BIOLOGICAL OVERVIEW

The Klarsicht (Klar) protein is a crucial factor in the regulation of bidirectional transport of lipid droplets (Welte, 1998). Lipid droplets in early embryos move bidirectionally along microtubules, and the balance of plus- and minus-end travel distances changes twice over a 2-h period, resulting in switches in the direction of net transport. In the absence of Klar, travel distances, travel velocities, and stall forces are greatly reduced, for both plus- and minus-end travel. Without Klar the motors for plus- and minus-end motion are active indiscriminately, engaging in a tug-of-war (Welte, 1998). Thus, Klar seems to be central for understanding how the activity of opposite-polarity motors is coordinated during bidirectional transport. In this system, Klar controls the minus-end motor cytoplasmic dynein (Gross, 2000; Gross, 2002) and an as yet unknown plus-end motor (Guo, 2005 and references therein).

Lack of Klar also disrupts the developmental regulation of droplet transport. In the wild type, these organelles are initially distributed throughout the periphery of the embryo (syncytial blastoderm, phase I), constantly moving back and forth along microtubules. At the beginning of cellularization (phase II), plus-end travel distances are up-regulated, causing net inward motion; the droplets accumulate basally, near microtubule plus-ends. One hour later (gastrulation, phase III), plus-end travel lengths decrease and droplets redistribute outward, apically. In embryos from klar mutant females (referred to as 'klar embryos'), this switch from net inward to net outward motion in phase III fails to occur because the balance of plus- and minus-end motion does not change correctly. Based on these phenotypic analyses, it was proposed that Klar may form a complex between the plus- and minus-end motors, controlling the response to transacting signals and coordinating motor activity (Welte, 1998; Guo, 2005 and references therein).

Klar is not only a central player in the mechanism of bidirectional transport of lipid droplets but also it controls motor activity for at least two other transport processes. In differentiating photoreceptors (R-cells) of wild-type animals, nuclei migrate first basally and then apically; in the absence of Klar, this switch in direction fails to occur, resulting in mispositioned nuclei (Fischer-Vize, 1994; Welte, 1998). In embryonic salivary glands, Klar controls minus-end-directed transport of secretory vesicles and thus modulates the growth of the apical membrane (Myat, 2002). Whether nuclei in photoreceptors and vesicles in salivary glands move bidirectionally like lipid droplets is not known, but both the minus-end motor dynein and the plus-end motor kinesin I are important for the correct positioning of photoreceptor nuclei. Whether Klar controls the motion of additional cargoes has not been determined (Guo, 2005 and references therein).

In its role in photoreceptor nuclear migration, Klarsicht is required for connecting the microtubule organizing center (MTOC) to the nucleus. In addition, in a genetic screen for klarsicht-interacting genes, Lam Dm₀, which encodes nuclear lamin, was found. Like Klarsicht, lamin is required for photoreceptor nuclear migration and for nuclear attachment to the MTOC. Moreover, perinuclear localization of Klarsicht requires lamin. It is proposed that nuclear migration requires linkage of the MTOC to the nucleus through an interaction between microtubules, Klarsicht, and lamin (Patterson, 2004).

Nuclear migration in the developing eye is critical for shaping each individual cell and thus for normal morphology of the entire compound eye. The Drosophila compound eye develops within the larval eye imaginal disc, an epithelial monolayer. Within the eye disc, the morphogenetic furrow marks the initiation of eye assembly. Rows of identical facets, or ommatidia, assemble posterior to the furrow, starting with the eight photoreceptors (R-cells), followed by the lens-secreting cone cells, and finally the pigment cells. Anterior to the furrow, cells are undifferentiated and their nuclei are positioned randomly within the monolayer. The nuclei dive basally within the furrow and posterior to the furrow, migrate apically as they are recruited into ommatidia (Patterson, 2004 and references therein).

