Monthly Archives

June 2024

NAD+ in Brain Aging and Neurodegenerative Disorders

By NuSchild Research

Abstract

NAD+ is a pivotal metabolite involved in cellular bioenergetics, genomic stability, mitochondrial homeostasis, adaptive stress responses, and cell survival. Multiple NAD+-dependent enzymes are involved in synaptic plasticity and neuronal stress resistance. Here, we review emerging findings that reveal key roles for NAD+ and related metabolites in the adaptation of neurons to a wide range of physiological stressors and in counteracting processes in neurodegenerative diseases, such as those occurring in Alzheimer’s, Parkinson’s, and Huntington diseases, and amyotrophic lateral sclerosis. Advances in understanding the molecular and cellular mechanisms of NAD+-based neuronal resilience will lead to novel approaches for facilitating healthy brain aging and for the treatment of a range of neurological disorders.
June 16, 2024
Love0

Metformin reverses TRAP1 mutation-associated alterations in mitochondrial function in Parkinson’s disease

By NuSchild Research

Abstract

The mitochondrial proteins TRAP1 and HTRA2 have previously been shown to be phosphorylated in the presence of the Parkinson’s disease kinase PINK1 but the downstream signalling is unknown. HTRA2 and PINK1 loss of function causes parkinsonism in humans and animals. Here, we identified TRAP1 as an interactor of HTRA2 using an unbiased mass spectrometry approach. In our human cell models, TRAP1 overexpression is protective, rescuing HTRA2 and PINK1-associated mitochondrial dysfunction and suggesting that TRAP1 acts downstream of HTRA2 and PINK1. HTRA2 regulates TRAP1 protein levels, but TRAP1 is not a direct target of HTRA2 protease activity. Following genetic screening of Parkinson’s disease patients and healthy controls, we also report the first TRAP1 mutation leading to complete loss of functional protein in a patient with late onset Parkinson’s disease. Analysis of fibroblasts derived from the patient reveal that oxygen consumption, ATP output and reactive oxygen species are increased compared to healthy individuals. This is coupled with an increased pool of free NADH, increased mitochondrial biogenesis, triggering of the mitochondrial unfolded protein response, loss of mitochondrial membrane potential and sensitivity to mitochondrial removal and apoptosis. These data highlight the role of TRAP1 in the regulation of energy metabolism and mitochondrial quality control. Interestingly, the diabetes drug metformin reverses mutation-associated alterations on energy metabolism, mitochondrial biogenesis and restores mitochondrial membrane potential. In summary, our data show that TRAP1 acts downstream of PINK1 and HTRA2 for mitochondrial fine tuning, whereas TRAP1 loss of function leads to reduced control of energy metabolism, ultimately impacting mitochondrial membrane potential. These findings offer new insight into mitochondrial pathologies in Parkinson’s disease and provide new prospects for targeted therapies.

June 16, 2024
Love0

Nicotinamide riboside kinases display redundancy in mediating nicotinamide mononucleotide and nicotinamide riboside metabolism in skeletal muscle cells

By NuSchild Research

Abstract

Objective

Augmenting nicotinamide adenine dinucleotide (NAD+) availability may protect skeletal muscle from age-related metabolic decline. Dietary supplementation of NAD+ precursors nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) appear efficacious in elevating muscle NAD+. Here we sought to identify the pathways skeletal muscle cells utilize to synthesize NAD+ from NMN and NR and provide insight into mechanisms of muscle metabolic homeostasis.

Methods

We exploited expression profiling of muscle NAD+ biosynthetic pathways, single and double nicotinamide riboside kinase 1/2 (NRK1/2) loss-of-function mice, and pharmacological inhibition of muscle NAD+ recycling to evaluate NMN and NR utilization.

