The TRRAP-HAT-Sp1 axis maintains brain homeostasis

The homeostasis of the brain is tightly controlled by the viability and functionality of various cell types, including neurons and glial cells, like oligodendrocytes, astrocytes as well as microglia. Defects of neurogenesis and maintenance of neural cells are associated with multiple neuropathologies, such as Intellectual Disability (ID) and Autism Spectrum Disorders (ASD) among other diseases. HAT and HDAC modulate brain functionality, e.g. memory formation, cognitive function, and neuroprotection, whereas the disturbance of the acetylation profiles has been related to multiple neuropathological diseases. However, how epigenetic regulation participates in the neurodevelopmental, neural differentiation and neurodegenerative processes remains largely unknown. In our studies, we have chosen the HAT adaptor, Trrap, to investigate how the disturbance of acetylation would affect brain functionality. We show that Trrap deletion in post-mitotic neurons results in neurodegeneration. In addition, Trrap deficiency in adult neural stem cells compromises their self-renewal and differentiation. With integrated transcriptomics, epigenomics, and proteomics we identify Sp1 as the master regulator controlled by Trrap-HAT and demonstrate that the Trrap-HAT-Sp1 axis ensures the proper expression of genes involved in microtubule dynamics. We find that Trrap mediates Sp1 binding through the maintenance of the acetylation profile on Sp1 and that acetylation of Sp1 plays an important role, dependent and independent of Trrap, in its transcription activation. Taken together, we demonstrate that Trrap, through its mediated acetylation, is involved in neuroprotection and neural differentiation via the regulation of Sp1 activity. My dissertation provides a novel insight into the role of epigenetic regulation of transcription factors in the maintenance of brain homeostasis and preventing neurodegeneration

EEF1A1 Deacetylation Enables Transcriptional Activation of Remyelination

Remyelination of the peripheral and central nervous systems (PNS and CNS, respectively) is a prerequisite for functional recovery after lesion. However, this process is not always optimal and becomes inefficient in the course of multiple sclerosis. Here we show that, when acetylated, eukaryotic elongation factor 1A1 (eEF1A1) negatively regulates PNS and CNS remyelination. Acetylated eEF1A1 (Ac- eEF1A1) translocates into the nucleus of myelinating cells where it binds to Sox10, a key transcription factor for PNS and CNS myelination and remyelination, to drag Sox10 out of the nucleus. We show that the lysine acetyltransferase Tip60 acetylates eEF1A1, whereas the histone deacetylase HDAC2 deacetylates eEF1A1. Promoting eEF1A1 deacetylation maintains the activation of Sox10 target genes and increases PNS and CNS remyelination efficiency. Taken together, these data identify a major mechanism of Sox10 regulation, which appears promising for future translational studies on PNS and CNS remyelination

Investigating the role of FUS, TDP-43 and DYNC1H1 mutations in the etiology of adult and childhood-onset motor neuron disease

Amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy with lower extremity predominance (SMA-LED) represent two distinctive types of motor neuron disease, the first of which ALS manifests usually as an adult-onset, rapidly progressive, terminal neurodegenerative disorder. Conversely, SMA-LED a childhood-onset disease is mainly characterised by lower limb wasting and intermittently compounded by intellectual disability. Firstly, this thesis represents investigations in the roles of both Fused in Sarcoma (FUS) and TAR-DNA binding protein (TDP-43), two mutated proteins in ALS. Secondly, this thesis explores the implications of SMA-LED mutations in the cytoplasmic dynein heavy chain (DYNC1H1) to investigate disease pathogenesis. Mutations found in RNA binding proteins FUS and TDP-43 are pathogenic in the adult-onset form of motor neuron disease ALS. These proteins are known to interact with DNA and it is suggested in the literature that FUS in particular has a protective role to repair DNA after lesions. FUS was shown to localise to sub-nuclear regions of UVA induced oxidative damage as well as nucleoli foci after induction of DNA single strand breaks through RNA polymerase II inhibition in mitotic cells and neurons. This re-localisation was inhibited by both caffeine and dipyridamole indicating FUS recruitment via phophodiesterases (PDEs). TDP-43 was found to vacate regions of oxidative damage and showed neuronal specific re-localisation to nucleoli in response to polymerase II inhibition that was not observed in mitotic cells. These data for the first time show that both FUS and TDP-43 re-localise after DNA damage events. FUS is shown to respond to transcriptional stress perhaps to protect nascent RNA, whereas the expulsion of TDP-43 may indicate a role in transcription coupled excision repair or heterochromatin remodelling. The SMA-LED autosomal dominant DYNC1H1 mutation p.R399G causes lower limb weakness and muscle atrophy as well as cognitive impairment but the pathogenesis remains unknown. p.R399G mutant fibroblasts exhibit a fragmented Golgi apparatus phenotype rescuable by HDAC6 inhibition in conjunction with a reduced localisation of the dynein complex to the Golgi membranes. Furthermore, western blot and immunoprecipitation assays showed decreased acetylation in mutant fibroblasts as well as an increased interaction between dynein and golgin160 in p.R399G mutants. These data for the first time show that mutations in dynein can modulate acetylation of microtubules and caffect dynein recruitment to the Golgi apparatus. This suggests a novel mechanism in which perturbed dynein-mediated regulation of microtubule acetylation and dynein-Golgi interaction underpin SMA-LED. Finally, the Dync1h1 mutant Arl SMA-LED mouse model recapitulates phenotypes of the human disease and presents with motor phenotypes. However, this mouse model is yet to be fully characterised and the extent of any aberrant brain development is unknown. Analysis of the model indicated defective cortical organisation in addition to defective cell migration seen in Arl/+ fibroblasts. Furthermore, collapse of the third ventricle and condensation of the corpus callosum was also observed in this model. These data show that the Arl p.Trp1206Arg mutation in the tail domain of Dync1h1 results in defective cellular migration as well as gross morphological brain deficits that mirror malformation of cortical development (MCD) in patients with SMA-LED. This is likely related to microtubule instability, Golgi abnormalities or impaired force production of dynein leading to suppressed cell migration and neuronal arborisation

The Caenorhabditis elegans Elongator Complex Regulates Neuronal α-tubulin Acetylation

Although acetylated α-tubulin is known to be a marker of stable microtubules in neurons, precise factors that regulate α-tubulin acetylation are, to date, largely unknown. Therefore, a genetic screen was employed in the nematode Caenorhabditis elegans that identified the Elongator complex as a possible regulator of α-tubulin acetylation. Detailed characterization of mutant animals revealed that the acetyltransferase activity of the Elongator is indeed required for correct acetylation of microtubules and for neuronal development. Moreover, the velocity of vesicles on microtubules was affected by mutations in Elongator. Elongator mutants also displayed defects in neurotransmitter levels. Furthermore, acetylation of α-tubulin was shown to act as a novel signal for the fine-tuning of microtubules dynamics by modulating α-tubulin turnover, which in turn affected neuronal shape. Given that mutations in the acetyltransferase subunit of the Elongator (Elp3) and in a scaffold subunit (Elp1) have previously been linked to human neurodegenerative diseases, namely Amyotrophic Lateral Sclerosis and Familial Dysautonomia respectively highlights the importance of this work and offers new insights to understand their etiology

Sirtuin2 in the CNS: Expression, functional roles, action mechanism and mutation-induced alteration of molecular /cell biological properties

Ph.DDOCTOR OF PHILOSOPH

The balance between NAD+ biosynthesis and consumption in ageing

Nicotinamide adenine dinucleotide (NAD+) is a vital coenzyme in redox reactions. NAD+ is also important in cellular signalling as it is consumed by PARPs, SARM1, sirtuins and CD38. Cellular NAD+ levels regulate several essential processes including DNA repair, immune cell function, senescence, and chromatin remodelling. Maintenance of these cellular processes is important for healthy ageing and lifespan. Interestingly, the levels of NAD+ decline during ageing in several organisms, including humans. Declining NAD+ levels have been linked to several age-related diseases including various metabolic diseases and cognitive decline. Decreasing tissue NAD+ concentrations have been ascribed to an imbalance between biosynthesis and consumption of the dinucleotide, resulting from, for instance, reduced levels of the rate limiting enzyme NAMPT along with an increased activation state of the NAD+-consuming enzymes PARPs and CD38. The progression of some age-related diseases can be halted or reversed by therapeutic augmentation of NAD+ levels. NAD+ metabolism has therefore emerged as a potential target to ameliorate age-related diseases. The present review explores how ageing affects NAD+ metabolism and current approaches to reverse the age-dependent decline of NAD+.publishedVersio

