Emerging insights into the function of very long chain fatty acids at cerebellar synapses

Very long chain-saturated and -polyunsaturated fatty acids (VLC-SFA and VLC-PUFA, respectively) are a functionally important class of fatty acids containing 28 carbons or more in their acyl chain. They are synthesized by the elongation of very long fatty acids-4 (ELOVL4) enzyme, expressed mainly in the brain, retina, skin, testes, and meibomian gland, where these fatty acids are found (Agbaga et al., 2008). Further, these organs exhibit tissue-specific VLC-PUFA and VLC-SFA biosynthesis and incorporation into complex lipids for specific functions. In the brain, skin, and Meibomian glands, the ELOVL4 mainly makes VLC-SFA, which are incorporated into complex sphingolipids. In the retina, the ELOVL4 makes VLC-PUFA that are incorporated into phosphatidylcholine, that are critical for visual function, while in testes and sperm, the VLC-PUFA are incorporated into sphingolipids that are critical for fertility (Yeboah et al., 2021).

Elongation of very long fatty acids-4 variants cause neurological disorders: Though VLC-SFA in the brain were reported over three decades ago to be associated with Zellweger spectrum disorders (Moser, 1987), little is known about their importance in neuronal function. The significance of VLC-SFA in brain function became clearer with the discovery that several mutations in the ELOVL4 gene cause distinct neurological diseases that vary according to the mutation and its inheritance pattern (Yeboah et al., 2021). Recessive ELOVL4 mutations that result in an inactive enzyme lead to neuroichthyosis, which manifests as severe seizures, intellectual disability, spasticity, ichthyosis, and early death (Aldahmesh et al., 2011). Heterozygous inheritance of any of several different ELOVL4 point mutations causes autosomal dominant spinocerebellar ataxia-34 (SCA34), a late-onset neurodegenerative disease of the cerebellum. This condition is characterized by cerebellar dysfunction manifested as gait ataxia, limb ataxia, dysarthria, and eye movement abnormalities (Ozaki et al., 2015; Yeboah et al., 2021). Some patients also show pyramidal tract signs, cognitive impairment, and erythrokeratodermia variabilis, a disorder of the skin. Magnetic resonance imaging of SCA34 patients shows age-related progressive cerebellar and pontine atrophy. These findings suggest that the cerebellum is particularly vulnerable to the disrupted function of ELOVL4 in the production of VLC-SFA.

In recent years, the number of patients diagnosed with SCA34 has steadily increased due to the growing use of genetic testing. The most common SCA34-causing ELOVL4 variants that have been reported are L168F, L168S, Q180P, T233M, I171T, and W246G (Yeboah et al., 2021). All of these mutations occur downstream of the enzyme’s catalytic site and are predicted to impair VLC-SFA biosynthesis to varying degrees, leading to different ages of onset. For instance, a recent report identified a patient with the SCA34-causing L168S variant who developed cerebellar ataxia and died within the first decade of life (Gyening et al., 2023a), whereas patients with the L168F variant do not develop ataxia until their fourth or fifth decade of life. As genetic testing becomes more accessible, it is likely that additional ELOVL4 variants associated with SCA34 will be identified, giving us a clearer understanding of the prevalence of different variants causing the disease.

Elongation of very long fatty acids-4 expression in the cerebellum: ELOVL4 is expressed in most neuronal cell types in the cerebellum, with particularly strong expression in cerebellar granule cells (Sherry et al., 2017). The neurons in the molecular layer, presumably the molecular layer interneurons, showed strong labeling, whereas the Purkinje cells showed moderate but clear staining for ELOVL4. This pattern of staining was consistent across different lobules of the cerebellar cortex. Many neurons in the deep cerebellar nuclei also showed intense labeling for ELOVL4. VLC-SFA-containing lipids are likely to follow the cellular expression pattern of ELOVL4, the enzyme responsible for their biosynthesis.

Rat model of spinocerebellar ataxia-34: To investigate the neuronal function of VLC-SFA and elucidate the pathophysiology of SCA34, we generated a Long Evans rat model of SCA34 by CRISPR/Cas-9-mediated knock-in of Elovl4 variant (c.736T>G, p.W246G) that causes SCA34 (Agbaga et al., 2020; Nagaraja et al., 2021). Unlike homozygous Elovl4 knock-out and homozygous STGD3 knock-in mice that die at birth due to dehydration because of the loss of skin VLC-FA, heterozygous (HET) and homozygous (MUT) W246G rats are viable and fertile. Both HET and MUT rats develop impaired motor coordination typical of human disease (Nagaraja et al., 2021), making this a bona fide animal model in which the pathophysiology of SCA34 can be investigated. Biochemical analysis of the skin revealed that the levels of VLC-SFA are reduced (but not abolished) in MUT rats, whereas there is no change in the levels of retinal VLC-PUFA (Agbaga et al., 2020). This is congruent with our recent in vitro data showing that the W246G ELOVL4 variant was defective in VLC-SFA biosynthesis but exhibited a gain of function in VLC-PUFA synthesis (Gyening et al., 2023b). Thus, the W246G mutant rat model represents a unique model for investigating the selective function of VLC-SFA in the brain while at the same time elucidating the neurological dysfunction underlying SCA34.

