XRCC1 is a molecular scaffold protein that assembles multi-protein complexes involved in DNA single-strand break repair1,2. Here, we show that biallelic mutations in human XRCC1 are associated with ocular motor apraxia, axonal neuropathy, and progressive cerebellar ataxia. XRCC1-mutant patient cells exhibit not only reduced rates of single-strand break repair but also elevated levels of protein ADP-ribosylation; a phenotype recapitulated in a related syndrome caused by mutations in the XRCC1 partner protein PNKP3-5 and implicating hyperactivation of poly (ADP-ribose) polymerase/s as a cause of cerebellar ataxia. Indeed, remarkably, genetic deletion of Parp1 rescued normal cerebellar ADP-ribose levels and reduced the loss of cerebellar neurons and ataxia in Xrcc1-defective mice, identifying a molecular mechanism by which endogenous single-strand breaks trigger neuropathology. Collectively, these data establish the importance of XRCC1 protein complexes for normal neurological function and identify PARP1 as a therapeutic target in DNA strand break repair-defective disease.

Cell migration is the consequence of the sum of positive and negative regulatory mechanisms. Although appropriate migration of neurons is a principal feature of brain development, the negative regulatory mechanisms remain obscure. We found that JNK1 was highly active in developing cortex and that selective inhibition of JNK in the cytoplasm markedly increased both the frequency of exit from the multipolar stage and radial migration rate and ultimately led to an ill-defined cellular organization. Moreover, regulation of multipolar-stage exit and radial migration in Jnk1(-/-) (also known as Mapk8) mice, resulted from consequential changes in phosphorylation of the microtubule regulator SCG10 (also called stathmin-2). Expression of an SCG10 mutant that mimics the JNK1-phosphorylated form restored normal migration in the brains of Jnk1(-/-) mouse embryos. These findings indicate that the phosphorylation of SCG10 by JNK1 is a fundamental mechanism that governs the transition from the multipolar stage and the rate of neuronal cell movement during cortical development.

f Cell migration is a fundamental biological function, critical during development and regeneration, whereas deregulated migration underlies neurological birth defects and cancer metastasis. MARCKS-like protein 1 (MARCKSL1) is widely expressed in nervous tissue, where, like Jun N-terminal protein kinase (JNK), it is required for neural tube formation, though the mechanism is unknown. Here we show that MARCKSL1 is directly phosphorylated by JNK on C-terminal residues (S120, T148, and T183). This phosphorylation enables MARCKSL1 to bundle and stabilize F-actin, increase filopodium numbers and dynamics, and retard migration in neurons. Conversely, when MARCKSL1 phosphorylation is inhibited, actin mobility increases and filopodium formation is compromised whereas lamellipodium formation is enhanced, as is cell migration. We find that MARCKSL1 mRNA is upregulated in a broad range of cancer types and that MARCKSL1 protein is strongly induced in primary prostate carcinomas. Gene knockdown in prostate cancer cells or in neurons reveals a critical role for MARCKSL1 in migration that is dependent on the phosphorylation state; phosphomimetic MARCKSL1 (MARCKSL1 S120D,T148D,T183D ) inhibits whereas dephospho-MARCKSL1 S120A,T148A,T183A induces migration. In summary, these data show that JNK phosphorylation of MARCKSL1 regulates actin homeostasis, filopodium and lamellipodium formation, and neuronal migration under physiological conditions and that, when ectopically expressed in prostate cancer cells, MARCKSL1 again determines cell movement. M ARCKS-like protein 1 (MARCKSL1) is an actin binding protein that is predominantly expressed in immature brain (1, 27). The MARCKSL1 homologue MARCKS has been more extensively studied and has been shown to bind actin with a stoichiometry of 1:2, thereby facilitating cross-linking (56; reviewed in reference 39). Binding to actin occurs via an effector domain (ED) that is 87% identical to the corresponding domain of MARCKSL1. Surprisingly, however, full-length MARCKSL1 does not cross-link F-actin (49; reviewed in reference 39), although the MARCKSL1 effector domain alone interacts with actin. This indicates that in a physiological context, another level of regulation is required for MARCKSL1 to regulate actin bundling. The only known critical function of MARCKSL1 is in early development of the nervous system, as genetic disruption of MARCKSL1 results in neural tube closure defects (5, 51), events that depend on coordinated control of actin functions, cell shape, and cell migration. MARCKSL1 is also associated with cell spreading (23); however, the mechanism whereby MARCKSL1 regulates F-actin in a cellular context has remained obscure.c-Jun N-terminal kinase 1 (JNK1) and JNK2, like MARCKSL1, are required for neural tube closure (20,36). However, the identity of the JNK effectors mediating this event remains unresolved. JNK activity is highly elevated in neuronal cells (8,42), and although initially unexpected (7,8,53), it is now accepted that a number of important physiological substrates fo...