Two Drosophila genes, klarsicht (previously known as marbles) and Glued, are essential for the apical migration of nuclei in differentiating R-cells (Fischer-Vize, 1994; Fan, 1997). Glued encodes the large subunit of dynactin, a protein complex that regulates the minus-end-directed microtubule motor dynein. The requirement for dynactin suggests that R-cell nuclear migration is a dynein- and microtubule-dependent process. Consistent with this idea, two other Drosophila genes, Bicaudal-D and Lis1, both of which may regulate dynein, are implicated in R-cell nuclear migration, although their mutant phenotypes are weak compared with klarsicht and Glued. Lis-1, a WD40 repeat protein, is the homolog of the human disease gene Lissencephaly-1. Lissencephaly, or smooth brain, is a disorder resulting from defects in neuronal migrations essential for normal human brain development. Neuronal migration requires nuclear migration, and the involvement of Lis-1 in Drosophila R-cell nuclear migration suggests that the two processes may be in part analogous. It is now clear that a connection between the MTOC and the nucleus is necessary for nuclear migration and that this connection is mediated by Klar and nuclear lamin. In addition to suggesting a specific role for Klar in nuclear migration, the results propose a general mechanistic explanation for the cytoplasmic effects of nuclear lamin, including human laminopathies (Patterson, 2004).

To understand the role of Klar in R-cell nuclear migration, Klar subcellular localization and the position of the MTOC was investigated in klar mutant eye discs. In addition, genetics was used to identify nuclear lamin, which functions in the same pathway with Klar. Klar was found to be perinuclear and associated with microtubules apical to the nucleus. In addition, in klar and Lam mutant discs, MTOCs form normally in R-cells, but are often not associated with the nucleus as they are in wild-type eyes. Finally, Lam⁺ was found to be required for Klar localization to the nuclear membrane. These observations, taken together with previous results, suggest a model for the function of Klar in nuclear migration where Klar, held in the nuclear envelope by nuclear lamin, links the nucleus to the MTOC (Patterson, 2004).

The interaction between Klar and lamin may be indirect, but it is likely to be specific, rather than a generalized failure of nuclear envelope assembly in Lam mutants. Although most R-cell nuclei fail to migrate apically even in weak, viable Lam mutants, >90% of nuclear envelopes are intact even in stronger, lethal Lam mutants (Patterson, 2004 and references therein).

It is proposed that one or more proteins may form a bridge between the KASH domain of Klar, present in the outer nuclear membrane, and nuclear lamin, in the inner nuclear envelope. The observation that in addition to its perinuclear localization, Klar is cytoplasmic (on apical microtubules) supports the idea that Klar is in the outer, as opposed to the inner, nuclear membrane. Similarly, C. elegans Anc-1 is present in the cytoplasm as well as the nuclear membrane, and a model has been proposed where the Anc-1 KASH domain is held in the outer nuclear membrane by an inner nuclear membrane protein, Unc-84 (Malone, 1999; Starr, 2002). Although nuclear lamin has not been shown directly to be required for Anc-1 nuclear membrane localization, nuclear envelope localization of Unc-84 requires lamin (Lee, 2002). For Syne-1, the vertebrate homolog of Anc-1, experiments where the detergent digitonin was used to allow antibody access to the outer but not the inner nuclear membrane provide direct evidence that the KASH domain is in the outer nuclear membrane (Zhen, 2002). There is, however, some conflicting data (Zhang, 2001; Mislow, 2001; Mislow, 2002; Patterson, 2004 and references therein).

It is speculated that the N-terminal portion of Klar is linked to microtubules by dynein. At present, it is not possible to test for colocalization of Klar and dynein because there are no available reagents that allow detection of dynein or dynactin in the eye disc. Nevertheless, there is much evidence to support an essential role for dynein in R-cell nuclear migration and Klar function. Dynactin, a regulator of dynein, is essential for R-cell nuclear migration in the eye; mutants in the p150 dynactin subunit (Glued) have a phenotype similar to that of klar mutants in the eye disc. In addition, dynein linkage could explain why Klar is localized to microtubules only apical to the nucleus; Klar that escapes the hold of the nuclear envelope, still attached to dynein, could walk along microtubules to the MTOC. Finally, Klar has been implicated as a regulator of dynein in Drosophila embryos (Welte, 1998). In addition to its role in R-cell nuclear migration, Klar is required for developmentally regulated migration of lipid storage vesicles during embryogenesis. Lipid droplets at the center of the cellular blastoderm embryo normally migrate cortically during gastrulation. In embryos from klar mutant mothers, the lipid droplets fail to migrate. A variety of data support a model where dynein transports the lipid droplets along microtubules, whose minus ends are at the cell periphery. The results of biophysical experiments has led to a model where Klar may attach the appropriate types of motor to lipid droplets, control the number of actively engaged motors on a droplet, or coordinate the activities of kinesins and dyneins bound simultaneously to the same droplet (Jackle, 1998; Welte, 1998; Gross, 2000). Notably, dynein is required for nuclear attachment to centrosomes (the MTOCs) during mitosis in the Drosophila embryo. Klar, however, is not essential for this process (Fischer-Vize, 1994; Patterson, 2004 and references therein).

