The Spinocerebellar Ataxia 34-Causing W246G ELOVL4 Mutation Does Not Alter Cerebellar Neuron Populations in a Rat Model.

Spinocerebellar ataxia 34 (SCA34) is an autosomal dominant disease that arises from point mutations in the fatty acid elongase, Elongation of Very Long Chain Fatty Acids 4 (ELOVL4), which is essential for the synthesis of Very Long Chain-Saturated Fatty Acids (VLC-SFA) and Very Long Chain-Polyunsaturated Fatty Acids (VLC-PUFA) (28-34 carbons long). SCA34 is considered a neurodegenerative disease. However, a novel rat model of SCA34 (SCA34-KI rat) with knock-in of the W246G ELOVL4 mutation that causes human SCA34 shows early motor impairment and aberrant synaptic transmission and plasticity without overt neurodegeneration. ELOVL4 is expressed in neurogenic regions of the developing brain, is implicated in cell cycle regulation, and ELOVL4 mutations that cause neuroichthyosis lead to developmental brain malformation, suggesting that aberrant neuron generation due to ELOVL4 mutations might contribute to SCA34. To test whether W246G ELOVL4 altered neuronal generation or survival in the cerebellum, we compared the numbers of Purkinje cells, unipolar brush cells, molecular layer interneurons, granule and displaced granule cells in the cerebellum of wildtype, heterozygous, and homozygous SCA34-KI rats at four months of age, when motor impairment is already present. An unbiased, semi-automated method based on Cellpose 2.0 and ImageJ was used to quantify neuronal populations in cerebellar sections immunolabeled for known neuron-specific markers. Neuronal populations and cortical structure were unaffected by the W246G ELOVL4 mutation by four months of age, a time when synaptic and motor dysfunction are already present, suggesting that SCA34 pathology originates from synaptic dysfunction due to VLC-SFA deficiency, rather than aberrant neuronal production or neurodegeneration.

Synapse-Specific Defects in Synaptic Transmission in the Cerebellum of W246G Mutant ELOVL4 Rats-a Model of Human SCA34.

Elongation of very long fatty acids-4 (ELOVL4) mediates biosynthesis of very long chain-fatty acids (VLC-FA; ≥28 carbons). Various mutations in this enzyme result in spinocerebellar ataxia-34 (SCA34). We generated a rat model of human SCA34 by knock-in of a naturally occurring c.736T>G, p.W246G mutation in the Elovl4 gene. Our previous analysis of homozygous W246G mutant ELOVL4 rats (MUT) revealed early-onset gait disturbance and impaired synaptic transmission and plasticity at parallel fiber-Purkinje cell (PF-PC) and climbing fiber-Purkinje cell (CF-PC) synapses. However, the underlying mechanisms that caused these defects remained unknown. Here, we report detailed patch-clamp recordings from Purkinje cells that identify impaired synaptic mechanisms. Our results show that miniature EPSC (mEPSC) frequency is reduced in MUT rats with no change in mEPSC amplitude, suggesting a presynaptic defect of excitatory synaptic transmission on Purkinje cells. We also find alterations in inhibitory synaptic transmission as miniature IPSC (mIPSC) frequency and amplitude are increased in MUT Purkinje cells. Paired-pulse ratio is reduced at PF-PC synapses but increased at CF-PC synapses in MUT rats, which along with results from high-frequency stimulation suggest opposite changes in the release probability at these two synapses. In contrast, we identify exaggerated persistence of EPSC amplitude at CF-PC and PF-PC synapses in MUT cerebellum, suggesting a larger readily releasable pool (RRP) at both synapses. Furthermore, the dendritic spine density is reduced in MUT Purkinje cells. Thus, our results uncover novel mechanisms of action of VLC-FA at cerebellar synapses, and elucidate the synaptic dysfunction underlying SCA34 pathology.SIGNIFICANCE STATEMENT Very long chain-fatty acids (VLC-FA) are an understudied class of fatty acids that are present in the brain. They are critical for brain function as their deficiency caused by mutations in elongation of very long fatty acids-4 (ELOVL4), the enzyme that mediates their biosynthesis, results in neurologic diseases including spinocerebellar ataxia-34 (SCA34), neuroichthyosis, and Stargardt-like macular dystrophy. In this study, we investigated the synaptic defects present in a rat model of SCA34 and identified defects in presynaptic neurotransmitter release and dendritic spine density at synapses in the cerebellum, a brain region involved in motor coordination. These results advance our understanding of the synaptic mechanisms regulated by VLC-FA and describe the synaptic dysfunction that leads to motor incoordination in SCA34.

