Luca Murru

LRRK2 kinase activity regulates synaptic vesicle trafficking and neurotransmitter release through modulation of LRRK2 macro-molecular complex

Mutations in Leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinsons disease (PD). LRRK2 is a complex protein that consists of multiple domains executing several functions, including GTP hydrolysis, kinase activity, and protein binding. Robust evidence suggests that LRRK2 acts at the synaptic site as a molecular hub connecting synaptic vesicles to cytoskeletal elements via a complex panel of protein-protein interactions. Here we investigated the impact of pharmacological inhibition of LRRK2 kinase activity on synaptic function. Acute treatment with LRRK2 inhibitors reduced the frequency of spontaneous currents, the rate of synaptic vesicle trafficking and the release of neurotransmitter from isolated synaptosomes. The investigation of complementary models lacking LRRK2 expression allowed us to exclude potential off-side effects of kinase inhibitors on synaptic functions. Next we studied whether kinase inhibition affects LRRK2 heterologous interactions. We found that the binding among LRRK2, presynaptic proteins and synaptic vesicles is affected by kinase inhibition. Our results suggest that LRRK2 kinase activity influences synaptic vesicle release via modulation of LRRK2 macro-molecular complex.

The Neurobiology of X-Linked Intellectual Disability

X-linked intellectual disability (XLID) affects 1% to 3% of the population. XLID subsumes several heterogeneous conditions, all of which are marked by cognitive impairment and reduced adaptive skills. XLID arises from mutations on the X chromosome; to date, 102 XLID genes have been identified. The proteins encoded by XLID genes are involved in higher brain functions, such as cognition, learning and memory, and their molecular role is the subject of intense investigation. Here, we review recent findings concerning a representative group of XLID proteins: the fragile X mental retardation protein; methyl-CpG-binding protein 2 and cyclin-dependent kinase-like 5 proteins, which are involved in Rett syndrome; the intracellular signaling molecules of the Rho guanosine triphosphatases family; and the class of cell adhesion molecules. We discuss how XLID gene mutations affect the structure and function of synapses.

Myosin IXa binds AMPAR and regulates synaptic structure, LTP, and cognitive function

Myosin IXa (Myo9a) is a motor protein that is highly expressed in the brain. However, the role of Myo9a in neurons remains unknown. Here, we investigated Myo9a function in hippocampal synapses. In rat hippocampal neurons, Myo9a localizes to the postsynaptic density (PSD) and binds the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) GluA2 subunit. Myo9a+/- mice displayed a thicker PSD and increased levels of PSD95 and surface AMPAR expression. Furthermore, synaptic transmission, long-term potentiation (LTP) and cognitive functions were impaired in Myo9a+/- mice. Together, these results support a key role for Myo9a in controlling the molecular structure and function of hippocampal synapses.

EEF2K/eEF2 pathway controls the excitation/inhibition balance and susceptibility to epileptic seizures

Abstract Alterations in the balance of inhibitory and excitatory synaptic transmission have been implicated in the pathogenesis of neurological disorders such as epilepsy. Eukaryotic elongation factor 2 kinase (eEF2K) is a highly regulated, ubiquitous kinase involved in the control of protein translation. Here, we show that eEF2K activity negatively regulates GABAergic synaptic transmission. Indeed, loss of eEF2K increases GABAergic synaptic transmission by upregulating the presynaptic protein Synapsin 2b and &agr;5‐containing GABAA receptors and thus interferes with the excitation/inhibition balance. This cellular phenotype is accompanied by an increased resistance to epilepsy and an impairment of only a specific hippocampal‐dependent fear conditioning. From a clinical perspective, our results identify eEF2K as a potential novel target for antiepileptic drugs, since pharmacological and genetic inhibition of eEF2K can revert the epileptic phenotype in a mouse model of human epilepsy.

