Elovl5 Expression in the Central Nervous System of the Adult Mouse

Abstract

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.

Keywords: Elovl5; PUFA; central nervous system; glia; neurons; spinocerebellar ataxia.

FIGURE 1

Expression of Elovl5 in the central nervous system. XGal staining (blue) indicates an ubiquitary expression of Elovl5 gene in forebrain (A), cerebellum and brainstem (B), spinal cord (C). MOB, Main Olfactory Bulb; CTX, Cortex; HPF, Hippocampus; MB, midbrain; TH, thalamus; HY, Hypothalamus; CNU, cerebral nuclei. Scalebars: (A,B) 500 μm and (C) 50 μm.

FIGURE 2

Elovl5 distribution in olfactory regions. (A) In the olfactory bulb, immunohistochemical labeling for Elovl5 (green) and DAPI (blue), shows a strong signal in mitral cells (mi) and in the glomerular layer (gl), while cells belonging to the granule cell layer (gcl) are negative. (B) The olfactory tubercle (OT) (shown at high magnification) shows a predominant expression of Elovl5 (green) in layer II and layer III neurons. (C) In layer II neurons of the accessory olfactory nucleus (AON) a moderate presence of the enzyme is detected. (D) The accessory olfactory bulb shows a similar signal as the one detected in the main bulb, with a high intensity in mitral cells (mi) and no signal in the granule cell layer (gcl). MOB, Main Olfactory Bulb; mi, mitral cells; gl, glomerular layer; opl, outer plexiform layer; gcl, granular cell layer; OT, Olfactory Tubercle; AON, Anterior Olfatory Nucleus; AOB, Accessory Olfactory Bulb. Scalebars: (A,C) 200 μm, (B) 100 μm, and (D) 50 μm.

FIGURE 3

Elovl5 expression in cortical and subcortical regions. (A) In the hippocampus, Elovl5 (green) is strongly expressed by CA2 and CA3, in a moderate way by CA1, while it is not present in dentate gyrus. Oriens, radiatum, and hilar interneurons are strongly labeled. Cell nuclei are counterstained by DAPI (blue). (B) In the neocortex Elovl5 labeling is present in a moderately strong way in layers II/III, IV and V. In layer VI, neurons display a weak intensity staining. (C) In the amygdala Elovl5 expression is more prominent in the cortical areas than in medial ones. (D) The claustrum is moderately positive for Elovl5 staining, while piriform and endopiriform cortex show a strong signal. HPF, hippocampal formation; CA, Cornu Ammonis; DG, dentate gyrus; NCTX, neocortex; AMY, Amygdala; AAA, Anterior amygdalar area; CEAm, Central amygdalar area, medial part; MEAad, Medial amygdalar area, anterodorsal part; MEApd, Medial amygdalar area, posterodorsal part; MEApv, Medial amygdalar area, posteroventral part; COApl, Cortical amygdalar area, posterior part; COApm, Cortical amygdalar area, posterior part, medial zone; PA, posterior amygdalar nucleus; BMAa; Basomedial amygdalar nucleus, anterior part; PIR, piriform cortex; CLA, claustrum; EPd, endopiriform nucleus, dorsal part. Scalebars: (A) 500 μm, (B,D) 100 μm, and (C) 250 μm.

FIGURE 4

Distribution of Elovl5 in thalamus. Elovl5 (green) expression in the thalamus has a region-specific intensity. The most intense labeling is shown by the reticular nucleus. LGd, dorsal part of lateral geniculate complex; LGv, ventral part of lateral geniculate complex; MG, medial geniculate complex; SPFp, subparafasciular nucleus, parvicellular part; PP, peripeduncular nucleus; ZI, zona incerta; VPM, Ventral posteromedial nucleus of thalamus; VPL, Ventral posterolateral nucleus of thalamus; RT, Reticular nucleus of thalamus. Scale bar: 200 μm.

FIGURE 5

Elovl5 expression in pons and medulla. (A) In the pons, the most prominent labeling for Elovl5 is shown by tegmental nuclei, while pontine nuclei, tegmental reticular nucleus and superior olivary complex displayed a moderately strong signal. Medium-low intensity is shown by pontine reticular nuclei. (B) In the medulla, very strong labeling for Elovl5 is shown by medial vestibular nucleus and spinal vestibular nucleus. A moderate to weak signal is detected in the nucleus of the solitary tract, intermediate reticular nucleus and gigantocellular reticular nucleus. A non-homogeneous pattern of expression is present in the paragigantocellular reticular nucleus and medullary reticular nucleus. (A) LDT, Laterodorsal Tegmentel Nucleus; PCN, Pontine Central Nuclei; PRNc, Pontine Reticular Nucleus, caudal part; PRNr, Pontine Reticular Nucleus, rostral part; TRN, Tegmental Reticular Nucleus; PN, Pontine Nuclei; SOC, Superior olivary Complex. (B) MV, Medial Vestibular Nucleus; SPIV, Spinal Vestibular Nucleus; NTS, Nucleus of Solitary Tract; IRN, Intermediate reticular Nucleus; GNR, Gigantocellular Reticular nucleus; PGNR, Paragigantocellular Reticular nucleus; MDRN, Medullary Reticular nucleus. Scale bars: (A) 200 μm and (B) 250 μm.

FIGURE 6

Distribution of Elovl5 in the cerebellum. (A) High magnification of the cerebellar cortex. Purkinje cells (Pcl) strongly express Elovl5; a moderate signal is present in sparse positive cells in white matter (wm), deeper part of the molecular layer (ml) and granular layer (gcl) (white arrow). (B) Prominent expression of Elovl5 (green) (DAPI in blue) by deep cerebellar nuclei (DCN). Scale bars: (A,B) 100 μm.

FIGURE 7

Elovl5 in the spinal cord. (A) Schematic representation of a spinal cord coronal section and areas magnified showed in panels (B–D). (B,C) Elovl5 (green) is strongly expressed by small cells in the spinal cord white matter and in a moderate way by motor neurons in the gray matter. (D) High magnification of large neurons of the dorsal horn, positive for Elovl5 staining (green). (B,C) wm, white matter; gm, gray matter. Scale bars: (B,C) 100 μm and (D) 50 μm.

FIGURE 8

Elovl5 expression in glial cells. (A–C) Widespread distribution of Elovl5 (red) in cultured AN2⁺ (green) oligodendrocyte precursor cells (A) and in maturing (B) and differentiated ramified (C) MBP⁺ (green in B-C) oligodendrocytes. (D) Elovl5 (green) expression in cultured Iba1⁺ (red) microglia. (E,F) Sagittal brain slices showing low and heterogeneous expression of Elovl5 (green) in GFAP⁺ (red) astrocytes. White arrowheads in (E) point to negative cells. Yellow arrowheads in (F) point to Elovl5⁺ cells. Scale bars: 10 μm.

FIGURE 9

Gene expression analysis of Elovl5 in glial cells. (A) Elovl5 expression in oligodendrocytes (OL, n = 4) compared to immature oligodendrocyte precursor cells (OPC, n = 4) (P < 0.05 Mann-Whitney U-test). (B) Elovl5 levels in microglial cells in resting (M0, n = 3), proinflammatory M1 phenotype (n = 3) and proregenerative M2 phenotype (n = 3). The expression levels were compared to the microglial cells in resting state. *P < 0.05.