Ablation of Sphingosine Kinase 1 Protects Cornea from Neovascularization in a Mouse Corneal Injury Model.

The purpose of this study was to investigate the role of sphingosine kinase 1 (SphK1), which generates sphingosine-1-phosphate (S1P), in corneal neovascularization (NV). Wild-type (WT) and Sphk1 knockout (Sphk1-/-) mice received corneal alkali-burn treatment to induce corneal NV by placing a 2 mm round piece of Whatman No. 1 filter paper soaked in 1N NaOH on the center of the cornea for 20 s. Corneal sphingolipid species were extracted and identified using liquid chromatography/mass spectrometry (LC/MS). The total number of tip cells and those positive for ethynyl deoxy uridine (EdU) were quantified. Immunocytochemistry was done to examine whether pericytes were present on newly forming blood vessels. Cytokine signaling and angiogenic markers were compared between the two groups using multiplex assays. Data were analyzed using appropriate statistical tests. Here, we show that ablation of SphK1 can significantly reduce NV invasion in the cornea following injury. Corneal sphingolipid analysis showed that total levels of ceramides, monohexosyl ceramides (HexCer), and sphingomyelin were significantly elevated in Sphk-/- corneas compared to WT corneas, with a comparable level of sphingosine among the two genotypes. The numbers of total and proliferating endothelial tip cells were also lower in the Sphk1-/- corneas following injury. This study underscores the role of S1P in post-injury corneal NV and raises further questions about the roles played by ceramide, HexCer, and sphingomyelin in regulating corneal NV. Further studies are needed to unravel the role played by bioactive sphingolipids in maintenance of corneal transparency and clear vision.

Improved fluorescent signal in expansion microscopy using fluorescent Fab fragment secondary antibodies.

Expansion microscopy (ExM) is a microscopic imaging approach that can achieve super-resolution visualization of fluorescently labeled biological samples using conventional fluorescence microscopy. The method is based on embedding of a fluorescently labeled biological sample in a hydrogel matrix followed by the physical expansion of the specimen, which is then viewed using a conventional fluorescent microscope. Variations of the method can be used to visualize endogenously expressed fluorescent proteins, such as GFP, fluorescently tagged antibodies, nucleic acids, or other fluorescently tagged molecules. A significant challenge of the method is that the physical expansion of the specimen produces a concommitant reduction in fluorescence intensity, which can make imaging difficult. We describe an approach for amplifying fluorescence signal following expansion of immunolabeled tissue sections by applying fluorescently labeled Fab fragment secondary antibodies to intensify fluorescent signal and enhance detection of labeling using conventional fluorescent microscopy. A method to increase immunofluorescence signal intensity of Expansion Microscopy specimens is described. Method utilizes commercially available reagents. Enhances ability to acquire useful images in expanded tissue samples.

Selective Ablation of Sod2 in Astrocytes Induces Sex-Specific Effects on Cognitive Function, d-Serine Availability, and Astrogliosis.

