9%) and then with 4% paraformaldehyde in 0.12 M phosphate buffer, pH = 7.2-7.4. Following perfusion, the brain and the spinal cord were removed and stored at 4 • C for 24 h immersed in the same fixative and later transferred to a solution made of 30% sucrose in 0.12 M phosphate buffer for few days. Perfused brains and spinal cords were embedded in paraffin, cut into 5 µm-thick sagittal slices and mounted on superfrost glass slides (Fisher Scientific, Göteborg, Sweden) with Tris-glycerol supplemented with 10% Mowiol (Calbiochem, LaJolla, CA, United States).
To assess Elovl5 expression in glial cells in vitro, postnatal day 2 (P2) pups were cryoanesthetized in melting ice. The experimental plan was designed according to the guidelines of the NIH, the European Communities Council (2010/63/EU) and the Italian Law for Care and Use of Experimental Animals (DL26/2014). The study was conducted according to the ARRIVE guidelines.

Quantitative RT-PCR
Total RNA from cell cultures was extracted with the Directzol RNA Miniprep kit (Zymo Research, Irvine, United States), and reverse transcribed to cDNA with the High-Capacity cDNA Archive kit (Thermo Fisher Scientific, Waltham, United States). Quantitative Real Time RT-PCR was performed as described in Sacco et al. (2010), with the pre-developed Taqman assay #Rn01446631_m1 (Thermo Fisher Scientific, Waltham, United States). A relative quantification approach was used, according to the 2-ddCT method. β-actin (Taqman assay #Rn00667869_m1; Thermo Fisher Scientific, Waltham, United States) was used to normalize expression levels.

XGal Staining
To assess Elovl5 expression we first mapped β-galactosidase activity in homozygous and heterozygous Elovl5 knock-out mouse line where lacZ was included as a reporter under the control of Elovl5 promoter. Floating slices (30 µm-thick brain sagittal slices) were incubated overnight at 37 • C with a solution containing 1 mg/ml X-gal, 0.02% Nonidet P-40, 0.01% sodium deoxycholate, 2 mM MgCl2, 5 mM potassium ferricyanide, and 5 mM potassium ferrocyanide (pH = 7.5). Subsequently, the sections were mounted on gelatin-coated slides and let air dry overnight. The next day the sections were counterstained with Nuclear Fast Red: mounted series were washed for 2 min in distilled water and then stained in 0.1% NFR (nuclear fast red) solution for 2 min. Sections were then rinsed again in distilled water for 2 min, and subsequently dehydrated using a series of alcohols: 50% (2 min), 75% (2 min), 90% (2 min), and 100% (2 min). Afterward, the gelatin-coated slides were immersed in xylene for 5 min and finally a clear glass coverslip was applied using a permanent mounting medium.

Immunofluorescence Reactions
Slides were incubated at 60 • C for 30 min to allow the embedded tissue to firmly adhere to the glass slide. Following, paraffin was removed by immerging three time the glass slides in xylol (3 min each) and the tissue was hydrated using a series of alcohols with decreasing graduation: 99% (three washes of 5 min each), 95% (three washes of 1 min each) 70% (three washes of 1 min each). Slides were washes twice in H 2 O (1 min each) and placed for 40 min at 95 • C in citrate buffer (pH = 6.0) for antigen retrieval. Following the antigen retrieval, sections were washed twice in PBS (two quick washes) and then incubated with blocking buffer (Normal Goat Serum 5%, PBS 0.12 M pH = 7.4 and 1% Triton X100). Following, slides were washed with PBS for three times and then incubated with a cocktail containing: monoclonal primary anti-Elovl5 antibody made in rabbit (1:300 NBP2-33500, Novus biologicals, Centennial, United States), 1.5% NGS and PBS+ overnight at room temperature (RT) in a humid dark chamber. The following morning the tissues was washed three times with PBS (5 min each) and incubated with biotinylated secondary antibody anti rabbit (1:200, Vector Labs, Burlingame, United States) for 2 h. Slides were then washed three times with PBS (5 min each) and incubated with streptavidin-HRP (1:100, PerkinElmer, Milano, IT), and PBS+ for 1 h at RT. Following the incubation, sections were washed three time in PBS (5 min each) and incubated with Tyramide-FITC (1:100 PerkinElmer, Milano, IT) for 5 min at RT in the humid dark chamber. After a 5-min incubation with DAPI (1:1,000, Fluka, Saint Louis, United States), when dry, glass coverslip was applied using Mowiol (Calbiochem, LaJolla, CA, United States).
For immunocytochemistry on cell cultures, after 7 days in vitro cells were fixed for 20 min at RT with 4% PFA in 0.1 M PB and labeled with anti-AN2 (rat homologue of NG2, 1:100; kind gift of Miltenyi Biotech GmbH, Bergisch Gladbach, DE, and