Glutamatergic synapses in neurodevelopmental disorders

ABSTRACT Neurodevelopmental disorders (NDDs) are a group of diseases whose symptoms arise during childhood or adolescence and that impact several higher cognitive functions such as learning, sociability and mood. Accruing evidence suggests that a shared pathogenic mechanism underlying these diseases is the dysfunction of glutamatergic synapses. We summarize present knowledge on autism spectrum disorders (ASD), intellectual disability (ID), Down syndrome (DS), Rett syndrome (RS) and attention‐deficit hyperactivity disorder (ADHD), highlighting the involvement of glutamatergic synapses and receptors in these disorders. The most commonly shared defects involve &agr;‐amino‐3‐hydroxy‐5‐methyl‐ 4‐isoxazole propionic acid receptors (AMPARs), N‐methyl‐d‐aspartate receptors (NMDARs) and metabotropic glutamate receptors (mGluRs), whose functions are strongly linked to synaptic plasticity, affecting both cell‐autonomous features as well as circuit formation. Moreover, the major scaffolding proteins and, thus, the general structure of the synapse are often deregulated in neurodevelopmental disorders, which is not surprising considering their crucial role in the regulation of glutamate receptor positioning and functioning. This convergence of defects supports the definition of neurodevelopmental disorders as a continuum of pathological manifestations, suggesting that glutamatergic synapses could be a therapeutic target to ameliorate patient symptomatology. Graphical abstract Core mechanisms involved in the pathogenesis of NDDs. ASD (Autism Spectrum Disorder): Neurexin and neuroligins are transmembrane proteins localised in the pre‐ and post‐synaptic compartments, respectively that interact each other in the synaptic cleft. Mutations in the gene encoding Neurexin‐1, Neuroligin‐3 and 4 have been found in patients affected by ASD. Neurexins regulate presynaptic vesicles release whereas neuroligins, through the interaction with PSD‐95, affect NMDARs functioning. SHANK3 protein belongs to a class of scaffolding molecules and it is implicated in autistic phenotype in patients affected by Phelan McDermid syndrome. This protein regulates different glutamate receptors (NMDARs, AMPARs and mGluR5) through direct or indirect interactions mediated by PSD‐95, SAPAP and Homer. Moreover, IGF‐1 pathway has been found associated to Shank3‐depletion defects. Mutations on the NMDARs subunit encoding genes GRIN2A and GRIN2B, are also directly affected by mutations in ASDs patients. ID (Intellectual Disability): FMRP protein, encoded by FMR1 gene, which mutations cause Fragile X syndrome, regulates mRNA translation of a great variety of proteins with crucial functions in excitatory post‐synapses such as STEP, PSD‐95 and APP. mGluR5 is strongly implicated in the regulation of FMRP functioning, and vice versa. OPHN1 and TSPAN7, which encoding genes are found mutated in patients affected by ID, are involved in the regulation of AMPARs trafficking. The MAGUK protein CASK, also affected in ID patients, has functions in both the pre and post‐synaptic compartments. In the first case it regulates Voltage Gated Calcium Channels (VGCC) and Neurexins; in the second it is implicated in NMDARs trafficking similarly to SAP102, another protein which encoding gene has been found mutated in ID patients. The presynaptic proteins Synaptophysin (SYP) and Synapsin (SYN) are both strongly implicated in the release of presynaptic vesicles containing the neurotransmitter glutamate and mutations in their coding genes have been linked to ID. DS (Down Syndrome): In DS patients the triplication of Chromosome 21 leads to different defects. The gene encoding for APP is localized in Chr 21 and its overexpression results in an Alzheimer‐like dementia. Nine genes contained in the chromosome 21 encode for proteins that interact with NMDARs directly or through the Calcineurin/Calmodulin (CaN/CaM) system. Moreover, neuronal and glial alterations in metabotropic glutamate receptors have been identified. RS (Rett Syndrome): MeCP2 protein is an epigenetic modulator. Studies on its function have elucidated its regulatory effect on PSD‐95, which appears to be reduced upon MeCP2 loss of function, and on GluA1‐containing AMPARs, which instead are increased. mGluRs have also been involved in RS; in particular, mGluR5‐dependent LTD has been found impaired in Mecp2 knock‐out mice. ADHD (Attention‐deficit hyperactivity disorder): ADHD is probably the most complex of NDDs with very little knowledge on specific mechanisms involving glutamate. Nowadays, several mutations on genes encoding for NMDARs subunits, GRIN2A and GRIN2B, and for mGluRs, GRM1, GRM5, GRM7 and GRM8, have been linked to this pathology. Figure. No caption available. HighlightsGlutamatergic synapse defects are common features in ASD, ID, DS, RS and ADHD.Shared NDDs pathological mechanisms converge on NMDARs and mGluRs.Different NDDs could be treated with similar therapeutic approaches.

New Role of ATM in Controlling GABAergic Tone During Development

The capacity to guarantee the proper excitatory/inhibitory balance is one of the most critical steps during early development responsible for the correct brain organization, function, and plasticity. GABAergic neurons guide this process leading to the right structural organization, brain circuitry, and neuronal firing. Here, we identified the ataxia telangiectasia mutated (ATM), a serine/threonine protein kinase linked to DNA damage response, as crucial in regulating neurotransmission. We found that reduced levels of ATM in the hippocampal neuronal cultures produce an excitatory/inhibitory unbalance toward inhibition as indicated by the higher frequency of miniature inhibitory postsynaptic current events and an increased number of GABAergic synapses. In vivo, the increased inhibition still persists and, even if a higher excitation is also present, a reduced neuronal excitability is found as indicated by the lower action potential frequency generated in response to high-current intensity stimuli. Finally, we found an elevated extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in heterozygous hippocampi associated with lower expression levels of the ERK1/2 phosphatase PP1. Given that the neurodegenerative condition associated with genetic mutations in the Atm gene, ataxia telangiectasia, presents a variable phenotype with impairment in cognition, our molecular findings provide a logical frame for a more clear comprehension of cognitive defects in the pathology, opening to novel therapeutic strategies.