The observation that the MTOC is normally apical to the R-cell nuclei, at the leading edge of nuclear movement, suggests that a force pulls on the MTOC from above. It is speculated that the mechanism for this force could be analogous to the means by which the nucleus of budding yeast are pulled into the bud neck before cell division. One pathway for migration of the nucleus into the bud neck involves dynein, anchored at the cell cortex to which the nucleus is moving. Cortically tethered dynein 'reels in' the nucleus by walking along microtubules whose plus ends are at the cortex, toward the MTOC, which is anchored to the nucleus. In support of this idea, microtubule plus-ends are present apically in R-cells (Mosley-Bishop, 1999), and dynactin is essential for R-cell nuclear migration (Patterson, 2004 and references therein).

Whether a force emanating from the apical membrane pulling on the MTOC would drive nuclear migration or serve as an anchor after the nucleus has migrated depends on where the MTOC initially forms. The gamma-tubulin antibody detects MTOCs only apically in differentiating cells. Transiently basal MTOCs associated with nuclei that are about to rise could have escaped detection. However, if the MTOC does form apically, then the force that drives nuclear migration would come from below the nucleus, that is, dynein, linked to the nuclear membrane by Klar and lamin, walking on microtubules up toward the MTOC (Patterson, 2004).

The model proposed whereby Klar forms a bridge between nuclear lamin in the inner nuclear membrane and cytoplasmic microtubules provides a general framework for explaining how nuclear lamin affects cytoplasmic events. Drosophila Lam mutations result in D/V polarity defects in eggs, and tracheal branching defects in embryos. Moreover, a variety of human diseases are the result of mutations in the LMNA gene, which encodes lamin A. The Drosophila Lam Dm₀ gene encodes type B lamin, whereas the Drosophila LamC gene encodes lamin C, which is most similar to human lamin A. The A/C- and B-type lamins are similar proteins, with some different structural features, and some expression pattern differences. LMNA-associated human diseases affect the heart, skeletal muscles, and the nervous system (Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, cardiomyopathy, and Charcot-Marie-Tooth disorder), and metabolism (Dunnigan-type lipodystrophy). The two main hypotheses as to how nuclear lamin defects can result in these disease phenotypes are that the mutations either result in nuclear envelope fragility or result in changes in gene expression. An alternative hypothesis is that the inner nuclear envelope interacts with the cytoplasm through proteins like Klar or Anc-1/Syne-1, which connect the inner nuclear envelope to the microtubule, or actin cytoskeletons, respectively (Patterson, 2004 and references therein).

Microtubule-driven nuclear rotations promote meiotic chromosome dynamics

At the onset of meiosis, each chromosome needs to find its homologue and pair to ensure proper segregation. In Drosophila, pairing occurs during the mitotic cycles preceding meiosis. This study shows that germ cell nuclei undergo marked movements during this developmental window. It was demonstrated that microtubules and Dynein drive nuclear rotations and are required for centromere pairing and clustering. It was further found that Klaroid (SUN) and Klarsicht (KASH) co-localize with centromeres at the nuclear envelope and are required for proper chromosome motions and pairing. Mud (NuMA in vertebrates) was identified as co-localizing with centromeres, Klarsicht and Klaroid. Mud is also required to maintain the integrity of the nuclear envelope and for the correct assembly of the synaptonemal complex. These findings reveal a mechanism for chromosome pairing in Drosophila, and indicate that microtubules, centrosomes and associated proteins play a crucial role in the dynamic organization of chromosomes inside the nucleus (Christophorou, 2015).