Images Acquisition and Processing
Slides were scanned with Slide-Scanner Axiscan Z1 (ZEISS, Oberkochen, DE) both at low and high magnification (5x and 20x). Images obtained were then modified and adapted in color, contrast and brightness with Zen Software 2.1 (ZEISS, Oberkochen, DE). All the structures were analyzed and recognized by using the Interactive Atlas Viewer [IAV; Allen Human Brain Atlas-Allen Institute for Brain Science. Mouse Brain Atlas (internet) 1 .]. Whereas, cell coverslipped cultures were examined using an E-800 Nikon microscope (Nikon, Melville, NY) connected to a color CCD Camera and a Leica TCS SP5 (Leica Microsystems, Wetzlar, DE) confocal microscope. Adobe Photoshop 6.0 (Adobe Systems, San Jose, CA, United States) was used to assemble the final plate.

Statistics
Statistical analyses were carried out with GraphPad Prism 7 (GraphPad sofware, Inc). The Shapiro-Wilk test was first applied to test for a normal distribution of the data. Since data were not normally distributed, Mann-Whitney U-test (to compare two groups) and Kruskal-Wallis test (for multiple 1 http://mouse.brain-map.org group comparisons) were used. In all instances, P < 0.05 was considered as statistically significant. Histograms represent mean ± standard error (SE).

Elovl5 Gene Expression in the Central Nervous System
In the lacZ reporter mouse, XGal staining revealed a widespread expression with some region-specific differences (Figure 1,  Supplementary Figure 1, and Table 1). It is important to note that the XGal staining observed in Elovl5 lacZ reporter mouse reflected the in-situ hybridization published in Allen Mouse Brain Atlas. Specifically, in the telencephalon, a prominent labeling was found in the olfactory bulb (mitral cell layer, Figure 1A), in the neocortex, in the hippocampus ( Figure 1A) and in the midbrain (Figure 1B). The thalamus showed a less pronounced, moderate XGal labeling, with the exception of a strong expression in the reticular nucleus ( Figure 1A). As previously shown (Hoxha et al., 2017), in the cerebellum Purkinje cells and deep cerebellar nuclei were the most expressing cell types ( Figure 1B). Interestingly, we also found several labeled cells in the cerebellar white matter, in the granular layer and in the deeper part of the molecular layer. In the spinal cord, we observed a pronounced labeling in the gray matter, in neurons of different cell body size (Figure 1C).

Elovl5 Protein Distribution in Brain Regions
Protein localization was analyzed in wild-type mice by immunofluorescence labeling throughout different brain regions in order to define the expression pattern in diverse cell populations ( Table 1). The intensity of immunoreactivity varied in different regions and neuronal populations.

Olfactory Areas
The olfactory regions presented a very typical labeling (Figure 2). In the main olfactory bulb, mitral cells were found strongly positive for Elovl5 staining. In the superficial part of the outer plexiform layer small cells, likely tufted cells, showed moderate Elovl5 labeling, whereas no positive cells were observed in the granular layer (Figure 2A). The accessory olfactory bulb presented a similar expression pattern with a strong staining in the mitral cells ( Figure 2D). In anterior olfactory nucleus positive cells for Elovl5 were found mainly in layer II (Figure 2C), whereas in the olfactory tuberculus in layers II and III ( Figure 2D).

Hippocampal Formation
Elovl5 immunolabeling was expected to be extremely strong in this region, because of the high signal obtained with the XGal staining ( Figure 1A). Immunofluorescence analysis confirmed a relatively strong labeling of pyramidal neurons, but with regional differences (Figure 3A). In fact, in all hippocampal fields Elovl5 labeling was clearly present in pyramidal neurons, with a gradient of intensity: the signal was stronger in CA2 and CA3 than in CA1 and subiculum, where it was just moderately intense. Several neurons of the strata oriens and radiatum were strongly labeled. Hilar neurons also showed a strong Elovl5 staining. Neurons of the dentate gyrus were negative, with the exception of a few strongly labeled cells in the subgranular zone.