Results

Skeletal muscle cells primarily rely on nicotinamide phosphoribosyltransferase (NAMPT), NRK1, and NRK2 for salvage biosynthesis of NAD+. NAMPT inhibition depletes muscle NAD+ availability and can be rescued by NR and NMN as the preferred precursors for elevating muscle cell NAD+ in a pathway that depends on NRK1 and NRK2. Nrk2 knockout mice develop normally and show subtle alterations to their NAD+ metabolome and expression of related genes. NRK1, NRK2, and double KO myotubes revealed redundancy in the NRK dependent metabolism of NR to NAD+. Significantly, these models revealed that NMN supplementation is also dependent upon NRK activity to enhance NAD+ availability.
June 16, 2024
Love0

SARM1 activation triggers axon degeneration locally via NAD+ destruction

By NuSchild Research

SARM1-driven axon degeneration

Axons, the long protrusions of nerve cells, are programmed to self-destruct under certain conditions that occur during development, stress, or disease states. Gerdts et al. outline a biochemical mechanism that controls such axon degeneration. The authors designed versions of SARM1 (sterile alpha and TIR motif—constraining 1) that could be activated or inhibited in cells. Their experiments showed that the activation of SARM1 was necessary and sufficient to cause axon destruction in cultured mouse neurons. SARM1-mediated destruction was associated with depletion of the metabolic cofactor NAD+ from cells.

Abstract

Axon degeneration is an intrinsic self-destruction program that underlies axon loss during injury and disease. Sterile alpha and TIR motif–containing 1 (SARM1) protein is an essential mediator of axon degeneration. We report that SARM1 initiates a local destruction program involving rapid breakdown of nicotinamide adenine dinucleotide (NAD+) after injury. We used an engineered protease-sensitized SARM1 to demonstrate that SARM1 activity is required after axon injury to induce axon degeneration. Dimerization of the Toll–interleukin receptor (TIR) domain of SARM1 alone was sufficient to induce locally mediated axon degeneration. Formation of the SARM1 TIR dimer triggered rapid breakdown of NAD+, whereas SARM1-induced axon destruction could be counteracted by increased NAD+ synthesis. SARM1-induced depletion of NAD+ may explain the potent axon protection in Wallerian degeneration slow (Wlds) mutant mice.
June 16, 2024
Love0

CD73 Protein as a Source of Extracellular Precursors for Sustained NAD+ Biosynthesis in FK866-treated Tumor Cells

By NuSchild Research

Abstract

NAD+ is mainly synthesized in human cells via the “salvage” pathways starting from nicotinamide, nicotinic acid, or nicotinamide riboside (NR). The inhibition with FK866 of the enzyme nicotinamide phosphoribosyltransferase (NAMPT), catalyzing the first reaction in the “salvage” pathway from nicotinamide, showed potent antitumor activity in several preclinical models of solid and hematologic cancers. In the clinical studies performed with FK866, however, no tumor remission was observed. Here we demonstrate that low micromolar concentrations of extracellular NAD+ or NAD+ precursors, nicotinamide mononucleotide (NMN) and NR, can reverse the FK866-induced cell death, this representing a plausible explanation for the failure of NAMPT inhibition as an anti-cancer therapy. NMN is a substrate of both ectoenzymes CD38 and CD73, with generation of NAM and NR, respectively. In this study, we investigated the roles of CD38 and CD73 in providing ectocellular NAD+ precursors for NAD+ biosynthesis and in modulating cell susceptibility to FK866. By specifically silencing or overexpressing CD38 and CD73, we demonstrated that endogenous CD73 enables, whereas CD38 impairs, the conversion of extracellular NMN to NR as a precursor for intracellular NAD+ biosynthesis in human cells. Moreover, cell viability in FK866-treated cells supplemented with extracellular NMN was strongly reduced in tumor cells, upon pharmacological inhibition or specific down-regulation of CD73. Thus, our study suggests that genetic or pharmacologic interventions interfering with CD73 activity may prove useful to increase cancer cell sensitivity to NAMPT inhibitors.
June 16, 2024
Love0

Enhancing NAD+ Salvage Pathway Reverts the Toxicity of Primary Astrocytes Expressing Amyotrophic Lateral Sclerosis-linked Mutant Superoxide Dismutase 1 (SOD1)

