Retromer dysfunction in amyotrophic lateral sclerosis.

Retromer is a heteropentameric complex that plays a specialized role in endosomal protein sorting and trafficking. Here, we report a reduction in the retromer proteins-vacuolar protein sorting 35 (VPS35), VPS26A, and VPS29-in patients with amyotrophic lateral sclerosis (ALS) and in the ALS model provided by transgenic (Tg) mice expressing the mutant superoxide dismutase-1 G93A. These changes are accompanied by a reduction of levels of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit GluA1, a proxy of retromer function, in spinal cords from Tg SOD1G93A mice. Correction of the retromer deficit by a viral vector expressing VPS35 exacerbates the paralytic phenotype in Tg SOD1G93A mice. Conversely, lowering Vps35 levels in Tg SOD1G93A mice ameliorates the disease phenotype. In light of these findings, we propose that mild alterations in retromer inversely modulate neurodegeneration propensity in ALS.

Complex Effects of the ZSCAN21 Transcription Factor on Transcriptional Regulation of α-Synuclein in Primary Neuronal Cultures and in Vivo.

α-Synuclein, a presynaptic neuronal protein encoded by the SNCA gene, is strongly implicated in Parkinson disease (PD). PD pathogenesis is linked to increased SNCA levels; however, the transcriptional elements that control SNCA expression are still elusive. Previous experiments in PC12 cells demonstrated that the transcription factor zinc finger and SCAN domain containing 21 (ZSCAN21) plays an important regulatory role in SNCA transcription. Currently, we characterized the role of ZSCAN21 in SNCA transcription in primary neuronal cultures and in vivo We found that ZSCAN21 is developmentally expressed in neurons in different rat brain regions. We confirmed its binding in the intron 1 region of SNCA in rat cortical cultures. Lentivirus-mediated silencing of ZSCAN21 increased significantly SNCA promoter activity, mRNA, and protein levels in such cultures. In contrast, ZSCAN21 silencing reduced SNCA in neurosphere cultures. Interestingly, ZSCAN21 overexpression in cortical neurons led to robust mRNA but negligible protein expression, suggesting that ZSCAN21 protein levels are tightly regulated post-transcriptionally and/or post-translationally in primary neurons. Efficient adeno-associated virus-mediated knockdown of ZSCAN21 in the postnatal and adult hippocampus, an area linked with non-motor PD symptoms, revealed no significant alterations in SNCA levels. Overall, our study demonstrates that ZSCAN21 is involved in the transcriptional regulation of SNCA in primary neuronal cultures, but the direction of the effect is variable, likely depending on neuronal maturation. However, the unaltered SNCA levels observed following ZSCAN21 down-regulation in the rat brain, possibly due to compensatory mechanisms, imply that ZSCAN21 is not a master regulator of SNCA in vivo.

Embryonic motor neuron programming factors reactivate immature gene expression and suppress ALS pathologies in postnatal motor neurons.

Aging is a major risk factor in amyotrophic lateral sclerosis (ALS) and other adult-onset neurodegenerative disorders. Whereas young neurons are capable of buffering disease-causing stresses, mature neurons lose this ability and degenerate over time. We hypothesized that the resilience of young motor neurons could be restored by re-expression of the embryonic motor neuron selector transcription factors ISL1 and LHX3. We found that viral re-expression of ISL1 and LHX3 reactivates aspects of the youthful gene expression program in mature motor neurons and alleviates key disease-relevant phenotypes in the SOD1G93A mouse model of ALS. Our results suggest that redeployment of lineage-specific neuronal selector transcription factors can be an effective strategy to attenuate age-dependent phenotypes in neurodegenerative disease.

Depletion of Mettl3 in cholinergic neurons causes adult-onset neuromuscular degeneration.

Motor neuron (MN) demise is a hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Post-transcriptional gene regulation can control RNA's fate, and defects in RNA processing are critical determinants of MN degeneration. N6-methyladenosine (m6A) is a post-transcriptional RNA modification that controls diverse aspects of RNA metabolism. To assess the m6A requirement in MNs, we depleted the m6A methyltransferase-like 3 (METTL3) in cells and mice. METTL3 depletion in embryonic stem cell-derived MNs has profound and selective effects on survival and neurite outgrowth. Mice with cholinergic neuron-specific METTL3 depletion display a progressive decline in motor behavior, accompanied by MN loss and muscle denervation, culminating in paralysis and death. Reader proteins convey m6A effects, and their silencing phenocopies METTL3 depletion. Among the m6A targets, we identified transactive response DNA-binding protein 43 (TDP-43) and discovered that its expression is under epitranscriptomic control. Thus, impaired m6A signaling disrupts MN homeostasis and triggers neurodegeneration conceivably through TDP-43 deregulation.