Neocortex, Claustrum, and Amygdala
The cerebral neocortex showed a layer-specific expression pattern ( Figure 3B). Cells showing moderately strong Elovl5 labeling were found in layer II/III, IV and V. In layer VI neurons displayed a weak intensity staining. In the corpus callosum, sparse positive cells were found. The piriform and endopiriform cortex showed a strong Elovl5 labeling (Figure 3C), whereas claustrum displayed a moderate intensity labeling ( Figure 3C). In the amygdala, neurons showed a moderate reactivity for Elovl5 in general: more prominent in the cortical regions than in medial ones ( Figure 3D).

Basal Ganglia
Caudate, putamen and nucleus accumbens presented a weak fluorescence signal for Elovl5 staining, with just few sparse cells with a very strong labeling. Whereas, the globus pallidus displayed a strong signal for Elovl5 immunofluorescence staining in both internal and external parts. The subthalamic nucleus was strongly labeled. The substantia nigra pars compacta and the ventral tegmental area showed a strong labeling ( Table 1).

Diencephalon Thalamus
The reticular nucleus of the thalamus showed the most prominent labeling for Elovl5, compared to the other nuclei (Figure 4). The peripeduncular nucleus also showed a strong labeling. The parvicellular part of subparafascicular nucleus, geniculate complex and ventral posteromedial and posterolateral nuclei of the thalamus presented instead moderate immunoreactivity to Elovl5 staining (Figure 4 and Table 1). In the zona incerta there was a strong labeling (Figure 4 and Table 1).

Hypothalamus
In the hypothalamus, the tuberal nucleus, retrochiasmatic area and anterior hypothalamic nucleus displayed the most prominent labeling for Elovl5 compared to the other structures belonging to this region. The lateral hypothalamic area showed a heterogenous expression pattern: the immunofluorescence signal was stronger in the rostral part of the nucleus while it had a weaker intensity in the caudal part (Table 1). Supramammillary and lateral mammillary nuclei, as well as lateral preoptic area presented a moderate immunoreactivity to Elovl5 antibody.

Brainstem
The Elovl5 expression pattern was very complex in the brainstem. The brainstem together with the cerebellum showed the highest intensely labeled cells (Table 1).

Midbrain
The inferior colliculus displayed weak or no labeling for Elovl5, whereas the superior colliculus showed a moderate signal. The most prominent labeling in this region was in the red nucleus. The periaqueductal gray and the midbrain reticular nucleus displayed moderate labeling (Table 1).

Pons
The tegmental nuclei showed the most prominent labeling for Elovl5 ( Figure 5A). Pontine nuclei, tegmental reticular nucleus and superior olivary complex displayed a strong presence of positive cells too (Figure 5A). The pontine reticular nuclei were found to be moderately and non-homogeneously positive for Elovl5 labeling (Figure 5A and Table 1).

Medulla
The medial vestibular nucleus and spinal vestibular nucleus presented a very strong labeling for Elovl5 ( Figure 5B). The nucleus of the solitary tract, intermediate reticular nucleus and gigantocellular reticular nucleus showed moderate to weak reactivity to Elovl5 antibody. The paragigantocellular reticular nucleus and medullary reticular nucleus, instead, displayed a peculiar pattern of expression: in both areas, the Elovl5 signal was not homogeneous (Figure 5B). In the paragigantocellular reticular nucleus, the parvicellular part showed a more prominent expression compared to the magnocellular one ( Figure 5B). In the medullary reticular nucleus the most intense signal was presented by neurons with bigger cell body compared to smaller ones, except for the fiber tract, in which small cells, probably oligodendrocytes, displayed an incredible high signal for Elovl5 labeling. The inferior olivary complex showed strong staining for Elovl5 (Table 1).

Cerebellum
In adult cerebellum the immunofluorescence labeling for Elovl5 reflected faithfully the XGal staining ( Figure 1B). Purkinje cells  showed a strong positivity in cell bodies ( Figure 6A) with no differences in the expression pattern between anterior and posterior lobules (Hoxha et al., 2017). The labeling was present also in some sparse neurons in the granular layer, in the deeper part of the molecular layer and cerebellar white matter ( Figure 6A). Interestingly, deep cerebellar nuclei displayed the most intense labeling ( Figure 6B).