Very long chain-saturated fatty acids function in the cerebellum: The initial studies in the cerebellum of W246G mutant rats using field potential recordings revealed deficits in synaptic transmission on Purkinje cells, the sole output neurons of the cerebellar cortex. Both basal synaptic transmission, as well as long-term potentiation (LTP) and long-term depression (LTD) were found to be impaired at parallel fiber to Purkinje cell (PF–PC) and climbing fiber to Purkinje cells (CF–PC) synapses, respectively, in MUT rats (Nagaraja et al., 2021). Interestingly, the thickness of various layers of the cerebellar cortex, the number of Purkinje cells, and various synaptic markers were unaltered in young adult MUT rats (Nagaraja et al., 2021). These results suggest that VLC-SFA deficiency in this animal model does not significantly impact cerebellar histology or synapse numbers in young adult animals but has major effects on synaptic function.

To gain an in-depth understanding of synaptic transmission deficits, we have recently completed and published a detailed study with patch-clamp recordings from Purkinje cells in acute cerebellar slices of wild-type and MUT rats (Nagaraja et al., 2023). We obtained information about presynaptic release properties at PF–PC and CF–PC synapses by evoking paired-pulse excitatory postsynaptic currents (EPSC) at these synapses. The paired-pulse ratio of PF–PC EPSC was reduced in the MUT cerebellum, which suggested an increase in the vesicular release probability at this synapse. To gain further evidence for this conclusion, we applied a low affinity, competitive antagonist of ionotropic glutamate receptors-kynurenic acid (KYN) while evoking EPSC at PF–PC synapses. The percentage inhibition of EPSC size brought about by KYN is inversely proportional to the amount of glutamate released per synapse. We found that the percentage reduction in PF–PC EPSC size by KYN was lower in MUT rats, which implies an increased evoked release of glutamate, congruent with elevated release probability. Parallel experiments revealed opposite alterations in the release probability at CF–PC synapses, as evidenced by an increase in the paired-pulse ratio and a higher percentage of inhibition of EPSC with KYN in the MUT cerebellum than in wild-type. These results suggest that VLC-SFAs regulate vesicular release probability in opposite ways at PF–PC and CF–PC synapses. The reasons for this difference are not currently known but may be related to the different release probabilities at the two synapses in unaltered conditions, with PF–PC synapses expressing low and CF–PC synapses showing high release probability. It is thus possible that VLC-SFA serve to determine the setpoint of this parameter at synapses, so their deficiency reduces the normal difference at these two synapse types. Notably, PF–PC and CF–PC synapses express different glutamate transporters, VGluT1 at PF–PC and VGluT2 at CF–PC synapses. It will be interesting to see if VLC-SFA have opposite effects on release probability at other VGluT1 and VGluT2-containing synapses, such as cortico-cortical and thalamocortical synapses formed on neocortical pyramidal neurons. We further investigated whether stimulus train-induced short-term synaptic plasticity is altered at PF-PC and CF–PC synapses in MUT rats. Unexpectedly, we found similar effects at both synapses in the form of exaggerated persistence of EPSC amplitude. These results suggest that the readily releasable pool is increased at the two synapses in the MUT cerebellum. Alternatively, there may be faster recycling of synaptic vesicles. Overall, these data suggest that VLC-SFA regulate release probability at PF–PC and CF–PC synapses in the opposite direction, whereas the impact on readily releasable vesicle pool or possibly recycling rate is convergent (Figure 1; Nagaraja et al., 2023).

Figure 1.

A schematic showing the layers and neuronal circuitry of the cerebellum.

The parallel fiber projections from granule cells and climbing fiber projections from inferior olive form excitatory synapses on Purkinje cells, whereas MLI form inhibitory synapses on these cells. The axons of the Purkinje cells make synapses on deep cerebellar nuclei neurons. SCA34 model rats show elevated release probability (Pr) at PF–PC synapses and reduced Pr at CF–PC synapses. There is also decreased density of Purkinje cell spines (depicted with broken lines). There is also increased frequency and amplitude of mIPSC on Purkinje cells by MLI. Created with BioRender.com. BC: Basket cell; CF: climbing fiber; DCN: deep cerebellar nuclei; GC: granule cell; GoC: Golgi cell; IO: inferior olive; MF: mossy fiber; mIPSC: miniature inhibitory postsynaptic current; MLI: molecular layer interneuron; PC: Purkinje cell; PCN: precerebellar nuclei; PF: parallel fiber; Pr: probability of release; SC: stellate cell; SCA34: spinocerebellar ataxia 34.

