Zhuo Huang

Rafael Lujan

Ivan Kadurin

Victor N Uebele

John J Renger

Annette C Dolphin

Mala M Shah
email: mala.shah@pharmacy.ac.uk

Pre-synaptic HCN1 channels regulate excitatory neurotransmission at select cortical synapses by altering Ca V 3.2 Ca 2+ channel activity

The Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels are subthreshold, voltage-gated ion channels that are highly expressed in hippocampal and cortical pyramidal cell dendrites, where they play an important role in regulating synaptic potential integration and plasticity. Here, we demonstrate that HCN1 subunits are also localized to the active zone of mature asymmetric synaptic terminals targeting mouse entorhinal cortical layer III pyramidal neurons. We found that HCN channels inhibit glutamate synaptic release by suppressing the activity of low threshold voltage-gated T-(Ca V 3.2) type Ca 2+ channels. In agreement, electron microscopy showed the co-localisation of pre-synaptic HCN1 and Ca V 3.2 subunit. This represents a novel mechanism by which HCN channels regulate synaptic strength and thereby neural information processing and network excitability.

Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels are low threshold, voltage-gated ion channels with very unusual biophysical properties [START_REF] Biel | Hyperpolarizationactivated cation channels: from genes to function[END_REF] .

These channels are open at potentials more negative to -50 mV and are important for regulating neuronal resting membrane potential (RMP). In addition, the channels are permeable to Na + and K + and form an inward current at rest, thereby depolarizing RMP. Interestingly, in the hippocampus and cortex, they are highly localized to pyramidal cell dendrites [START_REF] Lorincz | Polarized and compartment-dependent distribution of HCN1 in pyramidal cell dendrites[END_REF][START_REF] Notomi | Immunohistochemical localization of Ih channel subunits, HCN1-4, in the rat brain[END_REF] . Here, by regulating the RMP and in this way the biophysical properties of other ion channels as well as by modulating the membrane resistance, they influence excitatory post-synaptic potential (EPSP) kinetics and integration [START_REF] Huang | Loss of dendritic HCN1 subunits enhances cortical excitability and epileptogenesis[END_REF][START_REF] Magee | Dendritic lh normalizes temporal summation in hippocampal CA1 neurons[END_REF][START_REF] George | HCN hyperpolarizationactivated cation channels inhibit EPSPs by interactions with M-type K + channels[END_REF][START_REF] Tsay | HCN1 channels constrain synaptically evoked Ca 2+ spikes in distal dendrites of CA1 pyramidal neurons[END_REF] . These effects are likely to contribute substantially to synaptic plasticity and thus, processes such as information storage [START_REF] Nolan | A behavioral role for dendritic integration: HCN1 channels constrain spatial memory and plasticity at inputs to distal dendrites of CA1 pyramidal neurons[END_REF][START_REF] Wang | Alpha2A-adrenoceptors strengthen working memory networks by inhibiting cAMP-HCN channel signaling in prefrontal cortex[END_REF][START_REF] Brager | Plasticity of intrinsic excitability during longterm depression is mediated through mGluR-dependent changes in I h in hippocampal CA1 pyramidal neurons[END_REF][START_REF] Campanac | Downregulation of dendritic I h in CA1 pyramidal neurons after LTP[END_REF][START_REF] Fan | Activity-dependent decrease of excitability in rat hippocampal neurons through increases in I h[END_REF] . Indeed a reduction in HCN channel expression has been associated with enhanced learning [START_REF] Nolan | A behavioral role for dendritic integration: HCN1 channels constrain spatial memory and plasticity at inputs to distal dendrites of CA1 pyramidal neurons[END_REF][START_REF] Wang | Alpha2A-adrenoceptors strengthen working memory networks by inhibiting cAMP-HCN channel signaling in prefrontal cortex[END_REF] .