Cognitive decline is a debilitating aspect of aging and neurodegenerative diseases such as Alzheimer's disease are closely associated with mitochondrial dysfunction, increased reactive oxygen species, neuroinflammation, and astrogliosis. This study investigated the effects of decreased mitochondrial antioxidant response specifically in astrocytes on cognitive performance and neuronal function in C57BL/6J mice using a tamoxifen-inducible astrocyte-specific knockout of manganese superoxide dismutase (aSOD2-KO), a mitochondrial matrix antioxidant that detoxifies superoxide generated during mitochondrial respiration. We reduced astrocyte SOD2 levels in male and female mice at 11-12 months of age and tested in an automated home cage (PhenoTyper) apparatus for diurnal patterns, spatial learning, and memory function at 15 months of age. aSOD2-KO impaired hippocampal-dependent spatial working memory and decreased cognitive flexibility in the reversal phase of the testing paradigm in males. Female aSOD2-KO showed no learning and memory deficits compared with age-matched controls despite significant reduction in hippocampal SOD2 expression. aSOD2-KO males further showed decreased hippocampal long-term potentiation, but paired-pulse facilitation was unaffected. Levels of d-serine, an NMDA receptor coagonist, were also reduced in aSOD2-KO mice, but female knockouts showed a compensatory increase in serine racemase expression. Furthermore, aSOD2-KO mice demonstrated increased density of astrocytes, indicative of astrogliosis, in the hippocampus compared with age-matched controls. These data demonstrate that reduction in mitochondrial antioxidant stress response in astrocytes recapitulates age-related deficits in cognitive function, d-serine availability, and astrogliosis. Therefore, improving astrocyte mitochondrial homeostasis may provide a therapeutic target for intervention for cognitive impairment in aging.SIGNIFICANCE STATEMENT Diminished antioxidant response is associated with increased astrogliosis in aging and in Alzheimer's disease. Manganese superoxide dismutase (SOD2) is an antioxidant in the mitochondrial matrix that detoxifies superoxide and maintains mitochondrial homeostasis. We show that astrocytic ablation of SOD2 impairs hippocampal-dependent plasticity in spatial working memory, reduces long-term potentiation of hippocampal neurons and levels of the neuromodulator d-serine, and increases astrogliosis, consistent with defects in advanced aging and Alzheimer's disease. Our data provide strong evidence for sex-specific effects of astrocytic SOD2 functions in age-related cognitive dysfunction.

W246G Mutant ELOVL4 Impairs Synaptic Plasticity in Parallel and Climbing Fibers and Causes Motor Defects in a Rat Model of SCA34.

Spinocerebellar ataxia (SCA) is a neurodegenerative disorder characterized by ataxia and cerebellar atrophy. A number of different mutations gives rise to different types of SCA with characteristic ages of onset, symptomatology, and rates of progression. SCA type 34 (SCA34) is caused by mutations in ELOVL4 (ELOngation of Very Long-chain fatty acids 4), a fatty acid elongase essential for biosynthesis of Very Long Chain Saturated and Polyunsaturated Fatty Acids (VLC-SFA and VLC-PUFA, resp., ≥28 carbons), which have important functions in the brain, skin, retina, Meibomian glands, testes, and sperm. We generated a rat model of SCA34 by knock-in of the SCA34-causing 736T>G (p.W246G) ELOVL4 mutation. Rats carrying the mutation developed impaired motor deficits by 2 months of age. To understand the mechanism of these motor deficits, we performed electrophysiological studies using cerebellar slices from rats homozygous for W246G mutant ELOVL4 and found marked reduction of long-term potentiation at parallel fiber synapses and long-term depression at climbing fiber synapses onto Purkinje cells. Neuroanatomical analysis of the cerebellum showed normal cytoarchitectural organization with no evidence of degeneration out to 6 months of age. These results point to ELOVL4 as essential for motor function and cerebellar synaptic plasticity. The results further suggest that ataxia in SCA34 patients may arise from a primary impairment of synaptic plasticity and cerebellar network desynchronization before onset of neurodegeneration and progression of the disease at a later age.

The Elovl4 Spinocerebellar Ataxia-34 Mutation 736T>G (p.W246G) Impairs Retinal Function in the Absence of Photoreceptor Degeneration.

Elongation of very long chain fatty acids-4 (ELOVL4) is essential for synthesis of very long chain polyunsaturated and saturated fatty acids (VLC-PUFA and VLC-SFA, respectively) of chain length greater than 26 carbons. Mutations in the ELOVL4 gene cause several distinct neurodegenerative diseases including Stargardt-like macular dystrophy (STGD3), spinocerebellar ataxia 34 (SCA34), and a neuro-ichthyotic syndrome with severe seizures and spasticity, as well as erythrokeratitis variabilis (EKV), a skin disorder. However, the relationship between ELOVL4 mutations, its VLC-PUFA and VLC-SFA products, and specific neurological symptoms remains unclear. We generated a knock-in rat line (SCA34-KI) that expresses the 736T>G (p.W246G) form of ELOVL4 that causes human SCA34. Lipids were analyzed by gas chromatography and mass spectrometry. Retinal function was assessed using electroretinography. Retinal integrity was assessed by histology, optical coherence tomography, and immunolabeling. Analysis of retina and skin lipids showed that the W246G mutation selectively impaired synthesis of VLC-SFA, but not VLC-PUFA. Homozygous SCA34-KI rats showed reduced ERG a- and b-wave amplitudes by 90 days of age, particularly for scotopic responses. Anatomical analyses revealed no indication of neurodegeneration in heterozygote or homozygote SCA34-KI rats out to 6-7 months of age. These studies reveal a previously unrecognized role for VLC-SFA in regulating retinal function, particularly transmission from photoreceptors to the inner retina, in the absence of neurodegeneration. Furthermore, these findings suggest that the tissue specificity and symptoms associated with disease-causing ELOVL4 mutations likely arise from selective differences in the ability of the mutant ELOVL4 enzymes to support synthesis of VLC-PUFA and/or VLC-SFA.