Rotations of nuclei have been described previously in somatic cells; their function remains however unclear. In germ cells, meiotic chromosome movements are thought to be required for homologue pairing, removing chromosome entanglements, promoting maturation of recombination intermediates, or for assessing chromosome homology before synapsis, in different model organisms. In Drosophila, a high temporal correlation was found between nuclear rotations and chromosome pairing occurring mainly in 8-cell cysts. This work uncovered a second interesting correlation between the speed of nuclear rotation and the degree of centromere pairing and clustering. Indeed, mutations in klaroid affected the least nuclear rotations and disrupted the least centromere associations and synapsis. Rotations were slowed down more significantly in klarsicht, sas-4 and asl mutant germ cells. Accordingly, strong defects were observed in the initial pairing of centromeres and in synaptonemal complex formation. Finally, nuclear rotations were completely abolished in the absence of Dynein or dynamic microtubules. In dynein mutant germ cells, an average of six centromeres were distinguished during pre-meiotic pairing, which is higher than any mutants tested previously, including null alleles of c(3)G. Similarly, five centromeres on average were coundted during clustering in region 2a, a mutant phenotype that is comparable to the strongest orientation disruptor (ord) or c(3)G mutations (lateral and central elements of synaptonemal complex respectively). Nuclear rotations thus play an important role in homologue chromosome pairing and synaptonemal complex formation (Christophorou, 2015).

It was found that microtubules could be nucleated from the fusome, the nuclear envelope and the centrosome in region 1 germ cells. On the basis of these observations and centrosome mutant analysis, it is speculated that the whip-like movements of microtubules could be the main forces creating cytoplasmic flows, as observed in many biological systems and demonstrated theoretically. In addition, microtubules nucleated by the centrosomes could also push on the nucleus and the cell membrane, which could bias nuclear movement towards one direction of rotation as proposed for the migration of this same oocyte. These two forces depend on microtubules and dynein, and would act redundantly for efficient and unidirectional nuclear rotations. However, even in the absence of dynamic microtubules, centromeres ended up paired, albeit much later in region 2b. Synapsis, on the other hand, was completely disrupted. It is thus believed that, as in yeast and worms, these movements are there to facilitate pairing, synapsis or recombination, but that at least chromosome pairing could occur slowly without motions by redundant mechanisms. In flies, Spag4 is a second SUN-domain protein, but it is only expressed in male testis and is thus not likely to play a role during oogenesis. There is also a second KASH-domain protein called MSP-300/Nesprin, which interacts with the actin cytoskeleton. In the absence of microtubules, nuclei were not ‘rolling’ anymore; however, they still showed some back and forth ‘rocking’ movements. It will be interesting to investigate whether MSP300/Nesprin and the actin cytoskeleton are involved in these rocking movements (Christophorou, 2015).

This study found that although mud mutant ovaries showed only mild defects in centromere dynamics, significant genetic interactions were uncovered with klaroid and klarsicht in this same process. Striking features of Mud in this study were its co-localization with centromeres in interphasic germline cysts and the formation of polycomplexes in mud mutant cysts. The formation of polycomplexes was associated with a lack of nuclear membrane and diffused DNA in the cytoplasm, suggesting that Mud is required to maintain nuclear envelope integrity. It is proposed that the disappearance of the NE in mud cysts is the primary defect leading first to the de-localization of DNA into the cytoplasm and then the formation of polycomplexes. Polycomplexes could thus be the result of self-assembly of synaptonemal complex components polymerizing in the absence of chromatin. Polycomplexes were also observed in klaroid and klarsicht mutants although at a lower penetrance than in mud mutants. Interestingly, large distortions of the NE were also observed in muscle cell nuclei mutant for unc-84, which encodes a C. elegans SUN protein. These deformations were particularly strong in these cells, because muscle cell nuclei are subjected to mechanical stress. It is likely that rolling nuclei of 8-cell cysts are also exposed to some mechanical forces. Klarsicht, Klaroid and Mud may all participate in maintaining the integrity of the nuclear envelope in these conditions. In their absence, the NE is weakened and cannot resist mechanical forces, which also leads to synaptonemal complex assembly defects. In the most extreme cases the NE completely disappears causing the formation of polycomplexes. Interestingly, Mud initially localizes at the NE of all germline cells in region 1, but then becomes localized only to the cells remaining in meiosis in region 2a, and finally only specifically at the NE of the oocyte. This may hint that the meiotic nucleus is subjected to specific mechanical forces during oogenesis (Christophorou, 2015).