Emerging evidence, though, suggests that in addition to their dendritic localization, HCN1 channels may be present on certain axons and synaptic terminals in the hippocampus, cortex and other regions of the brain [START_REF] Notomi | Immunohistochemical localization of Ih channel subunits, HCN1-4, in the rat brain[END_REF][START_REF] Bender | Localization of HCN1 channels to presynaptic compartments: novel plasticity that may contribute to hippocampal maturation[END_REF][START_REF] Boyes | Functional presynaptic HCN channels in the rat globus pallidus[END_REF][START_REF] Lujan | Preferential localization of the hyperpolarization-activated cyclic nucleotide-gated cation channel subunit HCN1 in basket cell terminals of the rat cerebellum[END_REF] . Indeed, pre-synaptic HCN channel current, I h , has been indicated to affect inhibitory synaptic transmission in the rodent cerebellum, hippocampus and basal ganglia [START_REF] Boyes | Functional presynaptic HCN channels in the rat globus pallidus[END_REF][START_REF] Aponte | Hyperpolarization-activated cation channels in fast-spiking interneurons of rat hippocampus[END_REF][START_REF] Southan | Hyperpolarization-activated currents in presynaptic terminals of mouse cerebellar basket cells[END_REF] , though the mechanism by which this occurs is unknown. However, in contrast to invertebrate neurons where I h has been shown to influence excitatory synaptic release [START_REF] Beaumont | Temporal synaptic tagging by I h activation and actin: involvement in long-term facilitation and cAMP-induced synaptic enhancement[END_REF][START_REF] Beaumont | Enhancement of synaptic transmission by cyclic AMP modulation of presynaptic Ih channels[END_REF] , an explicit role for I h in regulating excitatory basal synaptic transmission in mammals has not yet been revealed. This is despite HCN subunit expression being detected in some immature mammalian glutamatergic pathways [START_REF] Bender | Localization of HCN1 channels to presynaptic compartments: novel plasticity that may contribute to hippocampal maturation[END_REF] and functional I h recorded from pre-synaptic immature terminals in the calyx of Held [START_REF] Cuttle | Modulation of a presynaptic hyperpolarization-activated cationic current (I h ) at an excitatory synaptic terminal in the rat auditory brainstem[END_REF] . In this study, we demonstrate that HCN1 channels are present at the active zone of mature glutamatergic cortical synaptic terminals establishing contact with entorhinal cortical (EC) layer III cells where they decrease neurotransmission by restricting Ca 2+ entry via pre-synaptic T (Ca V 3.2) -type Ca 2+ channels. Since changes in synaptic strength are pivotal to induction and maintenance of synaptic plasticity [START_REF] Stevens | Presynaptic function[END_REF] and given that EC layer III neurons are likely to be involved in memory formation and spatial navigation [START_REF] Brun | Impaired spatial representation in CA1 after lesion of direct input from entorhinal cortex[END_REF][START_REF] Remondes | Role for a cortical input to hippocampal area CA1 in the consolidation of a long-term memory[END_REF] , our results indicate that this is a novel mechanism by which HCN1 subunits may contribute to such physiological processes.

Results

HCN1 channels modulate glutamatergic synaptic release onto EC layer III cells exclusively

Synaptic release plays a central role in regulating individual neuronal as well as neural network excitability. Thus, elucidating the factors regulating synaptic transmission is critical for understanding how neural circuits function. In an earlier study [START_REF] Huang | Loss of dendritic HCN1 subunits enhances cortical excitability and epileptogenesis[END_REF] , we had observed that a loss of I h enhances synaptic transmission onto mature EC layer III pyramidal neurons, the principal cell type within layer III [START_REF] Shah | Seizureinduced plasticity of h channels in entorhinal cortical layer III pyramidal neurons[END_REF] . This is likely to have been, at least partly, due to enhanced feed forward excitation resulting from pyramidal cell hyperexcitability. However, since HCN subunits may be present at certain synaptic terminals [START_REF] Bender | Localization of HCN1 channels to presynaptic compartments: novel plasticity that may contribute to hippocampal maturation[END_REF][START_REF] Boyes | Functional presynaptic HCN channels in the rat globus pallidus[END_REF][START_REF] Lujan | Preferential localization of the hyperpolarization-activated cyclic nucleotide-gated cation channel subunit HCN1 in basket cell terminals of the rat cerebellum[END_REF][START_REF] Aponte | Hyperpolarization-activated cation channels in fast-spiking interneurons of rat hippocampus[END_REF][START_REF] Southan | Hyperpolarization-activated currents in presynaptic terminals of mouse cerebellar basket cells[END_REF][START_REF] Cuttle | Modulation of a presynaptic hyperpolarization-activated cationic current (I h ) at an excitatory synaptic terminal in the rat auditory brainstem[END_REF] , it is possible that this effect could be due to changes in synaptic release per se by altered pre-synaptic HCN channel function.

