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Loss-of-function mutations in Parkin are the most common causes of autosomal recessive Parkinson’s disease (PD). Many putative substrates of parkin have been reported; their pathogenic roles, however, remain obscure due to poor characterization, particularly in vivo. Here, we show that synaptotagmin, encoded by a PD-risk gene SYT11, is a physiological substrate of parkin and plays critical roles in mediating parkin-linked neurotoxicity. Unilateral overexpression of full-length, but not C2B-truncated, synaptotagmin in the substantia nigra pars compacta (SNpc) impairs ipsilateral striatal dopamine release, causes late-onset degeneration of dopaminergic neurons, and induces progressive contralateral motor abnormalities. Mechanistically, synaptotagmin impairs vesicle pool replenishment and thus dopamine release by inhibiting endocytosis. Furthermore, parkin deficiency induces synaptotagmin accumulation and PD-like neurotoxicity in mouse models, which is reversed by SYT11 knockdown in the SNpc or knockout of SYT11 restricted to dopaminergic neurons. Thus, PD-like neurotoxicity induced by parkin dysfunction requires synaptotagmin accumulation in SNpc dopaminergic neurons.

Parkinson’s disease (PD) is a complex neurodegenerative movement disorder characterized by the selective vulnerability of dopaminergic neurons in the substantia nigra pars compacta (SNpc), and the impairment of dopamine (DA) release in the striatum^{1,2,3,4,5,6,7}. Numerous PD-risk genes and environmental factors have been identified, but their functional connectivity is poorly understood, thus hindering the elucidation of a common pathogenic pathway for PD. Parkin (encoded by PARK2) is an E3 ubiquitin ligase that regulates proteasome-dependent protein degradation^1,3,8,9 and mitochondrial autophagy (mitophagy)^10,11,12,13. It has been shown that mutations in parkin affect either its E3 ligase activity or its interactions with E2 enzymes, causing familial recessive and sporadic early-onset PD^{1,8,12,14,15,16,17}. Abnormal accumulation of parkin substrates may contribute to DA neuron degeneration in PD associated with parkin mutations⁵. Although many putative substrates of parkin have been identified^{1,2,3,10,18,19,20}, due to a lack of unequivocal in vivo evidence, the involvement of these substrates in the pathogenesis of PD and the underlying mechanisms remain elusive.

A recent meta-analysis of PD populations based on five genome-wide association studies has identified 11 loci linked to a significant risk of PD, including synaptotagmin (SYT11) as one of five novel PD-risk genes²¹. A number of Syt proteins have been shown to be Ca²⁺ sensors for SNARE-mediated vesicle fusion during neurotransmitter release and hormone secretion²². Syt1 and Syt4 have also been shown to function in endocytosis^{23,24,25,26,27}. We recently found that Syt11, a non-Ca²⁺-binding Syt²⁸, inhibits endocytosis and thus vesicle recycling in neurons²⁹. Its role in neurodegenerative disease, however, has never been reported. Interestingly, Syt11 can be ubiquitinated by parkin in vitro and accumulates in Lewy bodies in the sporadic human PD patient brain tissue¹⁹. Here we show that Syt11 is a parkin substrate and mediates PD-like neurotoxicity and behavioral deficits.

Syt11 is a parkin substrate

To determine whether Syt11 is a parkin substrate, we used co-immunoprecipitation and found an interaction between Syt11 and parkin in HEK cells overexpressing YFP-parkin and Myc-Syt11 (Fig. 1a, b). We next verified that parkin ubiquitinates Syt11 in vitro¹⁹. The analysis of anti-Myc immunoprecipitates from the lysates of HEK cells transfected with Myc-Syt11, YFP-parkin, and FLAG-ubiquitin (Flag-Ub) revealed that the ubiquitination of Syt11 was markedly increased by the overexpression of parkin (Fig. 1c). In addition, Syt11 expression was greatly reduced by parkin overexpression, while the pathogenic parkin mutations found in PD patients^8,10,30,31, such as R42P (disrupting the ubiquitin-like domain) and RW (disrupting the RING1 domain), failed to mediate Syt11 degradation (Fig. 1d). Furthermore, the parkin-mediated decrease of Syt11 level was prevented by h pretreatment with the proteasome inhibitor MG (Fig. 1d), confirming that parkin-mediated Syt11 degradation occurs through the proteasome pathway.