Promoting adult hippocampal neurogenesis is expected to induce neuroplastic changes that improve mood and alleviate anxiety. However, the underlying mechanisms remain largely unknown and the hypothesis itself is controversial. Here we show that mice lacking Jnk1, or c-Jun N-terminal kinase (JNK) inhibitor-treated mice, display increased neurogenesis in adult hippocampus characterized by enhanced cell proliferation and survival, and increased maturation in the ventral region. Correspondingly, anxiety behaviour is reduced in a battery of tests, except when neurogenesis is prevented by AraC treatment. Using engineered retroviruses, we show that exclusive inhibition of JNK in adult-born granule cells alleviates anxiety and reduces depressive-like behaviour. These data validate the neurogenesis hypothesis of anxiety. Moreover, they establish a causal role for JNK in the hippocampal neurogenic niche and anxiety behaviour, and advocate targeting of JNK as an avenue for novel therapies against affective disorders.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) is a 5′-tyrosyl DNA phosphodiesterase important for the repair of DNA adducts generated by non-productive (abortive) activity of topoisomerase II (TOP2). TDP2 facilitates therapeutic resistance to topoisomerase poisons, which are widely used in the treatment of a range of cancer types. Consequently, TDP2 is an interesting target for the development of small molecule inhibitors that could restore sensitivity to topoisomerase-directed therapies. Previous studies identified a class of deazaflavin-based molecules that showed inhibitory activity against TDP2 at therapeutically useful concentrations, but their mode of action was uncertain. We have confirmed that the deazaflavin series inhibits TDP2 enzyme activity in a fluorescence-based assay, suitable for high-throughput screen (HTS)-screening. We have gone on to determine crystal structures of these compounds bound to a ‘humanized’ form of murine TDP2. The structures reveal their novel mode of action as competitive ligands for the binding site of an incoming DNA substrate, and point the way to generating novel and potent inhibitors of TDP2.

Genetic anomalies on the JNK pathway confer susceptibility to autism spectrum disorders, schizophrenia, and intellectual disability. The mechanism whereby a gain or loss of function in JNK signaling predisposes to these prevalent dendrite disorders, with associated motor dysfunction, remains unclear. Here we find that JNK1 regulates the dendritic field of L2/3 and L5 pyramidal neurons of the mouse motor cortex (M1), the main excitatory pathway controlling voluntary movement. In Jnk1-/- mice, basal dendrite branching of L5 pyramidal neurons is increased in M1, as is cell soma size, whereas in L2/3, dendritic arborization is decreased. We show that JNK1 phosphorylates rat HMW-MAP2 on T1619, T1622, and T1625 (Uniprot P15146) corresponding to mouse T1617, T1620, T1623, to create a binding motif, that is critical for MAP2 interaction with and stabilization of microtubules, and dendrite growth control. Targeted expression in M1 of GFP-HMW-MAP2 that is pseudo-phosphorylated on T1619, T1622, and T1625 increases dendrite complexity in L2/3 indicating that JNK1 phosphorylation of HMW-MAP2 regulates the dendritic field. Consistent with the morphological changes observed in L2/3 and L5, Jnk1-/- mice exhibit deficits in limb placement and motor coordination, while stride length is reduced in older animals. In summary, JNK1 phosphorylates HMW-MAP2 to increase its stabilization of microtubules while at the same time controlling dendritic fields in the main excitatory pathway of M1. Moreover, JNK1 contributes to normal functioning of fine motor coordination. We report for the first time, a quantitative Sholl analysis of dendrite architecture, and of motor behavior in Jnk1-/- mice. Our results illustrate the molecular and behavioral consequences of interrupted JNK1 signaling and provide new ground for mechanistic understanding of those prevalent neuropyschiatric disorders where genetic disruption of the JNK pathway is central.

ABSTACT. In neuroscience, diffraction limited two-photon (2P) microscopy is a cornerstone technique that permits minimally invasive optical monitoring of neuronal activity. However, most conventional 2P microscopes impose significant constraints on the size of the imaging field-of-view and the specific shape of the effective excitation volume, thus limiting the scope of biological questions that can be addressed and the information obtainable. Here, employing 'divergent beam optics' (DBO), we present an ultra-low-cost, easily implemented and flexible solution to address these limitations, offering a several-fold expanded three-dimensional field of view that also maintains single-cell resolution. We show that this implementation increases both the space-bandwidth product and effective excitation power, and allows for straight-forward tailoring of the point-spread-function. Moreover, rapid laser-focus control via an electrically tunable lens now allows near-simultaneous imaging of remote regions separated in three dimensions and permits the bending of imaging planes to follow natural curvatures in biological structures. Crucially, our core design is readily implemented (and reversed) within a matter of hours, and fully compatible with a wide range of existing 2P customizations, making it highly suitable as a base platform for further development. We demonstrate the application of our system for imaging neuronal activity in a variety of examples in mice, zebrafish and fruit flies.

The protein kinase JNK1 exhibits high activity in the developing brain, where it regulates dendrite morphology through the phosphorylation of cytoskeletal regulatory proteins. JNK1 also phosphorylates dendritic spine proteins, and Jnk1-/- mice display a long-term depression deficit. Whether JNK1 or other JNKs regulate spine morphology is thus of interest. Here, we characterize dendritic spine morphology in hippocampus of mice lacking Jnk1-/- using Lucifer yellow labelling. We find that mushroom spines decrease and thin spines increase in apical dendrites of CA3 pyramidal neurons with no spine changes in basal dendrites or in CA1. Consistent with this spine deficit, Jnk1-/- mice display impaired acquisition learning in the Morris water maze. In hippocampal cultures, we show that cytosolic but not nuclear JNK, regulates spine morphology and expression of phosphomimicry variants of JNK substrates doublecortin (DCX) or myristoylated alanine-rich C kinase substrate-like protein-1 (MARCKSL1), rescue mushroom, thin, and stubby spines differentially. These data suggest that physiologically active JNK controls the equilibrium between mushroom, thin, and stubby spines via phosphorylation of distinct substrates.