To test this, we initially recorded non-evoked miniature excitatory post-synaptic currents (mEPSC) in the presence of tetrodotoxin (TTX, 1 μM) and GABA receptor inhibitors at a fixed potential of -70 mV from mouse EC layer III neurons (Fig 1a)

using the whole-cell voltage clamp technique (as described previously [START_REF] Boyes | Functional presynaptic HCN channels in the rat globus pallidus[END_REF][START_REF] Aponte | Hyperpolarization-activated cation channels in fast-spiking interneurons of rat hippocampus[END_REF][START_REF] Southan | Hyperpolarization-activated currents in presynaptic terminals of mouse cerebellar basket cells[END_REF] , see Methods). To exclude the effects of post-synaptic HCN channels [START_REF] Fan | Activity-dependent decrease of excitability in rat hippocampal neurons through increases in I h[END_REF] and to reduce errors due to space-clamp [START_REF] Williams | Direct measurement of somatic voltage clamp errors in central neurons[END_REF] , 15 μM ZD7288 was incorporated in the patch pipette in all our experiments, unless otherwise stated. Intracellular ZD7288 enhanced the outward holding current, indicating that the post-synaptic neuronal resting membrane potential (RMP) was hyperpolarized (Supplementary Table 1). The mEPSC rise and decay times were also slowed down as would be expected from blocking postsynaptic HCN channels (see Refs. [START_REF] Huang | Loss of dendritic HCN1 subunits enhances cortical excitability and epileptogenesis[END_REF][START_REF] Magee | Dendritic lh normalizes temporal summation in hippocampal CA1 neurons[END_REF][START_REF] Shah | Seizureinduced plasticity of h channels in entorhinal cortical layer III pyramidal neurons[END_REF] ). Including ZD7288 in the patch pipette, though, had no effect on mEPSC frequency indicating this did not affect synaptic release per se (Supplementary Table 1). Further, substituting KMeSO 4 with CsMeSO 4 in the intracellular solution did not alter either somatic or dendritic mEPSC frequency, amplitude and kinetics (Supplementary Table 1), indicating that the errors due to space-clamp were not reduced further by blocking post-synaptic K + channels [START_REF] Williams | Direct measurement of somatic voltage clamp errors in central neurons[END_REF] .

To determine if functional HCN channels are present pre-synaptically, we bathapplied ZD7288 (15 μM) for 15 min only to exclude any potential non-specific effects of the compound on synaptic transmission [START_REF] Chevaleyre | Assessing the role of Ih channels in synaptic transmission and mossy fiber LTP[END_REF] . This concentration was deemed specific as it enhances wildtype dendritic excitability whilst having little effect on HCN1-/-dendrites (see Ref. In the above experiments, ZD7288 was included in the internal patch pipette solution.

However, even when this was not present in the internal solution, external application of ZD7288 still amplified wildtype mEPSC frequency by 108.3 ± 14.8% (n=6). In these experiments, the decay time constant increased significantly from 7.9 ± 0.6 (n=6) to 10.0 ± 0.5 (n=6, p < 0.05), as would be predicted by blocking post-synaptic HCN channels.

Since dendrites receive the majority of excitatory synaptic inputs [START_REF] Magee | Dendritic integration of excitatory synaptic input[END_REF] , we also recorded mEPSCs from EC layer III apical dendrites at a distance of 120 -150 μm from the soma (Fig 1c). The frequency, but not amplitude or kinetics, of dendritic mEPSCs was increased by application of ZD7288 by 69.7 ± 5.3% (n=5; Fig 1c). This effect was not significantly different (p=0.1) to that observed at the soma. The above results indicate that synapses targeting EC layer III neurons contain pre-synaptic I h . HCN1 subunits are likely to be predominantly expressed in the EC [START_REF] Notomi | Immunohistochemical localization of Ih channel subunits, HCN1-4, in the rat brain[END_REF][START_REF] Huang | Loss of dendritic HCN1 subunits enhances cortical excitability and epileptogenesis[END_REF] . We, therefore, compared HCN1-/-and wildtype mEPSCs recorded from layer III neurons. Since ZD7288 was present in the internal pipette solution, wildtype and HCN1-/-outward holding current and mEPSC kinetics were similar (Supplementary Table 1).