Following the determination of the presynaptic function of VLC-SFA at Purkinje cell excitatory synapses, we focused our attention on the postsynaptic apparatus. Both PF–PC and CF–PC synapses are formed on the dendritic spines of Purkinje cells. We used dye filling and calbindin staining to identify that the density of dendritic spines on Purkinje cells is reduced in the MUT cerebellum (Nagaraja et al., 2023). Furthermore, mEPSC frequency was also reduced, which together with findings on dendritic spines suggested reduced number of functional synapses on the MUT Purkinje cells. The amplitude of mEPSC was not changed, which indicates that the remaining spines and postsynaptic apparatus have unchanged AMPA receptor numbers. Interestingly, we found that the frequency and amplitude of miniature inhibitory postsynaptic current are elevated in MUT Purkinje cells, which suggest that VLC-SFA regulate both presynaptic and postsynaptic mechanisms at inhibitory synapses formed on these cells (Figure 1; Nagaraja et al., 2023).

Investigating the molecular and biophysical mechanisms underlying the above-mentioned synaptic deficits in MUT rats will help uncover the mechanism of action of VLC-SFA. We hypothesize that VLC-SFA are performing their synaptic roles by affecting neuronal membrane properties and/or by interacting with synaptic proteins. These fatty acids can reduce membrane fluidity due to their high hydrophobicity and stacking properties, which may aid in concentrating proteins in membrane microdomains. The long length of the fatty acyl chain can traverse through the lipid bilayer and thus potentially interfere with synaptic vesicle fusion with presynaptic plasma membrane. Similar to cholesterol, VLC-SFA may also interact with integral membrane proteins in the plane of the membrane to regulate their properties.

Connection between cerebellar very long chain-saturated fatty acids deficiency and ataxia: Based on the above-described advances made in identifying the deficits in SCA34 model rats, we posit the following functional mechanisms for ataxia in SCA34. The decrease in overall excitatory drive on Purkinje cells coupled with an increase in inhibitory synaptic transmission is likely to diminish the output of these cells, thereby compromising their ability to provide downstream guidance signals for motor coordination. Interestingly, molecular layer interneuron hyperactivity and increased inhibitory synapses on Purkinje cells have recently been reported in a mouse model of SCA1 (Pilotto et al., 2023). Strikingly, the chemogenetic suppression of inhibitory hyperactivity delayed Purkinje cell degeneration and reduced motor deficits in these animals. Thus, increased inhibitory synaptic transmission on Purkinje cells may be sufficient to cause ataxia in some hereditary ataxias. The alterations in short-term synaptic plasticity at PF–PC and CF–PC synapses observed in SCA34 rats are also likely to contribute to motor incoordination, as a precise interplay of synaptic responses at the two synapses is required for optimal Purkinje cell output in the control of motor performance. Motor learning highly depends on long-term synaptic plasticity, i.e., LTP and LTD at Purkinje cell synapses. The impairments of LTP and LTD at PF–PC and CF–PC synapses respectively in MUT cerebellum are likely to underlie impairments in motor learning in these animals.

Future directions and perspectives: We are just starting to understand the role of VLC-SFA-containing lipids in neuronal function. Further work is needed to identify the molecular mechanisms by which VLC-SFA regulate the release probability and vesicle dynamics at cerebellar synapses. Super-resolution imaging will provide useful insights into the disruption of the nanoscale distribution of proteins involved in synaptic vesicle trafficking and neurotransmitter release within specific presynaptic domains in MUT SCA34 rats. Deciphering the mechanisms underlying the impairment of LTP and LTD at PF–PC and CF–PC synapses in these animals would clarify the role of VLC-SFA in regulating these long-term forms of plasticity, which may involve both induction and expression mechanisms. There is also an important need to fully characterize the lipid species into which VLC-SFA are incorporated at cerebellar synapses, as different lipids compartmentalize into different subcellular locations and regulate different proteins. This can be achieved by using recent advances in lipidomics to analyze subcellular biochemical fractions as well as through spatial lipidomics. Even though lipids are challenging to study, the utilization of these new technologies will make it possible to make progress in the in-depth understanding of the function of VLC-SFA in the cerebellum. Finally, the role of VLC-SFA in brain regions beyond the cerebellum needs to be thoroughly investigated. Given that ELOVL4 is expressed in most brain regions, it likely has widespread roles at synapses. This is supported by the findings that biallelic inheritance of ELOVL4 loss-of-function variants in humans leads to neuroichthyosis, which involves widespread brain dysfunction, resulting in seizures, spastic paraplegia, and early death. Extensive research is required to determine whether all synapses equally depend on VLC-SFA for optimal function and to investigate both the general and synapse-specific roles of these fatty acids across different synapses in the brain.

We would like to thank Dr. David M. Sherry (University of Oklahoma Health Sciences Center, USA) for discussions and Dr. Kiran George (University of Oklahoma Health Sciences Center, USA) for figure illustrations.

This work was supported by NEI/NIH R01 EY030513; NIAMS/NIH R21-AR076035; Multi-PI Team Science grant from Presbyterian Health Foundation.

Additional file: Open peer review report 1 ^{(85.9KB, pdf)} .