Abnormal accumulation of lipid droplets in neurons induces the conversion of alpha-Synuclein to proteolytic resistant forms in a Drosophila model of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein (αSyn) aggregation and associated with abnormalities in lipid metabolism. The accumulation of lipids in cytoplasmic organelles called lipid droplets (LDs) was observed in cellular models of PD. To investigate the pathophysiological consequences of interactions between αSyn and proteins that regulate the homeostasis of LDs, a transgenic Drosophila model of PD was used in which human αSyn is specifically expressed in photoreceptor neurons. It was first found that overexpression of the LD-coating proteins Perilipin 1 or 2 (dPlin1/2), which limit the access of lipases to LDs, markedly increased triacylglyclerol (TG) loaded LDs in neurons. However, dPlin-induced-LDs in neurons are independent of lipid anabolic, and catabolic enzymes, indicating that alternative mechanisms regulate neuronal LD homeostasis. Interestingly, the accumulation of LDs induced by various LD proteins (dPlin1, dPlin2, CG7900 or KlarsichtLD-BD) was synergistically amplified by the co-expression of αSyn, which localized to LDs in both Drosophila photoreceptor neurons and in human neuroblastoma cells. Finally, the accumulation of LDs increased the resistance of αSyn to proteolytic digestion, a characteristic of αSyn aggregation in human neurons. It is proposed that αSyn cooperates with LD proteins to inhibit lipolysis and that binding of αSyn to LDs contributes to the pathogenic misfolding and aggregation of αSyn in neurons (Girard, 2021).

This study has investigated the mechanisms that regulate LD homeostasis in neurons, the contribution of αSyn to LD homeostasis, and whether αSyn-LD binding influences the pathogenic potential of αSyn. Expression of the LD proteins, dPlin1 and dPlin2, CG7900 or of the LD-binding domain of Klarsicht increased LD accumulation in Drosophila photoreceptor neurons and that this phenotype was amplified by co-expressing the PD-associated protein αSyn. Transfected and endogenous αSyn co-localized with PLINs on the LD surface in human neuroblastoma cells, as demonstrated by confocal microscopy and PLA assays. Neuronal accumulation of LDs was not dependent on the canonical enzymes of TG synthesis (Mdy, dFatp), Bmm/dATGL-dependent lipolysis or lipophagy inhibition. One possible explanation for LD accumulation is that LD proteins inhibit an unknown lipase in Drosophila photoreceptor neurons. Finally, it was observed that LD accumulation in photoreceptor neurons was associated with increased resistance of αSyn to proteinase K digestion, suggesting that LD accumulation might promote αSyn misfolding, an important step in the progression towards PD. Thus, this study has uncovered a potential novel role for LDs in the pathogenicity of αSyn in PD (Girard, 2021).

Understanding of the mechanisms of LD homeostasis in neurons under physiological or pathological conditions is far from complete. Neurons predominantly synthesize ATP through aerobic metabolism of glucose, rather than through FA β-oxidation, which likely explains the relative scarcity of LDs in neurons compared with glial cells. This study used the Drosophila adult retina that is composed of photoreceptor neurons and glial cells to explore the mechanism regulating LD homeostasis in the nervous system. The canonical mechanisms regulating TG turnover and LD formation are dependent on evolutionary conserved regulators of lipogenesis and lipolysis in the fly adipose tissue, called fat body, or in other non-fat cells, such as glial cells. Indeed, it has been shown that de novo TG-synthesis enzymes Dgat1/Mdy and dFatp, are required for LD biogenesis in the fat body and glial cells. This is in contrast to dPlin-induced neuronal accumulation of LDs (this study), which occurs through a mechanism, independent of Mdy- and dFatp-mediated de novo TG synthesis. One possibility is that LD biogenesis depends on Dgat2 in neurons. However, the fact that there are three Dgat2 paralogs encoded by the fly genome and that no triple mutant is available, precluded its functional analysis in the current study (Girard, 2021).

