Guanosine increases global SUMO1-ylation in the hippocampus of young and aged mice and improves the short-term memory of young mice.

The nucleoside guanosine is an endogenous neuromodulator associated with neuroprotection. The roles of guanosine during aging are still not fully elucidated. Guanosine modulates SUMOylation in neurons and astrocytes in vitro, but it is not known whether guanosine can modulate SUMOylation in vivo and improve cognitive functions during aging. SUMOylation is a post-translational protein modification with potential neuroprotective roles. In this follow-up study, we investigated whether guanosine could modulate SUMOylation in vivo and behavior in young and aged mice. Young (3-month-old) and aged (24-month-old) C57BL/6 mice were treated with guanosine (8 mg/kg intraperitoneal) daily for 14 days. Starting on day 8 of treatment, the following behavioral tests were performed: open field, novel object location, Y-maze, sucrose splash test, and tail suspension test. Treatment with guanosine did not change the locomotor activity of young or aged mice in the open-field test. Treatment with guanosine improved short-term memory only for young mice but did not change the working memory of either young or aged mice, as evaluated using object recognition and the Y-maze tests, respectively. Depressive-like behaviors, such as impaired grooming evaluated through the splash test, did not change in either young or aged mice. However, young mice treated with guanosine increased their immobility time in the tail suspension test, suggesting an effect on behavioral coping strategies. Global SUMO1-ylation was significantly increased in the hippocampus of young and aged mice after 14 days of treatment with guanosine, whereas no changes were detected in the cerebral cortex of either young or aged mice. Our findings demonstrate that guanosine also targets hippocampal SUMOylation in vivo, thereby contributing to a deeper understanding of its mechanisms of action. This highlights the involvement of SUMOylation in guanosine's modulatory and neuroprotective effects.

Circadian rhythm alterations affecting the pathology of neurodegenerative diseases.

The circadian rhythm is a nearly 24-h oscillation found in various physiological processes in the human brain and body that is regulated by environmental and genetic factors. It is responsible for maintaining body homeostasis and it is critical for essential functions, such as metabolic regulation and memory consolidation. Dysregulation in the circadian rhythm can negatively impact human health, resulting in cardiovascular and metabolic diseases, psychiatric disorders, and premature death. Emerging evidence points to a relationship between the dysregulation circadian rhythm and neurodegenerative diseases, suggesting that the alterations in circadian function might play crucial roles in the pathogenesis and progression of neurodegenerative diseases. Better understanding this association is of paramount importance to expand the knowledge on the pathophysiology of neurodegenerative diseases, as well as, to provide potential targets for the development of new interventions based on the dysregulation of circadian rhythm. Here we review the latest findings on dysregulation of circadian rhythm alterations in Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, spinocerebellar ataxia and multiple-system atrophy, focusing on research published in the last 3 years.

SUMOylation in peripheral tissues under low perfusion-related pathological states.

SUMOylation is described as a posttranslational protein modification (PTM) that is involved in the pathophysiological processes underlying several conditions related to ischemia- and reperfusion-induced damage. Increasing evidence suggests that, under low oxygen levels, SUMOylation might be part of an endogenous mechanism, which is triggered by injury to protect cells within the central nervous system. However, the role of ischemia-induced SUMOylation in the periphery is still unclear. This article summarizes the results of recent studies regarding SUMOylation profiles in several diseases characterized by impaired blood flow to the cardiorenal, gastrointestinal, and respiratory systems. Our review shows that although ischemic injury per se does not always increase SUMOylation levels, as seen in strokes, it seems that in most cases the positive modulation of protein SUMOylation after peripheral ischemia might be a protective mechanism. This complex relationship warrants further investigation, as the role of SUMOylation during hypoxic conditions differs from organ to organ and is still not fully elucidated.

Guanosine modulates SUMO2/3-ylation in neurons and astrocytes via adenosine receptors.