Sphingosine Kinase-1 Is Essential for Maintaining External/Outer Limiting Membrane and Associated Adherens Junctions in the Aging Retina.

Sphingosine-1-phosphate (S1P) produced by sphingosine kinases (SPHK1 and SPHK2) is a signaling molecule involved in cell proliferation and formation of cellular junctions. In this study, we characterized the retinas of Sphk1 knockout (KO) mice by electron microscopy and immunocytochemistry. We also tested cultured Müller glia for their response to S1P. We found that S1P plays an important role in retinal and retinal pigment epithelial (RPE) structural integrity in aging mice. Ultrastructural analysis of Sphk1 KO mouse retinas aged to 15 months or raised with moderate light stress revealed a degenerated outer limiting membrane (OLM). This membrane is formed by adherens junctions between neighboring Müller glia and photoreceptor cells. We also show that Sphk1 KO mice have reduced retinal function in mice raised with moderate light stress. In vitro assays revealed that exogenous S1P modulated cytoskeletal rearrangement and increased N-cadherin production in human Müller glia cells. Aged mice also had morphological degeneration of the RPE, as well as increased lipid storage vacuoles and undigested phagosomes reminiscent of RPE in age-related macular degeneration. These findings show that SPHK1 and S1P play a vital role in the structural maintenance of the mammalian retina and retinal pigmented epithelium by supporting the formation of adherens junctions.

Distribution of ELOVL4 in the Developing and Adult Mouse Brain.

ELOngation of Very Long chain fatty acids (ELOVL)-4 is essential for the synthesis of very long chain-fatty acids (fatty acids with chain lengths ≥ 28 carbons). The functions of ELOVL4 and its very long-chain fatty acid products are poorly understood at present. However, mutations in ELOVL4 cause neurodevelopmental or neurodegenerative diseases that vary according to the mutation and inheritance pattern. Heterozygous inheritance of different ELOVL4 mutations causes Stargardt-like Macular Dystrophy or Spinocerebellar Ataxia type 34. Homozygous inheritance of ELOVL4 mutations causes more severe disease characterized by seizures, intellectual disability, ichthyosis, and premature death. To better understand ELOVL4 and very long chain fatty acid function in the brain, we examined ELOVL4 expression in the mouse brain between embryonic day 18 and postnatal day 60 by immunolabeling using ELOVL4 and other marker antibodies. ELOVL4 was widely expressed in a region- and cell type-specific manner, and was restricted to cell bodies, consistent with its known localization to endoplasmic reticulum. ELOVL4 labeling was most prominent in gray matter, although labeling also was present in some cells located in white matter. ELOVL4 was widely expressed in the developing brain by embryonic day 18 and was especially pronounced in regions underlying the lateral ventricles and other neurogenic regions. The basal ganglia in particular showed intense ELOVL4 labeling at this stage. In the postnatal brain, cerebral cortex, hippocampus, cerebellum, thalamus, hypothalamus, midbrain, pons, and medulla all showed prominent ELOVL4 labeling, although ELOVL4 distribution was not uniform across all cells or subnuclei within these regions. In contrast, the basal ganglia showed little ELOVL4 labeling in the postnatal brain. Double labeling studies showed that ELOVL4 was primarily expressed by neurons, although presumptive oligodendrocytes located in white matter tracts also showed labeling. Little or no ELOVL4 labeling was present in astrocytes or radial glial cells. These findings suggest that ELOVL4 and its very long chain fatty acid products are important in many parts of the brain and that they are particularly associated with neuronal function. Specific roles for ELOVL4 and its products in oligodendrocytes and myelin and in cellular proliferation, especially during development, are possible.