The evolutionarily conserved and canonical TG lipase Bmm, otholog of mammalian adipose triglyceride lipase (ATGL) regulates lipolysis in the fat body. This study shows that Bmm regulates LD abundance in glial cells but not in photoreceptor neurons. Interestingly, in both bmm-mutant Drosophila (this study) and ATGL-mutant mice, neurons do not accumulate LDs. This suggests the existence of an unknown and possibly cell type specific lipase regulating the degradation of LDs in neurons. This is supported by the fact that the overexpression of dPlins proteins, which are known inhibitors of lipolysis, promotes LD accumulation in photoreceptor neurons. In further support of a neuron-specific TG lipase, the human hereditary spastic paraplegia gene DDHD2, a member of the iPLA1/PAPLA1 family, was proposed to be the main lipase regulating TG metabolism in the mammalian brain. A recent study, showed that Bmm plays a role in the somatic cells of the gonad and in neurons to regulate systemic TG breakdown. It was also suggested that Bmm may play a role in regulating LD turnover in neurons, although this was not directly tested in this study. The results using bmm knock-down and bmm mutants do not support a role of Bmm in the regulation of LD accumulation in photoreceptor neurons. However, the possibility cannot be excluded that Bmm would be required in a subpopulation of neurons to regulate LD content but this would require further analyses. Finally, the possibility cannot be excluded that the overexpression of LD proteins, such as dPlins but also CG7900 or the Klarsicht lipid-binding domain promotes LD accumulation by shielding and stabilizing LDs rather than limiting the access of lipases to LDs. Indeed, stabilization of LDs could well be an ancestral function of PLINs, as reported for yeast and Drosophila adipose tissue. Thus, inhibiting lipolysis and/or stabilizing LDs, allows the formation of LDs, which would be otherwise actively degraded in photoreceptor neurons. This opens avenues to further study LD homeostasis but also their pathophysiological role in diseases of the nervous system (Girard, 2021).

Earlier studies have observed the accumulation of LDs in cellular models of PD. For example, LDs form in SH-SY5Y cells exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a dopaminergic neurotoxin prodrug that causes PD-like symptoms in animal and cellular models. In addition, studies in yeast, rat dopaminergic neurons, and human induced pluripotent stem cells have proposed that αSyn expression induces lipid dysregulation and LD accumulation, but the underlying mechanisms remained unclear. Low levels of αSyn accumulation were hypothezised to perturb lipid homeostasis by enhancing unsaturated FA synthesis and the subsequent accumulation of DGs and TGs. The present study showed that αSyn expression alone did not enhance the accumulation of LDs but instead required concomitant overexpression of a LD protein. Moreover, αSyn expression alone had no effect on DG, TG, or LD content in Drosophila photoreceptor neurons, which indicates that αSyn-induced LDs are not driven by increased TG biosynthesis in this cellular context. Instead, the fact that endogenous αSyn and PLIN3 proteins co-localized at the LD surface in human neuroblastoma cells, suggests that LD-associated αSyn have a direct physiological function in promoting neutral lipid accumulation by inhibiting lipolysis. This hypothesis is supported by experiments in HeLa cells transfected with αSyn, loaded with fatty acids, in which the overexpression of αSyn protects LDs from lipolysis (Girard, 2021).

The results show that LDs contribute to αSyn conversion to proteinase K resistant forms, which indicates that LDs may be involved in the progression of PD pathology. This is an apparent discrepancy with the results in Fanning (2019), in which LDs protect from lipotoxicity cells expressing αSyn. In this study the authors used cellular models including yeast cells, and rat cortical neuron primary cultures exposed or not to oleic acid. In such cellular context, they propose that αSyn induces the accumulation of toxic diacylglycerol (DG), which is subsequently converted to TG and sequestered into LDs. LDs are thus protective by allowing the sequestration of toxic lipids. In the fly retina study, αSyn expression did not induce TG accumulation. In the Drosophila nervous system, toxic DG may not reach sufficient level to promote photoreceptor toxicity. Interestingly, this difference allowed study of the binding of αSyn to LD and examine their contribution to pathological conversion of αSyn. Indeed, the results suggest an alternative but not mutually exclusive role for LDs in promoting αSyn misfolding and conversion to a proteinase K-resistant form. The increased LD surface could provide a physical platform for αSyn deposition and conversion. In support of this hypothesis, it was previously proposed that αSyn aggregation is facilitated in the presence of synthetic phospholipid vesicles. Thus, the current results point to a direct role of LDs on αSyn resistance to proteinase K digestion (Girard, 2021).

This study showed that the accumulation of LD proteins, such as dPlins, is a prerequisite for the increased LD accumulation induced by αSyn in neurons. This raises the possibility that some physiological or pathological conditions will favor the expression and/or accumulation of LD proteins, which triggers the neuronal accumulation of LDs. Interestingly, it was proposed that age-dependent accumulation of fat and dPlin2 is dependent on the histone deacetylase (HDAC6) in Drosophila. Moreover, an accumulation of LD-containing cells (lipid-laden cells), associated with PLIN2 expression, was observed in meningeal, cortical and neurogenic brain regions of the aging mice. Finally, a recent expression study on all human perlipin proteins (PLIN1-5), found that PLIN2 accumulates, particularly in neurons, in brains of old subjects and of patients with Alzheimer disease. As an alternative putative mechanism regulating LD level, it was shown that targeted degradation of PLIN2 and PLIN3 occurs by chaperone-mediated autophagy (CMA). Thus, in aging tissue with decreased HDAC6 or reduced basal CMA, the accumulation of PLINs may initiate LD accumulation, hence favoring αSyn-induced LD production. In this study, mutations in the central autophagy gene Atg8 did not lead to LD accumulation in Drosophila retina. Thus a more systematic analysis will be required to identify the proteolytic mechanisms regulating dPlins degradation and LD accumulation in the aged Drosophila nervous system (Girard, 2021).