SUMOylation is a post-translational modification (PTM) whereby members of the Small Ubiquitin-like MOdifier (SUMO) family of proteins are conjugated to lysine residues in target proteins. SUMOylation has been implicated in a wide range of physiological and pathological processes, and much attention has been given to its role in neurodegenerative conditions. Due to its reported role in neuroprotection, pharmacological modulation of SUMOylation represents an attractive potential therapeutic strategy in a number of different brain disorders. However, very few compounds that target the SUMOylation pathway have been identified. Guanosine is an endogenous nucleoside with important neuromodulatory and neuroprotective effects. Experimental evidence has shown that guanosine can modulate different intracellular pathways, including PTMs. In the present study we examined whether guanosine alters global protein SUMOylation. Primary cortical neurons and astrocytes were treated with guanosine at 1, 10, 100, 300, or 500 µM at four time points, 1, 6, 24, or 48 h. We show that guanosine increases global SUMO2/3-ylation in neurons and astrocytes at 1 h at concentrations above 10 µM. The molecular mechanisms involved in this effect were evaluated in neurons. The guanosine-induced increase in global SUMO2/3-ylation was still observed in the presence of dipyridamole, which prevents guanosine internalization, demonstrating an extracellular guanosine-induced effect. Furthermore, the A1 adenosine receptor antagonist DPCPX abolished the guanosine-induced increase in SUMO2/3-ylation. The A2A adenosine receptor antagonist ZM241385 increased SUMOylation per se, but did not alter guanosine-induced SUMOylation, suggesting that guanosine may modulate SUMO2/3-ylation through an A1-A2A receptor interaction. Taken together, this is the first report to show guanosine as a SUMO2/3-ylation enhancer in astrocytes and neurons.

Sumoylation: Implications for Neurodegenerative Diseases.

The covalent posttranslational modifications of proteins are critical events in signaling cascades that enable cells to efficiently, rapidly and reversibly respond to extracellular stimuli. This is especially important in the CNS where the processes affecting synaptic communication between neurons are highly complex and very tightly regulated. Sumoylation regulates the function and fate of a diverse array of proteins and participates in the complex cell signaling pathways required for cell survival. One of the most complex signaling pathways is synaptic transmission.Correct synaptic function is critical to the working of the brain and its alteration through synaptic plasticity mediates learning, mental disorders and stroke. The investigation of neuronal sumoylation is a new and exciting field and the functional and pathophysiological implications are far-reaching. Sumoylation has already been implicated in a diverse array of neurological disorders. Here we provide an overview of current literature highlighting recent insights into the role of sumoylation in neurodegeneration. In addition we present a brief assessment of drug discovery in the analogous ubiquitin system and extrapolate on the potential for development of novel therapies that might target SUMO-associated mechanisms of neurodegenerative disease.

SUMO-regulated mitochondrial function in Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder characterized by cardinal motor signs such as rigidity, bradykinesia or rest tremor that arise from a significant death of dopaminergic neurons. Non-dopaminergic degeneration also occurs and it seems to induce the deficits in olfactory, emotional, and memory functions that precede the classical motor symptoms in PD. Despite the majority of PD cases being sporadic, several genes have previously been associated with the hereditary forms of the disease. The proteins encoded by some of these genes, including α-synuclein, DJ-1, and parkin, are modified by small ubiquitin-like modifier (SUMO), a post-translational modification that regulates a variety of cellular processes. Among the several pathogenic mechanisms proposed for PD is mitochondrial dysfunction. Recent studies suggest that SUMOylation can interfere with mitochondrial dynamics, which is essential for neuronal function, and may play a pivotal role in PD pathogenesis. Here, we present an overview of recent studies on mitochondrial disturbance in PD and the potential SUMO-modified proteins and pathways involved in this process. SUMOylation, a post-translational modification, interferes with mitochondrial dynamics, and may play a pivotal role in Parkinson's disease (PD). SUMOylation maintains α-synuclein (α-syn) in a soluble form and activates DJ-1, decreasing mitochondrial oxidative stress. SUMOylation may reduce the amount of parkin available for mitochondrial recruitment and decreases mitochondrial biogenesis through suppression of peroxisomal proliferator-activated receptor-γ co-activator 1 α (PGC-1α). Mitochondrial fission can be regulated by dynamin-related protein 1 SUMO-1- or SUMO-2/3-ylation. A fine balance for the SUMOylation/deSUMOylation of these proteins is required to ensure adequate mitochondrial function in PD.

Battling Alzheimer's Disease: Targeting SUMOylation-Mediated Pathways.

SUMO (small ubiquitin-like modifier) conjugation is a critically important control process in all eukaryotic cells, because it acts as a biochemical switch and regulates the function of hundreds of proteins in many different pathways. Although the diverse functional consequences and molecular targets of SUMOylation remain largely unknown, SUMOylation is becoming increasingly implicated in the pathophysiology of Alzheimer's disease (AD). Apart from the central SUMO-modified disease-associated proteins, such as amyloid precursor protein, amyloid ß, and tau, SUMOylation also regulates several other processes underlying AD. These are involved in inflammation, mitochondrial dynamics, synaptic transmission and plasticity, as well as in protective responses to cell stress. Herein, we review current reports on the involvement of SUMOylation in AD, and present an overview of potential SUMO targets and pathways underlying AD pathogenesis.