Regulation of α-synuclein expression in cultured cortical neurons.

Alpha-synuclein (SNCA) is a predominantly neuronal protein involved in the control of neurotransmitter release. The levels of SNCA expression are closely linked to the pathogenesis of Parkinson's disease; however, the biochemical pathways and transcriptional elements that control SNCA expression are not well understood. We previously used the model system of neurotrophin-mediated PC12 cell neuronal differentiation to examine these phenomena. Although these studies were informative, they were limited to the use of a cell line; therefore, in the current work, we have turned our attention to cultured primary rat cortical neurons. In these cultures, SNCA expression increased with time in culture, as the neurons mature. Luciferase assays based on transient transfections of fusion constructs encoding components of the transcriptional control region of SNCA identified various promoter areas that have a positive or negative effect on SNCA transcription. Intron 1, previously identified by us as an important regulatory region in the PC12 cell model, cooperates with regions 5' to exon 1 to mediate gene transcription. Using selective pharmacological tools, we find that tyrosine kinase receptors and the phosphatidyl-inositol 3 kinase signaling pathway are involved in mediating these effects. The exogenous application of the neurotrophin brain-derived neurotrophic factor (BDNF) is sufficient on its own to promote the transcriptional activation of SNCA through this pathway, but a neutralizing antibody against BDNF failed to affect SNCA transcription in maturing cultures, suggesting that BDNF is not the main factor involved in maturation-induced SNCA transcription in this model. Further in vivo studies are needed to establish the role of neurotrophin signaling in the control of SNCA transcription.

Correction: Loss of ß-Glucocerebrosidase Activity Does Not Affect Alpha-Synuclein Levels or Lysosomal Function in Neuronal Cells.

[This corrects the article DOI: 10.1371/journal.pone.0060674.].

Sumoylation regulates the assembly and activity of the SMN complex.

SMN is a ubiquitously expressed protein and is essential for life. SMN deficiency causes the neurodegenerative disease spinal muscular atrophy (SMA), the leading genetic cause of infant mortality. SMN interacts with itself and other proteins to form a complex that functions in the assembly of ribonucleoproteins. SMN is modified by SUMO (Small Ubiquitin-like Modifier), but whether sumoylation is required for the functions of SMN that are relevant to SMA pathogenesis is not known. Here, we show that inactivation of a SUMO-interacting motif (SIM) alters SMN sub-cellular distribution, the integrity of its complex, and its function in small nuclear ribonucleoproteins biogenesis. Expression of a SIM-inactivated mutant of SMN in a mouse model of SMA slightly extends survival rate with limited and transient correction of motor deficits. Remarkably, although SIM-inactivated SMN attenuates motor neuron loss and improves neuromuscular junction synapses, it fails to prevent the loss of sensory-motor synapses. These findings suggest that sumoylation is important for proper assembly and function of the SMN complex and that loss of this post-translational modification impairs the ability of SMN to correct selective deficits in the sensory-motor circuit of SMA mice.

New Insights on the Role of N 6-Methyladenosine RNA Methylation in the Physiology and Pathology of the Nervous System.

RNA modifications termed epitranscriptomics represent an additional layer of gene regulation similar to epigenetic mechanisms operating on DNA. The dynamic nature and the increasing number of RNA modifications offer new opportunities for a rapid fine-tuning of gene expression in response to specific environmental cues. In cooperation with a diverse and versatile set of effector proteins that "recognize" them, these RNA modifications have the ability to mediate and control diverse fundamental cellular functions, such as pre-mRNA splicing, nuclear export, stability, and translation. N 6-methyladenosine (m6A) is the most abundant of these RNA modifications, particularly in the nervous system, where recent studies have highlighted it as an important post-transcriptional regulator of physiological functions from development to synaptic plasticity, learning and memory. Here we review recent findings surrounding the role of m6A modification in regulating physiological responses of the mammalian nervous system and we discuss its emerging role in pathological conditions such as neuropsychiatric and neurodegenerative disorders.

Functional dissection of the alpha-synuclein promoter: transcriptional regulation by ZSCAN21 and ZNF219.