To further confirm that parkin also regulates endogenous Syt11 expression, we overexpressed YFP-parkin in hippocampal neurons and collected the transfected cells with a fluorescence-activated cell sorter. Western blot analysis revealed that the endogenous Syt11 expression in primary neurons was also decreased by parkin overexpression (Supplementary Fig. 1). Importantly, knockdown (KD) of parkin expression using a lentivirus carrying parkin shRNA (shParkin) induced the accumulation of Syt11 in the SNpc (Fig. 1e–j and Supplementary Fig. 12a–c), while the scrambled shRNA served as a control (parkin_Ipsi/Contra:  ± , P = ; Syt11_Ipsi/Contra =  ± , P = ; n = 3), verifying the role of parkin in regulating Syt11 levels in vivo. Together with the previous in vitro data¹⁹, our findings demonstrate that Syt11 is a parkin substrate, while parkin acts as the E3 ligase to directly regulate Syt11 protein levels through ubiquitin-dependent proteasome degradation.

Syt11 accumulation induces behavioral deficits

Since parkin deficiency and its pathogenic mutations induce the abnormal accumulation of Syt11 (Fig. 1), we next asked whether increasing Syt11 levels alone is sufficient to trigger PD-related toxicity in the mouse brain. Sytcarrying lentivirus was delivered to the SNpc in the right hemisphere by stereotaxic injection (Fig. 2a). The results showed efficient infection of dopaminergic neurons in the SNpc, as evidenced by GFP expression in tyrosine hydroxylase (TH)-positive cells (Fig. 2b). Parkin expression remained largely unchanged in mice overexpressing Syt11 (Fig. 2d and Supplementary Fig. 2). Since Syt11 was unilaterally overexpressed in the SNpc region, we used methamphetamine (METH)-induced rotational asymmetry^20,32 as a functional behavioral readout to assess the progression and severity of motor abnormalities induced by Syt11 overexpression. Mice injected with virus carrying GFP-only served as controls and they showed no defect of asymmetric rotation (Fig. 2c–h). Strikingly, unilateral overexpression (OE) of Syt11 induced a progressive increase in METH-induced contralateral rotation and a gradually shortened latency to rotation during 3–8 weeks after virus injection (Fig. 2c–h). Footprint gait analysis^33,34, an assay that does not depend on pharmacological stimulation, also showed an unbalanced gait in SytOE mice 1 month after virus injection (Fig. 2i–l). Mice with unilateral SytOE showed greater variation of the contralateral stride length (Fig. 2i), which was similar on average to that of the ipsilateral side (contralateral,  ± ; ipsilateral,  ± , P = ), but with a larger standard deviation and range (Fig. 2j, k). Consistently, SytOE mice also showed greater contralateral than ipsilateral front/hindpaw overlap (Fig. 2l). In contrast, both stride length and paw overlap were similar on both sides of the control virus-injected mice (Fig. 2m–p). Collectively, these findings indicate that Syt11 overexpression in this brain region is sufficient to induce locomotor deficits.

Syt11 accumulation impairs DA release

We next made amperometric recordings with electrochemical carbon fiber electrodes (CFEs) in striatal slices to determine whether SytOE in the SNpc impairs DA release from nigrostriatal terminals^32,35. When a local electrical stimulus (Estim) was applied to the striatal slice, there was a transient increase in amperometric current (I_amp, with an amplitude of ~ pA) with a subsequent decay to baseline, representing a transient increase in extracellular DA concentration (~ μM, Fig. 3a and Supplementary Fig. 3). Strikingly, unilateral overexpression of Syt11 in the SNpc markedly decreased the DA release in the ipsilateral striatum compared with that in the contralateral striatum of the same mice at 1 month after virus injection (Fig. 3a–c). In contrast, DA release in the ipsilateral striatum remained intact when control virus was used (Fig. 3d–f). In addition, SytOE inhibited secretion in neuroendocrine adrenal chromaffin cells (Supplementary Fig. 4). Together, these results demonstrate that Syt11 overexpression in the SNpc in vivo is sufficient to cause a defect of DA release in the striatum.

Syt11 accumulation leads to late onset loss of DA neurons

To investigate whether Syt11 overexpression also leads to the loss of dopaminergic neurons in the SNpc, we performed stereological counting of TH-positive neurons in the SytOE and contralateral sides of the SNpc^20,33. Interestingly, SytOE failed to induce a clear loss of dopaminergic neurons in the SNpc at one month after injection (Supplementary Fig. 5), despite the robust motor impairment (Fig. 2) and DA release defects (Fig. 3a–f) at this stage. However, we observed ~35% loss of DA neurons in the SNpc with SytOE at 3 months after injection, while no significant loss of TH-positive neurons was found when control virus was used (Fig. 3g, h). We also found positive terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining in the SytOE, but not in the contralateral SNpc (Fig. 3g), suggesting that these DA neurons degenerate through apoptosis. Consistently, the reduced fluorescence intensity of TH staining also revealed a substantial reduction of TH-positive neurites in the ipsilateral striatum (Fig. 3i, j). Thus, our data indicate that Syt11 overexpression also induces the progressive loss of dopaminergic neurons, which is preceded by depression of DA release.