Interestingly, the average somatic, but not dendritic, HCN1-/-mEPSC amplitudes were smaller than in wildtypes (Supplementary Table 1). Importantly, though, HCN1-/-mEPSC frequency was significantly greater than in wildtypes (Fig 1b,c, Supplementary Table 1). Moreover, omitting ZD7288 from the internal pipette solution did not affect HCN1-/-mEPSC frequency or kinetics (Supplementary Table 1). Additionally, 15 min bath application of ZD7288 or zatebradine had no effect on somatic or dendritic HCN1-/-mEPSC frequency, amplitude or shape (Fig 1b,c; Supplementary Fig 1b). These findings confirm that the enhanced wildtype mEPSC frequency caused by pharmacological blockers of I h is likely to be due to HCN channel block and not non-specific effects of the compounds [START_REF] Chevaleyre | Assessing the role of Ih channels in synaptic transmission and mossy fiber LTP[END_REF] . These results also indicate that pre-synaptic HCN1 channels are likely to be involved in regulating excitatory spontaneous release from synapses localised to EC layer III neurons.

We then recorded miniature inhibitory synaptic currents (mIPSCs) to determine if HCN channels were also present pre-synaptically on interneurons. In contrast to mEPSCs, somatic EC layer III mIPSCs were unaffected by 15 HCN1 subunits are present at the active zone of selective terminals.

To determine if HCN1 subunits are located on EC synaptic terminals, we carried out electron microscopy studies using a pre-embedding immunogold method (see Methods). We counted 2752 particles in the medial EC from sections obtained from 3 wildtype mice. 72.2 ± 0.8% of HCN1 immunogold particles were observed along the extra-synaptic plasma membrane of dendritic shafts and spines (Fig 3a,e), confirming that HCN channels are largely present in EC dendrites. Interestingly, 27.8 ± 1.0% of immunogold particles were located at pre-synaptic sites (Fig 3b,e).

We further analysed the distribution of pre-synaptic particles. Of 153 immunogold particles that were detected on synaptic terminals, the majority ((129/153, 84%) of HCN1 immunogold particles were localized at terminals establishing asymmetrical excitatory synapses with pyramid dendritic spines (Fig 3b Enhanced Ca 2+ dependence of HCN1-/-mEPSCs How do pre-synaptic HCN1 subunits regulate synaptic transmission at synapses targeting EC layer III neurons? Since acute treatment of ZD7288 in wildtype neurons results in enhanced mEPSC frequency, it suggests that pre-synaptic HCN1 subunits do not affect synaptogenesis. Nevertheless, we compared the number of synapses in superficial EC layers (layers I-III) between wildtypes and HCN1-/-mice using electron microscopy. From sections obtained from 3 wildtype and 4 HCN1-/-mice, no obvious differences in the number of synapses were detected (Supplementary Fig

4)

, indicating that HCN1 subunits on synaptic terminals are likely to reduce synaptic transmission by directly affecting release probability.

One possible mechanism by which pre-synaptic HCN1 channels may regulate synaptic transmission is by limiting Ca 2+ influx, as has been suggested to occur in dendrites [START_REF] Tsay | HCN1 channels constrain synaptically evoked Ca 2+ spikes in distal dendrites of CA1 pyramidal neurons[END_REF] . Recent studies suggest that spontaneous as well as action potential-driven synaptic release may be dependent upon Ca 2+ entry via voltage-gated Ca 2+ channels (VGCC) [START_REF] Sun | A dual-Ca2+-sensor model for neurotransmitter release in a central synapse[END_REF][START_REF] Daw | Asynchronous transmitter release from cholecystokinin-containing inhibitory interneurons is widespread and target-cell independent[END_REF][START_REF] Hefft | Asynchronous GABA release generates long-lasting inhibition at a hippocampal interneuron-principal neuron synapse[END_REF][START_REF] Neher | Multiple roles of calcium ions in the regulation of neurotransmitter release[END_REF][START_REF] Groffen | Doc2b Is a High-Affinity Ca 2+ Sensor for Spontaneous Neurotransmitter Release[END_REF] . We, therefore, tested if the mEPSCs recorded from layer III soma were