Alpha-synuclein (SNCA) is an abundant neuronal protein involved in synaptic neurotransmission. SNCA expression levels have been strongly implicated in Parkinson's disease pathogenesis. We have previously demonstrated that in the PC12 cell line elements in intron 1 may mediate SNCA transcriptional regulation in response to neurotrophins. We have now identified transcription factor (TF) binding sites in intron 1 and the 5'-promoter of SNCA. A binding site for the TF zinc finger and SCAN domain containing (ZSCAN)21 in the 5'-region of intron 1 is required for intron 1 transcriptional activity. Small interfering RNA against ZSCAN21 inhibits activation in the luciferase assay and diminishes SNCA protein levels in naïve and neurotrophin-treated PC12 cells and in primary cultured cortical neurons, demonstrating that ZSCAN21 is a novel transcriptional regulator of SNCA in neuronal cells. The 5'-promoter of SNCA has a complex architecture, including multiple binding sites for the TF zinc finger protein (ZNF)219, which functions as both an activator and a repressor. Targeting ZSCAN21 or other TFs controlling SNCA transcriptional activity may provide novel therapeutic avenues not only for Parkinson's disease but also for other synucleopathies.

Deletion of Ripk3 Prevents Motor Neuron Death In Vitro but not In Vivo.

Increasing evidence suggests that necroptosis, a form of programmed cell death (PCD), contributes to neurodegeneration in several disorders, including ALS. Supporting this view, investigations in both in vitro and in vivo models of ALS have implicated key molecular determinants of necroptosis in the death of spinal motor neurons (MNs). Consistent with a pathogenic role of necroptosis in ALS, we showed increased mRNA levels for the three main necroptosis effectors Ripk1, Ripk3, and Mlkl in the spinal cord of mutant superoxide dismutase-1 (SOD1G93A) transgenic mice (Tg), an established model of ALS. In addition, protein levels of receptor-interacting protein kinase 1 (RIPK1; but not of RIPK3, MLKL or activated MLKL) were elevated in spinal cord extracts from these Tg SOD1G93A mice. In postmortem motor cortex samples from sporadic and familial ALS patients, no change in protein levels of RIPK1 were detected. Silencing of Ripk3 in cultured MNs protected them from toxicity associated with SOD1G93A astrocytes. However, constitutive deletion of Ripk3 in Tg SOD1G93A mice failed to provide behavioral or neuropathological improvement, demonstrating no similar benefit of Ripk3 silencing in vivo. Lastly, we detected no genotype-specific myelin decompaction, proposed to be a proxy of necroptosis in ALS, in either Tg SOD1G93A or Optineurin knock-out mice, another ALS mouse model. These findings argue against a role for RIPK3 in Tg SOD1G93A-induced neurodegeneration and call for further preclinical investigations to determine if necroptosis plays a critical role in the pathogenesis of ALS.

A novel cell death pathway that is partially caspase dependent, but morphologically non-apoptotic, elicited by proteasomal inhibition of rat sympathetic neurons.

Proteasomal dysfunction has been linked to neurodegeneration. Pharmacological proteasomal inhibitors may have pro-survival or pro-death effects in neuronal cells. We have previously found that application of such agents to mouse sympathetic neurons leads to activation of the intrinsic apoptotic pathway. We show here that in rat sympathetic neurons proteasomal inhibition leads to a form of death that is morphologically non-apoptotic, with features of autophagy. The intrinsic apoptotic pathway is activated in a delayed fashion compared with mouse neurons, and is in part responsible for death, as evidenced by the partial protective effects of bcl-xL and the general caspase inhibitor Boc-aspartyl-fluoromethylketone. Death is accompanied by induction of Bim and caspase activation, but caspase 3 activation is lacking; 3-methyl-adenine inhibits macroautophagy, but has a relatively small pro-survival effect. We conclude that a complex array of pro- and anti-apoptotic effects elicited by proteasomal inhibition in rat sympathetic neurons leads to partial engagement of the intrinsic apoptotic pathway and a morphologically non-apoptotic, autophagic form of death. The species difference with mouse neurons is underscored by the fact that proteasomal inhibitors are protective against apoptosis elicited by nerve growth factor deprivation in rat, but not mouse, sympathetic neurons. The type of death described herein may be relevant to neurodegenerative diseases, where morphological evidence for apoptosis has been scant.

Alpha-synuclein dimerization in erythrocytes of patients with genetic and non-genetic forms of Parkinson's Disease.