Spinal Cord
In the spinal cord, Elovl5 showed a well distinguishable celltype expression pattern (Figures 7A-D). In fact, by analyzing the gray matter, motor neurons belonging to laminae VIII and IX presented a moderate intensity signal for Elovl5 staining ( Figure 7B). From laminae I to VII, instead, cells displayed very weak labeling. The most prominent expression seemed to belong to glial cells in the white matter (Figures 7C,D).

Elovl5 Expression in Glial Cells
In each analyzed region of the CNS Elovl5 immunoreactivity was not restricted to neurons, but rather comprises glial cells, whose immunophenotyping was in most cases hampered by the slice treatments required for anti-Elovl5 immunostaining (see section "Materials and Methods"). Thus, in order to identify Elovl5 positive glial cell types, double immunofluorescence staining was performed on primary cell cultures and on sagittal mouse brain sections. Elovl5 was expressed by virtually all immature oligodendrocyte precursor cells (identified by the expression of the chondroitin sulfate proteoglycan AN2; Boda et al., 2011) and by oligodendrocytes (expressing the myelin basic protein, MBP) in vitro (Figures 8A-C). Notably, Elovl5 mRNA level appeared remarkably increased in oligodendrocytes compared to immature OPCs ( Figure 9A, P < 0.05 Mann-Whitney U-test), suggesting Elovl5 participation in oligodendroglia maturation. Elovl5 immunolabeling was also found in microglial cells, identified by expression of ionized calcium-binding adapter molecule 1 (Iba1) ( Figure 8D). In these cells, Elovl5 mRNA expression showed a non-significant tendency to increase when microglia were polarized toward a proinflammatory M1 phenotype, compared to resting microglia ( Figure 9B). In contrast, microglia polarization toward a proregenerative M2 phenotype was not associated to changes in Elovl5 transcript levels ( Figure 9B). Finally, while most astrocytes showed no or little immunolabeling for Elovl5, a subpopulation of glial fibrillary acidic protein (GFAP) positive astrocytes was found positively marked (Figures 8E,F).