Small Changes Huge Impact: The Role of Protein Posttranslational Modifications in Cellular Homeostasis and Disease

Posttranslational modifications (PTMs) modulate protein function in most eukaryotes and have a ubiquitous role in diverse range of cellular functions. Identification, characterization, and mapping of these modifications to specific amino acid residues on proteins are critical towards understanding their functional significance in a biological context. The interpretation of proteome data obtained from the high-throughput methods cannot be deciphered unambiguously without a priori knowledge of protein modifications. An in-depth understanding of protein PTMs is important not only for gaining a perception of a wide array of cellular functions but also towards developing drug therapies for many life-threatening diseases like cancer and neurodegenerative disorders. Many of the protein modifications like ubiquitination play a decisive role in various drug response(s) and eventually in disease prognosis. Thus, many commonly observed PTMs are routinely tracked as disease markers while many others are used as molecular targets for developing target-specific therapies. In this paper, we summarize some of the major, well-studied protein alterations and highlight their importance in various chronic diseases and normal development. In addition, other promising minor modifications such as SUMOylation, observed to impact cellular dynamics as well as disease pathology, are mentioned briefly

Novel HDAC6 inhibitors increase tubulin acetylation and rescue axonal transport of mitochondria in a model of Charcot-Marie-Tooth Type 2F

Disruption of axonal transport causes a number of rare, inherited axonopathies and is heavily implicated in a wide range of more common neurodegenerative disorders, many of them age- related. Acetylation of α-tubulin is one important regulatory mechanism, influencing microtubule stability and motor protein attachment. Of several strategies so far used to enhance axonal transport, increasing microtubule acetylation through inhibition of the deacetylase enzyme HDAC6 has been one of the most effective. Several inhibitors have been developed and tested in animal and cellular models but better drug candidates are still needed. Here we report the development and characterisation of two highly potent HDAC6 inhibitors, which show low toxicity, promising pharmacokinetic properties, and enhance microtubule acetylation in the nanomolar range. We demonstrate their capacity to rescue axonal transport of mitochondria in a primary neuronal culture model of the inherited axonopathy Charcot- Marie-Tooth Type 2F, caused by a dominantly acting mutation in heat shock protein beta 1.Funding for this work was provided by Takeda Development Centre Europe Ltd. M.P.C. is funded by the John and Lucille van Geest Foundation

Lysine deacetylases and mitochondrial dynamics in neurodegeneration

Lysine acetylation is a key post-translational modification known to regulate gene transcription, signal transduction, cellular transport and metabolism. Lysine deacetylases (KDACs), including classical KDACs (a.k.a. histone deacetylases; HDACs) and sirtuins (SIRTs), are emerging therapeutic targets in neurodegeneration. Given the strong link between abnormal mitochondrial dynamics and neurodegenerative disorders (e.g. in Alzheimer, Parkinson and Huntington diseases), here we examine the evidence for KDAC-mediated regulation of mitochondrial biogenesis, fission-fusion, movement and mitophagy. Mitochondrial biogenesis regulation was reported for SIRT1, SIRT3, and class IIa KDACs, mainly via PGC-1alpha modulation. SIRT1 or SIRT3 overexpression rescued mitochondrial density and fission-fusion balance in neurodegeneration models. Mitochondrial fission decreased with pan-classical-KDAC inhibitors and increased with nicotinamide (pan-sirtuin-inhibitor/activator depending on concentration and NAD(+) conversion). Mitochondrial movement increased with HDAC6 inhibition, but this is not yet reported for the other tubulin deacetylase SIRT2. Inhibition of HDAC6 or SIRT2 was reported neuroprotective. Mitophagy is assisted by the HDAC6 ubiquitin-binding and autophagosome-lysosome fusion promoting activities, and was also associated with SIRT1 activation. In summary, KDACs can potentially modulate multiple components of mitochondrial dynamics, however, several key points require clarification. The SIRT1-biogenesis connection relies heavily in controversial caloric restriction (CR) regimes or CR-mimetic drugs, and appears cell-type dependent, recommending caution before linking SIRT1 activation with general neuroprotection. Future studies should clarify mitochondrial fission-fusion regulation by KDACs, and the interplay between HDAC6 and SIRT1 in mitophagy. Also, further studies are required to ascertain whether HDAC6 inhibition to enhance mitochondrial trafficking does not compromise autophagy or clearance of misfolded proteins in neurodegenerative disorders