Syt11 inhibits endocytosis and vesicle replenishment

An independent study from our lab has shown that syt11 inhibits dynamin-dependent endocytosis by limiting the membrane invagination in dorsal root ganglion and hippocampal neurons^27,29, so we next determined whether the impaired DA transmission was also due to the inhibitory role of Syt11 in endocytosis in dopaminergic neurons. Alexa Fluor-conjugated transferrin uptake was measured to evaluate clathrin-mediated endocytosis (CME) in Sytoverexpressing dopaminergic neurons in the SNpc of TH-driven GFP transgenic mice^29,36. We found that, compared with the contralateral side, transferrin uptake was markedly suppressed in TH-positive neurons in the SytOE SNpc (Fig. 4a and Supplementary Fig. 6), indicating that CME was slowed due to SytOE. To further verify the inhibitory role of Syt11 in neuronal endocytosis, we used total internal reflection fluorescence (TIRF) imaging of clathrin-DsRed in the somata of hippocampal neurons with or without Syt11 overexpression. Strikingly, most of the assembled clathrin clusters were clamped at the plasma membrane, and thus the CME events were markedly reduced in SytOE neurons (Fig. 4b and Supplementary Fig. 7). Furthermore, we used live imaging of the synaptophysin-pHluorin reporter, an assay for stimulated synaptic endocytosis/exocytosis (Fig. 4c), to monitor the function of Syt11 in endocytosis and vesicle recycling. Consistently, SytOE neurons showed a robust reduction in endocytic rate with a significantly longer time constant, confirming the inhibitory role of Syt11 in synaptic endocytosis and vesicle recycling of hippocampal neurons (Fig. 4d–f). These findings indicate an inhibitory action of Syt11 on endocytosis in DA neurons.

DA release in response to paired-pulse stimulation was used to further assess the role of Syt11 in vesicle recycling in DA neurons. As expected, we found a reduced paired-pulse ratio of DA release in ipsilateral striatal slices overexpressing Syt11 (Fig. 5a and Supplementary Fig. 8), indicating that the vesicle replenishment³⁵ was also inhibited. Burst stimulation using a train of 10 pulses at 20 Hz also revealed a reduced releasable vesicle pool in the striatal DA terminals on the SytOE side (Fig. 5b and Supplementary Fig. 9a–c). In addition, we re-examined the vesicle recycling rate after depleting the releasable vesicle pools and confirmed the slowed vesicle replenishment by Syt11 (Fig. 5c and Supplementary Fig. 9). Furthermore, we crossed homozygous floxed Sytnull mice with heterozygous DA transporter-driven Cre-knockin mice (DAT-Cre)^37,38,39 to produce DA neuron-restricted Syt11 conditional knockout (SytcKO) mice (Fig. 5d). As expected, immunostaining showed a nearly complete loss of Syt11 in TH-positive neurons in the ventral midbrain, while Syt11 expression in TH-negative neurons remained intact (Supplementary Fig. 10a, b), indicating a specific knockout of Syt11 in dopaminergic neurons. Consistently, western blots showed a dramatic reduction of Syt11 expression in the whole ventral midbrain (Supplementary Fig. 10c–e). Strikingly, amperometric recordings demonstrated increased DA release (Fig. 5e), accelerated vesicle replenishment (Fig. 5f), and enlarged releasable vesicle pools (Fig. 5g, h) in the striatum of SytcKO mice, confirming the inhibitory role of Syt11 in vesicle recycling in DA neurons.

Structure–function analysis revealed that the C2B domain of Syt11 is critical for its inhibitory role in endocytosis²⁹. We next investigated whether this domain is also essential for Syt11 to mediate PD-related neurotoxicity (Fig. 6a). Strikingly, unilateral overexpression of the C2B-truncated form of Syt11 in the SNpc (Fig. 6b–d) failed to impair the ipsilateral striatal DA release (Fig. 6e, f) and the contralateral motor stability (Fig. 6g–k), indicating the critical role of the C2B domain in the Sytmediated pathogenesis of PD. Collectively, these findings suggest that Syt11 plays an important role in DA transmission by regulating endocytosis and the vesicle-recycling process, while the upregulation/accumulation of Syt11 causes the reduction of releasable vesicle pools, impairment of DA release in the striatum, and thus the progression of PD.

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