DISCUSSION
The present study provides for the first time, to our knowledge, a comprehensive description of Elovl5 cell-and region-specific localization in the adult mouse brain. By immunohistochemistry, we showed that there are no rostro-caudal and ventro-dorsal differences in the expression of Elovl5 throughout the brain. Interestingly, big soma projecting neurons (e.g., mitral cells, cerebellar Purkinje cells, and cortical/hippocampal pyramidal cells) and glial cells showed a prominent expression of Elovl5.
XGal staining and immunohistochemistry revealed subtle discrepancies between Elovl5 promoter activity and Elovl5 protein expression. Indeed, we observed that the XGal staining was very strong and homogeneous in the hippocampal pyramidal neurons, while the fluorescent signal was only moderately intense in CA2 and CA3 pyramidal cells and lower in the other hippocampal fields. On the other hand, in the thalamus, XGal analysis showed a low signal, while the fluorescent signal was stronger in all thalamic nuclei. These results suggest the existence of transcriptional or post-transcriptional regulation of Elovl5 expression. Notably, it is reported that estrogens can act as negative regulators of miRNAs which downregulate Elovl5 at a post-trascriptional level (Zhang et al., 2017).
Elovl5 distribution in distinct CNS areas and in specific cell types suggests a unique role of this enzyme in the local synthesis of PUFAs subserving specific neuronal/glial processes and CNS functions. In the olfactory areas of the telencephalon, mitral cells showed the most prominent expression of Elovl5 protein, in contrast with a lack of labeling in the granular layer. Furthermore, strong positivity for Elovl5 was found in pyriform cortex, olfactory tubercle and anterior olfactory nucleus, which are part of the olfactory system. These findings are in line with the loss of the sense of smell found in SCA38 patients and in Elovl5 −/− mice (Borroni et al., 2016;Hoxha et al., 2017), suggesting a crucial role of Elovl5 in olfaction, in line with the reported importance of PUFAs availability for a correct olfactory discrimination and a proper olfactory tissue integrity (Greiner et al., 2001;Le Bon et al., 2018;Khoury et al., 2020).
A pattern similar to the olfactory bulb was also observed in the cerebellar cortex. Purkinje cells were strongly positive for Elovl5, whereas granular cells were negative. This expression pattern well correlates with the gait abnormality and balance deficits observed in SCA38 patients with ELOVL5 mutation and in the Elovl5 −/− mouse model (Borroni et al., 2016;Hoxha et al., 2017). By studying this pathological mouse model, it has been shown that the absence of Elovl5 in Purkinje cells led to a shrinkage of the dendritic tree causing molecular layer atrophy (Hoxha et al., 2017). This evidence suggests an important role of Elovl5 for the correct maintenance of Purkinje cell dendritic morphology, a necessary requirement for cerebellar function.
In the hippocampus, Elovl5 showed a peculiar expression pattern: labeling was specific for pyramidal cells and interneurons of the strata oriens and radiatum. The involvement of PUFAs in hippocampal synaptic plasticity has been largely described in the literature (Fukaya et al., 2007;Cutuli et al., 2014;Thomazeau et al., 2017) suggesting that the Elovl5 expression in this region is required for proper functioning and refinement of the circuits.
The characterization of Elovl5 expression in the brainstem was challenging because of the huge density of cells and nuclei. In general, the most prominent labeling was shown by nuclei functionally and anatomically connected to the cerebellum (e.g., the red nucleus in the midbrain and the vestibular nuclei in the medulla).
An interesting finding is that Elovl5 expression is not restricted to neurons but is extended to glial cells. The expression by both mature and immature oligodendrocytes suggests that Elovl5 downstream products might be important players in myelination and other oligodendroglia functions.
Along with oligodendrocytes, microglial cells showed a prominent expression of Elovl5, consistent with the observation that some Elovl5 downstream products, omega-3 PUFAs, play a major role in the resolution of neuroinflammation and in enhancing beneficial immune response (Ebert et al., 2009;Chen et al., 2014;Joffre et al., 2019). Astrocytes instead showed no or little Elovl5 immunolabeling. However, a subpopulation of astrocytes was found positively marked, suggesting a local regulation of fatty acid production that could be possibly exploited for signaling or expansion of local membranes in astrocytes (Sakers et al., 2017). Furthermore, it has been recently shown that, while undergoing a neurogenic program, some astrocytes need to upregulate genes encoding for enzymes essential for lipid metabolisms, including Elovl5 (Magnusson et al., 2020).
The expression pattern of Elovl5 in the CNS is similar to the expression of Elovl4 (Sherry et al., 2017). Mutations of ELOVL5 or ELOVL4 cause spinocerebellar ataxia 38 and 34, respectively (Ozaki et al., 2015;Borroni et al., 2016) with common neurological symptoms which might be explained by the regional expression similarity between the two enzymes. Interestingly, both enzymes are expressed in neurons and glial cells. Elovl5 is expressed by oligodendroglial cells, microglia and is heterogeneously in vivo expressed by astrocytes while Elovl4 is reported to be weakly expressed by astrocytes and presumably by oligodendrocytes (Sherry et al., 2017).
However, their cell-specific expression differs in some brain areas. In the cerebellum Elovl5 is expressed by Purkinje cells but not granule cells while Elovl4 is expressed mainly by granule cells with a low positivity in Purkinje cells (Sherry et al., 2017). Furthermore, in the olfactory bulb Elovl5 is expressed by mitral cells but not by granule cells while Elovl4 is expressed the most by granule cells with a low expression by mitral cells (Sherry et al., 2017). It is not clear how this difference in expression determines the onset of some neurological symptoms which are specific for SCA38 or SCA34, for instance the hyposmia in the SCA38 patients (Borroni et al., 2016).
Overall, the region-/cell-specific expression of Elovl5 suggests a special requirement of local production of VLCF with signaling or structural functions, in specific neuronal and glial cell types. However, further functional studies are needed to dissect the context-dependent role of Elovl5 and of its downstream products in neural cell physiology.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
All experimental procedures on adult mice and on pups have been approved by the Ethical Committee of the University of Torino and authorized by the Italian Ministry of Health (authorization numbers: 161/2016-PR and 510/2020-PR).

AUTHOR CONTRIBUTIONS
IB, FT, and EH supervised the experiments, data analysis, wrote the manuscript, and contributed to the conception of the work and to the discussion of the results. IB, FM, EB, and EH performed the experiments and data analysis. All authors approved the final version of the manuscript.