HCN1 channels inhibit mEPSC frequency by limiting T-type Ca 2+ channel activity

prevented by pre-treatment with three different inhibitors of the low voltage-activated T-type Ca 2+ channels: mibefradil (10 μM) as well as the more potent and selective inhibitors TTA-P2 [START_REF] Dreyfus | Selective T-type calcium channel block in thalamic neurons reveals channel redundancy and physiological impact of I T window[END_REF][START_REF] Shipe | Design, synthesis, and evaluation of a novel 4aminomethyl-4-fluoropiperidine as a T-type Ca 2+ channel antagonist[END_REF][START_REF] Uebele | T-type calcium channels regulate cortical plasticity invivo. [corrected][END_REF] (1μM) and TTA-A2 [START_REF] Shipe | Design, synthesis, and evaluation of a novel 4aminomethyl-4-fluoropiperidine as a T-type Ca 2+ channel antagonist[END_REF][START_REF] Uebele | T-type calcium channels regulate cortical plasticity invivo. [corrected][END_REF] (500 nM) (Fig 5a,c). The latter two have been shown to have no effects on other VGCC in either heterologous expression systems [START_REF] Shipe | Design, synthesis, and evaluation of a novel 4aminomethyl-4-fluoropiperidine as a T-type Ca 2+ channel antagonist[END_REF][START_REF] Uebele | T-type calcium channels regulate cortical plasticity invivo. [corrected][END_REF] or neurons [START_REF] Dreyfus | Selective T-type calcium channel block in thalamic neurons reveals channel redundancy and physiological impact of I T window[END_REF] . These findings suggest that inhibition of HCN1 channels 3). As mibefradil may inhibit R-type Ca 2+ channels too [START_REF] Randall | Contrasting biophysical and pharmacological properties of T-type and R-type calcium channels[END_REF] , we tested the effects of SNX-482 (200 nM) on HCN1-/-mEPSCs. 15 min application of SNX-482 had no effect on these (Fig 5d). To verify that T-type Ca 2+ channels expression is not modified in HCN1-/-mice, we did Western blot analysis using Ca V 3.1 and Ca V 3.2 antibodies. We were unable to detect any Ca V 3.1 protein expression in wildtype or HCN1-/-EC. However, bands corresponding to the size of Ca V 3.2 proteins expressed in tsA-201 cells were present in wildtype and HCN1-/-EC samples (Supplementary Fig 6a ,7a). Semi-quantitative analysis indicated that there was no difference in expression of this between wildtype and HCN1-/-tissue (Supplementary Fig 6a ,7a). These findings re-affirm that a decrease in pre-synaptic HCN channel function leads to increased contribution of T-type Ca 2+ channel activity in modulating basal glutamatergic synaptic release.

Co-localisation of HCN1 and Ca V 3.2 subunits on EC synaptic terminals.

Since T-type Ca 2+ channel inhibitors prevent the effects of ZD7288, it raises the possibility that these and HCN1 channels co-exist on the same pre-synaptic terminals.

To investigate this, we took advantage of Ca V 3.1 and Ca V 3.2 antibodies to examine the localization of Ca V 3 and HCN1 subunits in wildtype EC. For these particular experiments, the anti-HCN1 antibody was visualized using an immunoperoxidase reaction and the anti-Ca v 3 antibody by a silver-intensified immunogold reaction (see Methods). Consistent with the Western blot data, Ca V 3.1 labelling could not be detected. However, remarkably, Ca V 3.2 immunogold particles were present on asymmetrical synaptic terminals (Fig 6a) in sections obtained from 3 wildtype mice.