By NuSchild Research

Abstract

Nicotinamide adenine dinucleotide (NAD+) participates in redox reactions and NAD+-dependent signaling pathways. Although the redox reactions are critical for efficient mitochondrial metabolism, they are not accompanied by any net consumption of the nucleotide. On the contrary, NAD+-dependent signaling processes lead to its degradation. Three distinct families of enzymes consume NAD+ as substrate: poly(ADP-ribose) polymerases, ADP-ribosyl cyclases (CD38 and CD157), and sirtuins (SIRT1–7). Because all of the above enzymes generate nicotinamide as a byproduct, mammalian cells have evolved an NAD+ salvage pathway capable of resynthesizing NAD+ from nicotinamide. Overexpression of the rate-limiting enzyme in this pathway, nicotinamide phosphoribosyltransferase, increases total and mitochondrial NAD+ levels in astrocytes. Moreover, targeting nicotinamide phosphoribosyltransferase to the mitochondria also enhances NAD+ salvage pathway in astrocytes. Supplementation with the NAD+ precursors nicotinamide mononucleotide and nicotinamide riboside also increases NAD+ levels in astrocytes. Amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Superoxide dismutase 1 (SOD1) mutations account for up to 20% of familial ALS and 1–2% of apparently sporadic ALS cases. Primary astrocytes isolated from mutant human superoxide dismutase 1-overexpressing mice as well as human post-mortem ALS spinal cord-derived astrocytes induce motor neuron death in co-culture. Increasing total and mitochondrial NAD+ content in ALS astrocytes increases oxidative stress resistance and reverts their toxicity toward co-cultured motor neurons. Taken together, our results suggest that enhancing the NAD+ salvage pathway in astrocytes could be a potential therapeutic target to prevent astrocyte-mediated motor neuron death in ALS.
June 16, 2024
Love0

Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase

By NuSchild Research

Abstract

Yeast Sir2 is a heterochromatin component that silences transcription at silent mating loci1, telomeres2 and the ribosomal DNA3,4, and that also suppresses recombination in the rDNA5 and extends replicative life span6. Mutational studies indicate that lysine 16 in the amino-terminal tail of histone H4 and lysines 9, 14 and 18 in H3 are critically important in silencing, whereas lysines 5, 8 and 12 of H4 have more redundant functions7,8,9. Lysines 9 and 14 of histone H3 and lysines 5, 8 and 16 of H4 are acetylated in active chromatin and hypoacetylated in silenced chromatin, and overexpression of Sir2 promotes global deacetylation of histones9,10, indicating that Sir2 may be a histone deacetylase. Deacetylation of lysine 16 of H4 is necessary for binding the silencing protein, Sir3 (ref. 8). Here we show that yeast and mouse Sir2 proteins are nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylases, which deacetylate lysines 9 and 14 of H3 and specifically lysine 16 of H4. Our analysis of two SIR2 mutations supports the idea that this deacetylase activity accounts for silencing, recombination suppression and extension of life span in vivo. These findings provide a molecular framework of NAD-dependent histone deacetylation that connects metabolism, genomic silencing and ageing in yeast and, perhaps, in higher eukaryotes.

June 16, 2024
Love0

CaMKII regulates proteasome phosphorylation and activity and promotes memory destabilization following retrieval

By NuSchild Research

Abstract

Numerous studies have suggested that memories “destabilize” and require de novo protein synthesis in order to reconsolidate following retrieval, but very little is known about how this destabilization process is regulated. Recently, ubiquitin–proteasome mediated protein degradation has been identified as a critical regulator of memory trace destabilization following retrieval, though the specific mechanisms controlling retrieval-induced changes in ubiquitin–proteasome activity remain equivocal. Here, we found that proteasome activity is increased in the amygdala in a CaMKII-dependent manner following the retrieval of a contextual fear memory. We show that in vitro inhibition of CaMKII reversed retrieval-induced increases in proteasome activity. Additionally, in vivo pharmacological blockade of CaMKII abolished increases in proteolytic activity and activity related regulatory phosphorylation in the amygdala following retrieval, suggesting that CaMKII was “upstream” of protein degradation during the memory reconsolidation process. Consistent with this, while inhibiting CaMKII in the amygdala did not impair memory following retrieval, it completely attenuated the memory impairments that resulted from post-retrieval protein synthesis blockade. Collectively, these results suggest that CaMKII controls the initiation of the memory reconsolidation process through regulation of the proteasome.