Differential regulation of GABAB receptor trafficking by different modes of N-methyl-D-aspartate (NMDA) receptor signaling.

Inhibitory GABAB receptors (GABABRs) can down-regulate most excitatory synapses in the CNS by reducing postsynaptic excitability. Functional GABABRs are heterodimers of GABAB1 and GABAB2 subunits and here we show that the trafficking and surface expression of GABABRs is differentially regulated by synaptic or pathophysiological activation of NMDA receptors (NMDARs). Activation of synaptic NMDARs using a chemLTP protocol increases GABABR recycling and surface expression. In contrast, excitotoxic global activation of synaptic and extrasynaptic NMDARs by bath application of NMDA causes the loss of surface GABABRs. Intriguingly, exposing neurons to extreme metabolic stress using oxygen/glucose deprivation (OGD) increases GABAB1 but decreases GABAB2 surface expression. The increase in surface GABAB1 involves enhanced recycling and is blocked by the NMDAR antagonist AP5. The decrease in surface GABAB2 is also blocked by AP5 and by inhibiting degradation pathways. These results indicate that NMDAR activity is critical in GABABR trafficking and function and that the individual subunits can be separately controlled to regulate neuronal responsiveness and survival.

SUMOylation effects on neural stem cells self-renewal, differentiation, and survival.

SUMO (small ubiquitin-like modifier) conjugation or SUMOylation, a post-translational modification, is a crucial regulator of protein function and cellular processes. In the context of neural stem cells (NSCs), SUMOylation has emerged as a key player, affecting their proliferation, differentiation, and survival. By modifying transcription factors, such as SOX1, SOX2, SOX3, SOX6, Bmi1, and Nanog, SUMOylation can either enhance or impair their transcriptional activity, thus impacting on NSCs self-renewal. Moreover, SUMOylation regulates neurogenesis and neuronal differentiation by modulating key proteins, such as Foxp1, Mecp2, MEF2A, and SOX10. SUMOylation is also crucial for the survival and proliferation of NSCs in both developing and adult brains. By regulating the activity of transcription factors, coactivators, and corepressors, SUMOylation acts as a molecular switch, inducing cofactor recruitment and function during development. Importantly, dysregulation of NSCs SUMOylation has been implicated in various disorders, including embryonic defects, ischemic cerebrovascular disease, glioma, and the harmful effects of benzophenone-3 exposure. Here we review the main findings on SUMOylation-mediated regulation of NSCs self-renewal, differentiation and survival. Better understanding NSCs SUMOylation mechanisms and its functional consequences might provide new strategies to promote neuronal differentiation that could contribute for the development of novel therapies targeting neurodegenerative diseases.

Renal protection after hemorrhagic shock in rats: Possible involvement of SUMOylation.

Hemorrhagic shock (HS), a leading cause of preventable death, is characterized by severe blood loss and inadequate tissue perfusion. Reoxygenation of ischemic tissues exacerbates organ damage through ischemia-reperfusion injury. SUMOylation has been shown to protect neurons after stroke and is upregulated in response to cellular stress. However, the role of SUMOylation in organ protection after HS is unknown. This study aimed to investigate SUMOylation-mediated organ protection following HS. Male Wistar rats were subjected to HS (blood pressure of 40 ± 2 mmHg, for 90 min) followed by reperfusion. Blood, kidney, and liver samples were collected at various time points after reperfusion to assess organ damage and investigate the profile of SUMO1 and SUMO2/3 conjugation. In addition, human kidney cells (HK-2), treated with the SUMOylation inhibitor TAK-981 or overexpressing SUMO proteins, were subjected to oxygen and glucose deprivation to investigate the role of SUMOylation in hypoxia/reoxygenation injury. The animals presented progressive multiorgan dysfunction, except for the renal system, which showed improvement over time. Compared to the liver, the kidneys displayed distinct patterns in terms of oxidative stress, apoptosis activation, and tissue damage. The global level of SUMO2/3 in renal tissue was also distinct, suggesting a differential role. Pharmacological inhibition of SUMOylation reduced cell viability after hypoxia-reoxygenation damage, while overexpression of SUMO1 or SUMO2 protected the cells. These findings suggest that SUMOylation might play a critical role in cellular protection during ischemia-reperfusion injury in the kidneys, a role not observed in the liver. This difference potentially explains the renal resilience observed in HS animals when compared to other systems.