Epilepsy and intellectual disability linked protein Shrm4 interaction with GABA B Rs shapes inhibitory neurotransmission

Shrm4, a protein expressed only in polarized tissues, is encoded by the KIAA1202 gene, whose mutations have been linked to epilepsy and intellectual disability. However, a physiological role for Shrm4 in the brain is yet to be established. Here, we report that Shrm4 is localized to synapses where it regulates dendritic spine morphology and interacts with the C terminus of GABAB receptors (GABABRs) to control their cell surface expression and intracellular trafficking via a dynein-dependent mechanism. Knockdown of Shrm4 in rat severely impairs GABABR activity causing increased anxiety-like behaviour and susceptibility to seizures. Moreover, Shrm4 influences hippocampal excitability by modulating tonic inhibition in dentate gyrus granule cells, in a process involving crosstalk between GABABRs and extrasynaptic δ-subunit-containing GABAARs. Our data highlights a role for Shrm4 in synaptogenesis and in maintaining GABABR-mediated inhibition, perturbation of which may be responsible for the involvement of Shrm4 in cognitive disorders and epilepsy.

Pharmacological Modulation of AMPAR Rescues Intellectual Disability-Like Phenotype in Tm4sf2-/y Mice.

Abstract Intellectual disability affects 2‐3% of the worlds population and typically begins during childhood, causing impairments in social skills and cognitive abilities. Mutations in the TM4SF2 gene, which encodes the TSPAN7 protein, cause a severe form of intellectual disability, and currently, no therapy is able to ameliorate this cognitive impairment. We previously reported that, in cultured neurons, shRNA‐mediated down‐regulation of TSPAN7 affects AMPAR trafficking by enhancing PICK1‐GluA2 interaction, thereby increasing the intracellular retention of AMPAR. Here, we found that loss of TSPAN7 function in mice causes alterations in hippocampal excitatory synapse structure and functionality as well as cognitive impairment. These changes occurred along with alterations in AMPAR expression levels. We also found that interfering with PICK1‐GluA2 binding restored synaptic function in Tm4sf2−/y mice. Moreover, potentiation of AMPAR activity via the administration of the ampakine CX516 reverted the neurological phenotype observed in Tm4sf2−/y mice, suggesting that pharmacological modulation of AMPAR may represent a new approach for treating patients affected by TM4SF2 mutations and intellectual disability.

Oligophrenin-1 regulates number, morphology and synaptic properties of adult-born inhibitory interneurons in the olfactory bulb.

Among the X-linked genes associated with intellectual disability, Oligophrenin-1 (OPHN1) encodes for a Rho GTPase-activating protein, a key regulator of several developmental processes, such as dendrite and spine formation and synaptic activity. Inhibitory interneurons play a key role in the development and function of neuronal circuits. Whether a mutation of OPHN1 can affect morphology and synaptic properties of inhibitory interneurons remains poorly understood. To address these open questions, we studied in a well-established mouse model of X-linked intellectual disability, i.e. a line of mice carrying a null mutation of OPHN1, the development and function of adult generated inhibitory interneurons in the olfactory bulb. Combining quantitative morphological analysis and electrophysiological recordings we found that the adult generated inhibitory interneurons were dramatically reduced in number and exhibited a higher proportion of filopodia-like spines, with the consequences on their synaptic function, in OPHN1 ko mice. Furthermore, we found that olfactory behaviour was perturbed in OPHN1 ko mice. Chronic treatment with a Rho kinase inhibitor rescued most of the defects of the newly generated neurons. Altogether, our data indicated that OPHN1 plays a key role in regulating the number, morphology and function of adult-born inhibitory interneurons and contributed to identify potential therapeutic targets.

Hyperactivity of Rac1-GTPase pathway impairs neuritogenesis of cortical neurons by altering actin dynamics

The small-GTPase Rac1 is a key molecular regulator linking extracellular signals to actin cytoskeleton dynamics. Loss-of-function mutations in RAC1 and other genes of the Rac signaling pathway have been implicated in the pathogenesis of Intellectual Disability (ID). The Rac1 activity is negatively controlled by GAP proteins, however the effect of Rac1 hyperactivity on neuronal networking in vivo has been poorly studied. ArhGAP15 is a Rac-specific negative regulator, expressed in the main subtypes of pyramidal cortical neurons. In the absence of ArhGAP15, cortical pyramidal neurons show defective neuritogenesis, delayed axonal elongation, reduced dendritic branching, both in vitro and in vivo. These phenotypes are associated with altered actin dynamics at the growth cone due to increased activity of the PAK-LIMK pathway and hyperphosphorylation of ADF/cofilin. These results can be explained by shootin1 hypo-phosphorylation and uncoupling with the adhesion system. Functionally, ArhGAP15−/− mice exhibit decreased synaptic density, altered electroencephalographic rhythms and cognitive deficits. These data suggest that both hypo- and hyperactivation of the Rac pathway due to mutations in Rac1 regulators can result in conditions of ID, and that a tight regulation of Rac1 activity is required to attain the full complexity of the cortical networks.