Introduction

The formation of long-term fear memories requires de novo gene transcription and protein translation in neurons during memory consolidation (Johansen et al., 2011, McGaugh, 2000). While once thought to be permanent, it is now widely supported that upon retrieval once consolidated memories “destabilize” and require new protein synthesis in order to “restabilize”, a process referred to as memory reconsolidation (Alberini and Ledoux, 2013, Nader et al., 2000, Tronson and Taylor, 2007). This reconsolidation process is thought to be dynamic, allowing modification of previously formed memories. Consistent with this, numerous studies have shown that reconsolidation can strengthen, weaken or change the specific content of a memory (De Oliveira Alvares et al., 2013, Inda et al., 2011, Lee, 2008, Lee, 2010, Monfils et al., 2009, Schiller et al., 2010, Sierra et al., 2013), which highlights the therapeutic potential of the reconsolidation process in alleviating fear associated with traumatic memories.
While most studies have focused on the mechanisms that regulate the restabilization or protein synthesis-dependent phase of the reconsolidation process, few have examined the mechanisms that regulate memory trace destabilization. NMDA receptor activation appears to initiate the destabilization process as inhibition of NMDA receptor activity in the amygdala prior to retrieval prevents the memory impairments that result from post-retrieval administration of the protein synthesis inhibitor anisomycin (Ben Mamou et al., 2006, Lopez et al., 2015, Wang et al., 2009). Downstream of NMDA receptors, ubiquitin–proteasome mediated protein degradation has been consistently implicated as a critical regulator of memory trace destabilization since blocking functional proteasome activity prevents memories from undergoing reconsolidation and can attenuate reconsolidation-dependent memory modification (Jarome et al., 2011, Lee, 2008, Lee et al., 2008). However, though NMDA receptor activity can result in increased proteasome activity in vitro and in vivo (Bingol and Schuman, 2006, Jarome et al., 2011), it is hypothesized that this occurs through a second messenger and not as a direct result of calcium influx (Jarome & Helmstetter, 2013). To date, the molecule(s) that links NMDA receptor activation to protein degradation during the destabilization process remains equivocal.
One molecule that is directly activated by increased intracellular calcium levels is the calcium–calmodulin dependent protein kinase II (CaMKII), which has well described roles in the memory consolidation process (Bejar et al., 2002, Mayford et al., 1996, Rodrigues et al., 2004, Yasuda and Mayford, 2006). Interestingly, the role of CaMKII in the reconsolidation of fear memories has never been examined. Additionally, studies examining the role of CaMKII in the reconsolidation of memory for other behavioral tasks have found mixed results, with some indicating normal memory retention following post-retrieval inhibition of CaMKII signaling (Arguello et al., 2014, Da Silva et al., 2013, Sakurai et al., 2007). One intriguing explanation for these mixed results is that CaMKII regulates protein degradation upstream of its potential (but not proven) regulation of protein synthesis during the reconsolidation process (Jarome & Helmstetter, 2013). Consistent with this, CaMKII can regulate proteasome activity and phosphorylation in vitro and in vivo (Bingol et al., 2010, Djakovic et al., 2009, Djakovic et al., 2012, Hamilton et al., 2012, Jarome et al., 2013), though this relationship has never been examined during memory reconsolidation. Here, using a combination of biochemical, pharmacological and behavioral approaches, we directly tested whether CaMKII controls memory trace destabilization through its regulation of the proteasome.
June 16, 2024
Love0

Acetylation Targets Mutant Huntingtin to Autophagosomes for Degradation

By NuSchild Research

Summary

Huntington’s disease (HD) is an incurable neurodegenerative disease caused by neuronal accumulation of the mutant protein huntingtin. Improving clearance of the mutant protein is expected to prevent cellular dysfunction and neurodegeneration in HD. We report here that such clearance can be achieved by posttranslational modification of the mutant Huntingtin (Htt) by acetylation at lysine residue 444 (K444). Increased acetylation at K444 facilitates trafficking of mutant Htt into autophagosomes, significantly improves clearance of the mutant protein by macroautophagy, and reverses the toxic effects of mutant huntingtin in primary striatal and cortical neurons and in a transgenic C. elegans model of HD. In contrast, mutant Htt that is rendered resistant to acetylation dramatically accumulates and leads to neurodegeneration in cultured neurons and in mouse brain. These studies identify acetylation as a mechanism for removing accumulated protein in HD, and more broadly for actively targeting proteins for degradation by autophagy.