Worldwide prevalence of sleep problems in community-dwelling older adults: A systematic review and meta-analysis.

The understanding of the prevalence of sleep problems in older adults can provide a broad and reliable perspective into the occurrence of such issues among older adults. This systematic review and meta-analysis aimed to estimate the worldwide prevalence of sleep problems in community-dwelling older adults. Studies that provide information on the prevalence of sleep problems in community-dwelling older adults (≥60 years) were screened between December 2022 and March 2023. A total of 20,379 studies were identified in database searches, from which 252 were included in this review. These studies covered the last 35 years (from 1988 to 2023) and pooled a sample of 995,544 participants from 36 countries. The most frequent sleep problem worldwide was obstructive sleep apnea (46.0%), followed by poor sleep quality (40.0%), other sleep problems (37.0%), insomnia (29.0%), and excessive daytime sleepiness (19.0%). No significant difference in the prevalence estimates of all sleep problems was observed between the sexes. This systematic review and meta-analysis showed a high prevalence of some sleep problems, mainly obstructive sleep apnea, poor sleep quality, and other sleep problems. Our estimates can be useful for managers and policymakers in planning healthcare strategies for sleep problems aimed at the older population.

Molecular targets underlying SUMO-mediated neuroprotection in brain ischemia.

SUMOylation (small ubiquitin-like modifier conjugation) is an important post-translational modification which is becoming increasingly implicated in the altered protein dynamics associated with brain ischemia. The function of SUMOylation in cells undergoing ischemic stress and the identity of small ubiquitin-like modifier (SUMO) targets remain in most cases unknown. However, the emerging consensus is that SUMOylation of certain proteins might be part of an endogenous neuroprotective response. This review brings together the current understanding of the underlying mechanisms and downstream effects of SUMOylation in brain ischemia, including processes such as autophagy, mitophagy and oxidative stress. We focus on recent advances and controversies regarding key central nervous system proteins, including those associated with the nucleus, cytoplasm and plasma membrane, such as glucose transporters (GLUT1, GLUT4), excitatory amino acid transporter 2 glutamate transporters, K+ channels (K2P1, Kv1.5, Kv2.1), GluK2 kainate receptors, mGluR8 glutamate receptors and CB1 cannabinoid receptors, which are reported to be SUMO-modified. A discussion of the roles of these molecular targets for SUMOylation could play following an ischemic event, particularly with respect to their potential neuroprotective impact in brain ischemia, is proposed.

Targeting α-synuclein post-translational modifications in Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease characterized by the progressive loss of dopaminergic neurons in the nigrostriatal pathway. Although the exact mechanisms underlying PD are still not completely understood, it is well accepted that α-synuclein plays key pathophysiological roles as the main constituent of the cytoplasmic inclusions known as Lewy bodies. Several post-translational modifications (PTMs), such as the best-known phosphorylation, target α-synuclein and are thus implicated in its physiological and pathological functions. In this review, we present (1) an overview of the pathophysiological roles of α-synuclein, (2) a descriptive analysis of α-synuclein PTMs, including phosphorylation, ubiquitination, SUMOylation, acetylation, glycation, truncation, and O-GlcNAcylation, as well as (3) a brief summary on α-synuclein PTMs as potential biomarkers for PD. A better understanding of α-synuclein PTMs is of paramount importance for elucidating the mechanisms underlying PD and can thus be expected to improve early detection and monitoring disease progression, as well as identify promising new therapeutic targets.

A nationwide study on pain manifestations and sleep problems in community-dwelling older adults: findings from ELSI-Brazil.