Approximately 84% of these were present on axons pre-labelled with HCN1. HCN1 and Ca V 3.2 co-labelling was noticeable on spines and dendrites too (Supplementary channel activity. T-type Ca 2+ channels are low threshold VGCCs that typically activate at potentials more positive to -75 mV [START_REF] Crunelli | The 'window' T-type calcium current in brain dynamics of different behavioural states[END_REF][START_REF] Talavera | Biophysics and structure-function relationship of Ttype Ca 2+ channels[END_REF] . Interestingly, native as well as heterogously-expressed Ca V 3.1-3.3 channels inactivate at potentials below -55 mV-60 mV [START_REF] Crunelli | The 'window' T-type calcium current in brain dynamics of different behavioural states[END_REF][START_REF] Talavera | Biophysics and structure-function relationship of Ttype Ca 2+ channels[END_REF] . Hence, there is a small "window" during which these channels are active.

Since T-type Ca 2+ channel blockers had no effect on basal wildtype mEPSC frequency (Supplementary Fig 5 ) and the basal frequency between Ca V 3.2-/-and wildtype was similar (Supplementary Table 4), despite Ca V 3.2 immunogold particles being present on wildtype synaptic terminals (Fig 6b), it implies that these channels are inactive at rest. A reduction in HCN channel activity hyperpolarizes the membrane [START_REF] Biel | Hyperpolarizationactivated cation channels: from genes to function[END_REF][START_REF] Huang | Loss of dendritic HCN1 subunits enhances cortical excitability and epileptogenesis[END_REF] and thus one possibility is that RMP hyperpolarization relieves T-type Ca 2+ channel inactivation. To investigate this hypothesis, we lowered external K + concentration from 2.5 mM to 1.75 mM, which, using the Nernst equation, is estimated to hyperpolarize the RMP by ~10 mV. Indeed, reducing external K + concentration resulted in EC layer III neuronal hyperpolarization by 7.3 ± 0.9 mV (n=3). Further, switching from 2.5 mM to 1.75 mM K + significantly enhanced wildtype mEPSC frequency within 10 min by 91.6 ± 14.1% (n=6, p < 0.01, Fig 7a,c).

This was restored to near control levels by adding mibefradil [START_REF] Randall | Contrasting biophysical and pharmacological properties of T-type and R-type calcium channels[END_REF] (n=6, p < 0.01). In addition, square of the coefficient of variation (CV 2 ) decreased significantly to 0.7 ± 0.1 (p < 0.01). In contrast, ZD7288 (15 μM) had no effect on PPR in HCN1-/-neurons (PPR ratio before and after ZD7288 = 1.1 ± 0.2 and 1.1 ± 0.1, n=6, p = 0.92). The lowered PPR and CV 2 in wildtypes indicate that synaptic release was enhanced by pharmacological block of HCN channels. These findings, therefore, demonstrate that pre-synaptic HCN channels affect multiple modes of synaptic transmission.

Discussion

The significant findings of this study are that functional HCN1 channels are present on glutamatergic synaptic terminals synapsing onto EC layer III neurons where they reduce spontaneous and evoked neurotransmitter release. These effects are at least partly due to altered availability of low-threshold, voltage-gated T-type Ca 2+ channel.

In support, electron microscopy showed that HCN1 and Ca V 3.2 subunits co-exist on EC pre-synaptic terminals. As EC layer III neurons have been suggested to play significant roles in processes such as spatial navigation [START_REF] Brun | Impaired spatial representation in CA1 after lesion of direct input from entorhinal cortex[END_REF] and learning and memory [START_REF] Remondes | Role for a cortical input to hippocampal area CA1 in the consolidation of a long-term memory[END_REF] and HCN channel function has been associated with modification of neural network rhythms and memory formation [START_REF] Nolan | A behavioral role for dendritic integration: HCN1 channels constrain spatial memory and plasticity at inputs to distal dendrites of CA1 pyramidal neurons[END_REF] , the dynamic modulation of synaptic inputs onto these neurons by HCN and T-type Ca 2+ channels may contribute to the regulation and modulation of these physiological states.