Based on a combination of the current results and these observations, a model is proposed of LD homeostasis in healthy and diseased neurons. In healthy neurons, relatively few LDs are detected due to a combination of low basal rate of TG synthesis, active lipolysis and limited LD shielding capacity. In pathological conditions such as PD, possibly in combination with an age-dependent ectopic fat accumulation and Plin proteins increased expression, αSyn and Plins could cooperate to limit lipolysis and promote the accumulation of LDs in neurons. This could set a vicious cycle in which αSyn enhances Plin-dependent LD stabilization, which, in turn, would increase αSyn conversion to a proteinase K-resistant form, culminating in αSyn aggregation and formation of cytoplasmic inclusion bodies. Collectively, these results raise the possibility that αSyn binding to LDs could be an important step in the pathogenesis of PD (Girard, 2021).

GENE STRUCTURE

The single klar gene gives rise to at least three messages and three Klar protein isoforms. A proximal promoter before exon 0 and a distal promoter before exon G are ~80 kb apart. The originally published klar cDNA (exon 0-18) encodes isoform alpha that can partially rescue the klar nuclear migration defect in photoreceptors (Mosley-Bishop, 1999). The message for the droplet-specific isoform beta likely starts at the proximal promoter and ends with exon 15X that encodes the LD domain. Isoform gamma prominent in ovaries is encoded by a message transcribed from the distal promoter. No gross defects have been detected in ovaries when this isoform is disrupted by class II alleles. Other isoforms may exist. A comprehensive description of all Klar isoforms and of their expression pattern will be important for understanding the full role Klar plays in transport regulation (Guo, 2005).

cDNA clone length - 8312 bp

Bases in 5' UTR - 662

Exons - 18

Bases in 3' UTR - 861

PROTEIN STRUCTURE

Amino Acids - 2262

Structural Domains

klarsicht encodes a large protein, unique except for its small N-terminal KASH (Klarsicht, Anc-1, Syne-1 homology) domain, which localizes proteins to the nuclear membrane (Mosley-Bishop, 1999; Apel, 2000; Zhang, 2001; Starr, 2002; Zhen, 2002). The KASH-domain-containing protein Anc-1 and its vertebrate homolog, Syne-1 (also known as Myne-1, Nesprin, and NUANCE) are dystrophin-related proteins that anchor the nucleus to the actin cytoskeleton (Apel, 2000; Mislow, 2001: Mislow, 2002; Zhang, 2001; Starr, 2002; Zhen, 2002; Starr, 2003). In addition to its role in nuclear migration in the eye, klar is required for the developmentally regulated migrations of lipid droplets during embryogenesis. In this role, it has been proposed that Klar regulates dynein and also the plus-end-directed motor kinesin (Patterson, 2004 and references therein).

Comparing the klar genomic region and the reported klar cDNA (Mosley-Bishop, 1999) predicts 19 exons (exon 0 to exon 18). The only obviously conserved region in the predicted Klar protein (~250 kDa) is the C-terminal 60-amino acid Klarsicht, ANC-1, Syne-1 homology (KASH) domain (Starr, 2002). KASH domains are present in many proteins, including the actin-binding proteins ANC-1 and Syne-1, and the dystrophin-related Msp300. ANC-1 and Syne-1 are perinuclear and tether nuclei to the actin cytoskeleton (Apel, 2000; Starr, 2002). For Syne-1, the KASH domain is both necessary and sufficient to target the protein to the nuclear envelope (Zhang, 2001). A Myc-tagged Klar protein expressed ectopically in photoreceptors is also perinuclear (Mosley-Bishop, 1999), but the localization of endogenous Klar is unknown, in photoreceptors or any other tissue (Guo, 2005).

date revised: 15 December 2023

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