BACKGROUND: Variations of α-synuclein levels or species have been reported in Parkinson's Disease (PD). There has been little systematic examination of erythrocytes, a rich source of α-synuclein. METHODS: Erythrocyte membranes were obtained from PD patients (mutation carriers in the α-synuclein gene (A53T-PD) and glucocerebrosidase gene (GBA-PD) (n=18 each), and patients without known mutations (GU-PD, n=56)), and age-/sex-matched controls (n=56). Levels of monomeric and dimeric α-synuclein were assessed using Western immunoblotting. RESULTS: A statistically significant increase of α-synuclein dimer and dimer to monomer ratio was found in GBA-PD and GU-PD. In contrast, dimer levels of A53T-PD were not different from controls. No difference was found in α-synuclein monomer levels. CONCLUSIONS: The increased α-synuclein dimer in GBA-PD and GU-PD is suggestive of an apparent systemic dysfunction causing the dimerization, and potentially oligomerization, of α-synuclein. These results may have implications for PD pathogenesis and biomarker development.

α-Synuclein dimerization in erythrocytes of Gaucher disease patients: correlation with lipid abnormalities and oxidative stress.

Several observations suggest that disturbed homeostasis of α-Synuclein (α-Syn) may provide a link between Gaucher disease (GD) and Parkinson's disease (PD). We recently reported increased dimerization of α-Syn in the red blood cell (RBC) membrane of patients with GD. Several studies indicate a crucial relationship between lipids, oxidative stress and α-Syn status. Here we investigated the relationship between the observed increased dimerization of α-Syn in the cell membranes of RBCs, cells devoid of lysosomes and lacking lysosomal enzyme synthesis, and the lipid abnormalities and oxidative stress already described in GD. Correlation studies showed that in GD the α-Syn dimer/monomer ratio is positively correlated with the levels of glucosylceramide (GlcCer) and the glucosylceramide/ceramide (GlcCer/Cer) ratio and negatively with the levels of malonyldialdehyde (MDA) and plasmalogens. In conclusion, we have shown that the increased tendency of α-Syn to form dimers in the RBC membrane of patients with GD, is correlated with both the level of lipids, including GlcCer, the primary lipid abnormality in GD, and the increased oxidative stress observed in this disorder. The study of other tissues, and in particular brain, will be important in order to elucidate the significance of these findings regarding the link between GD and PD.

Loss of ß-glucocerebrosidase activity does not affect alpha-synuclein levels or lysosomal function in neuronal cells.

To date, a plethora of studies have provided evidence favoring an association between Gaucher disease (GD) and Parkinson's disease (PD). GD, the most common lysosomal storage disorder, results from the diminished activity of the lysosomal enzyme ß-glucocerebrosidase (GCase), caused by mutations in the ß-glucocerebrosidase gene (GBA). Alpha-synuclein (ASYN), a presynaptic protein, has been strongly implicated in PD pathogenesis. ASYN may in part be degraded by the lysosomes and may itself aberrantly impact lysosomal function. Therefore, a putative link between deficient GCase and ASYN, involving lysosomal dysfunction, has been proposed to be responsible for the risk for PD conferred by GBA mutations. In this current work, we aimed to investigate the effects of pharmacological inhibition of GCase on ASYN accumulation/aggregation, as well as on lysosomal function, in differentiated SH-SY5Y cells and in primary neuronal cultures. Following profound inhibition of the enzyme activity, we did not find significant alterations in ASYN levels, or any changes in the clearance or formation of its oligomeric species. We further observed no significant impairment of the lysosomal degradation machinery. These findings suggest that additional interaction pathways together with aberrant GCase and ASYN must govern this complex relation between GD and PD.

Increased dimerization of alpha-synuclein in erythrocytes in Gaucher disease and aging.

Gaucher disease (GD) patients and carriers of glucocerebrosidase mutations are at an increased risk for Parkinson's disease (PD). The presynaptic protein alpha-synuclein (AS) is linked to PD. In the current work we examined biochemical properties of AS in GD patients. We generated membrane-enriched lysates from erythrocytes of 27 patients with GD and 32 age- and sex-matched controls and performed Western immunoblotting with antibodies against AS. Levels of monomeric AS did not differ between GD patients and controls and did not change as a function of age. However, the ratio of dimeric to monomeric AS was significantly increased in GD patients, and showed a significant positive correlation with age. Therefore, two major risk factors for PD, aging and GD status, are associated with an increased AS dimer to monomer ratio in erythrocytes. This ratio needs to be validated in further studies as a potential biomarker for PD risk.