PURPOSE: Sleep problems are common and affect approximately 36-70% of older adults worldwide and can be associated with negative outcomes such as pain. There is believed to be a bidirectional relationship between sleep problems and pain, modulated by inflammation and stress. The objective was to investigate the association between self-reported sleep problems and pain manifestations. METHODS: A cross-sectional study using data from the second wave of the Brazilian Longitudinal Study of Aging (2019-2021) was conducted. The exposure variables were self-reported sleep problems: poor sleep quality, insomnia (initial, intermediate, and final), and daytime sleepiness. The outcomes were self-reported pain manifestations: frequent pain, moderate/intense/strong pain, and pain-related disability. Logistic regressions were performed to verify the association between exposures and outcomes. RESULTS: A total of 6875 community-dwelling older adults participated in this study (71.1 ± 8.3 years; 54.4% female). Older adults with self-reported poor sleep quality, initial, intermediate and final insomnia, and daytime sleepiness had, respectively, 1.99 (95% CI 1.57-2.53), 1.47 (95% CI 1.11-1.97), 1.65 (95% CI 1.27-2.14), 1.69 (95% CI 1.29-2.22), and 1.76 (95% CI 1.35-2.29) greater odds of reporting frequent pain. The odds of moderate/intense/strong pain were higher in older adults that reported poor sleep quality (OR: 2.21; 95% CI 1.08-4.51). Older adults with self-reported poor sleep quality, initial, intermediate and final insomnia, and daytime sleepiness had, respectively, 1.84 (95% CI 1.11-3.02), 1.73 (95% CI 1.14-2.62), 1.80 (95% CI 1.19-2.73), 1.58 (95% CI 1.07-2.34), and 1.63 (95% CI 1.11-2.39) greater odds of reporting pain-related disability. CONCLUSION: Self-reported sleep problems are associated with pain manifestations in older adults. The results may help in the proposition of programs and public health policies.

A nationwide study on sleep complaints and associated factors in older adults: ELSI-Brazil.

Sleep problems, such as difficulty falling asleep, staying asleep, early awakening with failure to continue sleep, and altered sleep-wake cycle, are common in the general population. This cross-sectional study with 6,929 older adults (≥ 60 years) aimed to estimate the prevalence of different types of sleep problems, their associated factors, and the population-attributable fraction of associated factors among older adults. The outcome variables consisted of self-reported sleep problems: insomnia (initial, intermediate, late, and any type of insomnia), poor sleep quality, and daytime sleepiness. The independent variables were sociodemographic and behavioral characteristics and health conditions. The prevalence proportions were initial insomnia (49.1%), intermediate insomnia (49.2%), late insomnia (45.9%), any type of insomnia (58.6%), poor sleep quality (15.6%), and daytime sleepiness (38.4%). Female sex, presence of two or more chronic diseases, not eating the recommended amount of fruits and vegetables, and regular and bad/very bad self-rated health were positively associated with the sleep problems investigated. Consuming alcohol once a month or more was inversely associated with initial insomnia. Population attributable fraction estimates ranged from 3% to 19% considering two or more chronic diseases, not eating the recommended amount of fruits and vegetables, and regular and bad/very bad self-rated health. High prevalence of self-reported sleep problems was evinced in older adults. These results can be useful to guide public health services in the creation of informational, evaluative, and screening strategies for sleep problems in older Brazilian adults.

Oxygen/glucose deprivation induces a reduction in synaptic AMPA receptors on hippocampal CA3 neurons mediated by mGluR1 and adenosine A3 receptors.

Hippocampal CA1 pyramidal neurons are highly sensitive to ischemic damage, whereas neighboring CA3 pyramidal neurons are less susceptible. It is proposed that switching of AMPA receptor (AMPAR) subunits on CA1 neurons during an in vitro model of ischemia, oxygen/glucose deprivation (OGD), leads to an enhanced permeability of AMPARs to Ca(2+), resulting in delayed cell death. However, it is unclear whether the same mechanisms exist in CA3 neurons and whether this underlies the differential sensitivity to ischemia. Here, we investigated the consequences of OGD for AMPAR function in CA3 neurons using electrophysiological recordings in rat hippocampal slices. Following a 15 min OGD protocol, a substantial depression of AMPAR-mediated synaptic transmission was observed at CA3 associational/commissural and mossy fiber synapses but not CA1 Schaffer collateral synapses. The depression of synaptic transmission following OGD was prevented by metabotropic glutamate receptor 1 (mGluR1) or A(3) receptor antagonists, indicating a role for both glutamate and adenosine release. Inhibition of PLC, PKC, or chelation of intracellular Ca(2+) also prevented the depression of synaptic transmission. Inclusion of peptides to interrupt the interaction between GluA2 and PICK1 or dynamin and amphiphysin prevented the depression of transmission, suggesting a dynamin and PICK1-dependent internalization of AMPARs after OGD. We also show that a reduction in surface and total AMPAR protein levels after OGD was prevented by mGluR1 or A(3) receptor antagonists, indicating that AMPARs are degraded following internalization. Thus, we describe a novel mechanism for the removal of AMPARs in CA3 pyramidal neurons following OGD that has the potential to reduce excitotoxicity and promote neuroprotection.

SUMO-modifying Huntington's disease.