Keyword

  1. MOLNEURO

Introduction

Accumulation and aggregation of mutant proteins is a hallmark of several neurodegenerative disorders such as Parkinson’s, Alzheimer’s, and Huntington’s disease (HD) (). One of the major therapeutic challenges in the field of neurodegeneration has been to improve the degradation of accumulated mutant proteins. While the ubiquitin-proteosome system (UPS) represents an important defense against abnormal protein accumulation, aggregation-prone proteins appear to be poor substrates for proteosomal degradation and better targets for autophagic-lysosomal degradation (). In terms of the mode of cargo delivery to the lysosome, three forms of autophagic degradation have been described so far—microautophagy, chaperone-mediated autophagy, and macroautophagy (). This latter form, whereby cytosolic constituents and organelles are engulfed by multilamellar vesicles which then fuse to the lysosome, has been implicated in a wide array of neurological disorders including HD ().
Huntington’s disease is a devastating neurodegenerative disorder characterized by progressive and severe motor and cognitive impairment; death ensues about 15 years after the onset of symptoms (). The mutation is inherited as autosomal dominant and causes expansion of a stretch of glutamines near the N terminus of huntingtin, a protein of uncertain function whose mutant form accumulates as nuclear and cytoplasmic inclusions in HD brain (). In a conditional mouse model of HD, it was found that elimination of mutant Huntingtin (Htt) expression not only halted symptomatic progression but also led to regression of the disease-like symptoms (). Initial experiments in human HD brain found aberrant accumulation of huntingtin in late endosomal structures, suggesting dependence on autophagy (). Recent findings showed that activation of autophagy by systemic administration of rapamycin may be sufficient to partially ameliorate symptoms in an HD mouse model (). While these and other studies demonstrate neuroprotection by the inhibition of the ubiquitous protein kinase mTOR and nonspecific activation of autophagy, it remains unclear whether autophagy can be selectively activated in order to remove disease proteins of interest.
In this study, we demonstrate a link between acetylation of a nonhistone protein and targeted degradation by autophagy. Modification of mutant huntingtin by acetylation promotes its targeting into autophagosomes and facilitates specific degradation of the mutant protein by the autophagic-lysosomal pathway. Furthermore, we show that acetylation and clearance of mutant huntingtin leads to neuroprotection in primary neurons and a transgenic C. elegans model of HD, highlighting the importance of selective targeting of disease proteins to autophagosomes for degradation.
June 16, 2024
Love0

CD38 is critical for social behaviour by regulating oxytocin secretion

By NuSchild Research

Abstract

CD38, a transmembrane glycoprotein with ADP-ribosyl cyclase activity, catalyses the formation of Ca2+ signalling molecules, but its role in the neuroendocrine system is unknown. Here we show that adult CD38 knockout (CD38-/-) female and male mice show marked defects in maternal nurturing and social behaviour, respectively, with higher locomotor activity. Consistently, the plasma level of oxytocin (OT), but not vasopressin, was strongly decreased in CD38-/- mice. Replacement of OT by subcutaneous injection or lentiviral-vector-mediated delivery of human CD38 in the hypothalamus rescued social memory and maternal care in CD38-/- mice. Depolarization-induced OT secretion and Ca2+ elevation in oxytocinergic neurohypophysial axon terminals were disrupted in CD38-/- mice; this was mimicked by CD38 metabolite antagonists in CD38+/+ mice. These results reveal that CD38 has a key role in neuropeptide release, thereby critically regulating maternal and social behaviours, and may be an element in neurodevelopmental disorders.

June 15, 2024
Love0