Small ubiquitin-like modifiers, SUMOs, are proteins that are conjugated to target substrates and regulate their functions in a post-translational modification called SUMOylation. In addition to its physiological roles, SUMOylation has been implicated in several neurodegenerative diseases, such as Alzheimer's, Parkinson's, and Huntington's diseases (HD). HD is a neurodegenerative monogenetic autosomal dominant disorder caused by a mutation in the CAG repeat of the huntingtin (htt) gene, which expresses a mutant Htt protein more susceptible to aggregation and toxicity. Besides Htt, other SUMO ligases, enzymes, mitochondrial and autophagic components are also important for the progression of the disease. Here we review the main aspects of Htt SUMOylation and its role in cellular processes involved in the pathogenesis of HD.

Restraint stress potentiates neuropeptide Y-mediated impairment on spatial memory in rats.

Memory is the ability to store, retrieve and use information that requires a progressive time-dependent stabilization process known as consolidation to be established. The hippocampus is essential for processing all the information that forms memory, especially spatial memory. Neuropeptide Y (NPY) affects memory, so in this study we investigated the participation and recruitment of NPY receptors during spatial memory consolidation in rats. Using the water maze test, we show that NPY (1 pmol) injected into the dorsal hippocampus impaired memory consolidation and that previous restraint stress (30 min) potentiates NPY effects, i.e. further impaired memory consolidation. Using selective antagonists for NPY Y1 and Y2 receptors we demonstrate that both receptors play a key role on spatial memory consolidation. Our data suggest that NPY modulates aversive and adaptive memory formation by NPY receptors activation.

Effects of amyloid-ß on protein SUMOylation and levels of mitochondrial proteins in primary cortical neurons.

Defining the molecular changes that underlie Alzheimer's disease (AD) is an important question in neuroscience. Here, we examined changes in protein SUMOylation, and proteins involved in mitochondrial dynamics, in an in vitro model of AD induced by application of amyloid-ß 1-42 (Aß1-42) to cultured neurons. We observed Aß1-42-induced decreases in global SUMOylation and in levels of the SUMO pathway enzymes SENP3, PIAS1/2, and SAE2. Aß exposure also decreased levels of the mitochondrial fission proteins Drp1 and Mff and increased activation of caspase-3. To examine whether loss of SENP3 is cytoprotective we knocked down SENP3, which partially prevented the Aß1-42-induced increase in caspase-3 activation. Together, these data support the hypothesis that altered SUMOylation may play a role in the mechanisms underlying AD.

CaV2.2 (N-type) voltage-gated calcium channels are activated by SUMOylation pathways.

SUMOylation is an important post-translational modification process involving covalent attachment of SUMO (Small Ubiquitin-like MOdifier) protein to target proteins. Here, we investigated the potential for SUMO-1 protein to modulate the function of the CaV2.2 (N-type) voltage-gated calcium channel (VGCC), a protein vital for presynaptic neurotransmitter release. Co-expression of SUMO-1, but not the conjugation-deficient mutant SUMO-1ΔGG, increased heterologously-expressed CaV2.2 Ca2+ current density, an effect potentiated by the conjugating enzyme Ubc9. Expression of sentrin-specific protease (SENP)-1 or Ubc9 alone, had no effect on recombinant CaV2.2 channels. Co-expression of SUMO-1 and Ubc9 caused an increase in whole-cell maximal conductance (Gmax) and a hyperpolarizing shift in the midpoint of activation (V1/2). Mutation of all five CaV2.2 lysine residues to arginine within the five highest probability (>65 %) SUMOylation consensus motifs (SCMs) (construct CaV2.2-Δ5KR), produced a loss-of-function mutant. Mutagenesis of selected individual lysine residues identified K394, but not K951, as a key residue for SUMO-1-mediated increase in CaV2.2 Ca2+ current density. In synaptically-coupled superior cervical ganglion (SCG) neurons, SUMO-1 protein was distributed throughout the cell body, axons and dendrites and presumptive presynaptic terminals, whilst SUMO-1ΔGG protein was largely confined to the cell body, in particular, the nucleus. SUMO-1 expression caused increases in paired excitatory postsynaptic potential (EPSP) ratio at short (20-120 ms) inter-stimuli intervals in comparison to SUMO-1ΔGG, consistent with an increase in residual presynaptic Ca2+ current and an increase in release probability of synaptic vesicles. Together, these data provide evidence for CaV2.2 VGCCs as novel targets for SUMOylation pathways.