Although HCN1 and HCN2 subunits are both expressed in the EC [START_REF] Notomi | Immunohistochemical localization of Ih channel subunits, HCN1-4, in the rat brain[END_REF] , our results suggest that HCN1 subunits are likely to predominantly contribute to the formation of pre-synaptic HCN channels in glutamatergic synapses in the EC. Indeed, HCN1 subunit immunoreactivity was detected on the active zone of wildtype excitatory synaptic terminals in the adult EC ( Notwithstanding, our results provide strong support for the involvement of HCN1 channels in modulating EC excitatory synaptic transmission.

Remarkably, our results suggest that the presence of pre-synaptic HCN channels depolarizes the RMP, thereby restricting Ca 2+ entry via pre-synaptic T-type Ca 2+ channel activity and preventing glutamate release (Figs 4567). This suggests that presynaptic HCN channels may provide an additional regulatory mechanism for controlling synaptic release at these synapses. Hence, by affecting T-type Ca 2+ channel activity, modulation of HCN channel activity by protein kinases or phosphatases will alter synaptic transmission at these synapses. Indeed, EC synaptic terminals may have functional metabotropic glutamate receptors [START_REF] Evans | Activation of presynaptic group III metabotropic receptors enhances glutamate release in rat entorhinal cortex[END_REF] , activation of which could potentially reduce HCN channel activity by altering kinase activity and induce plasticity [START_REF] Brager | Plasticity of intrinsic excitability during longterm depression is mediated through mGluR-dependent changes in I h in hippocampal CA1 pyramidal neurons[END_REF] .

Altered T-type Ca 2+ channel activity by HCN channels has been suggested to occur in hippocampal dendrites too [START_REF] Tsay | HCN1 channels constrain synaptically evoked Ca 2+ spikes in distal dendrites of CA1 pyramidal neurons[END_REF] and it is, therefore, tempting to speculate that this might be a universal mechanism by which these channels affect neuronal excitability.

Fascinatingly, though, in invertebrate neurons, I h has been shown to increase excitatory synaptic release in a "non-ionic" way via a pathway involving actin depolymerization [START_REF] Beaumont | Temporal synaptic tagging by I h activation and actin: involvement in long-term facilitation and cAMP-induced synaptic enhancement[END_REF] . It is possible that this might occur in some mammalian interneurons too [START_REF] Aponte | Hyperpolarization-activated cation channels in fast-spiking interneurons of rat hippocampus[END_REF][START_REF] Southan | Hyperpolarization-activated currents in presynaptic terminals of mouse cerebellar basket cells[END_REF] . This therefore suggests that HCN channels in synaptic terminals may have multiple roles: a "non-ionic" purpose whereby they modulate release by processes that involve changes in the cytoskeleton and a more conventional function in which they influence synaptic transmission by modifying the terminal RMP as well as the membrane resistivity, which has been shown to occur post-synaptically in dendrites [START_REF] Huang | Loss of dendritic HCN1 subunits enhances cortical excitability and epileptogenesis[END_REF][START_REF] Shah | Seizureinduced plasticity of h channels in entorhinal cortical layer III pyramidal neurons[END_REF] . Which role they adopt may depend on their location within a synapse as well as their proximity to other proteins and ion channels.

Interestingly, the expression of HCN channel subunits and their effects on synaptic release were restricted to a subset of EC glutamatergic synaptic terminals (Figs 123). This suggests that there may be factors that control expression of HCN channels in specific synapses and not others. These synapses may belong to a particular cell type. Indeed, synapses targeting EC layer III neurons can originate from a number of sources, including other EC layer III neurons, layer V and pre-subicular neurons.

Since HCN subunits have not been detected in the temporoammonic (TA) pathway, EC layer III pyramid axons, or terminals synapsing onto CA1 dendrites (which belong to the TA pathway) [START_REF] Bender | Localization of HCN1 channels to presynaptic compartments: novel plasticity that may contribute to hippocampal maturation[END_REF] , it is likely that HCN channels are present on those synaptic terminals originating from EC layer V or pre-subiculum. Further work is, therefore, required to resolve whether HCN subunit expressing synapses belong to a particular cell type and whether these synaptic terminals contain or lack proteins that regulate HCN channel expression. In addition, it remains to be determined if other cortical synapses express functional pre-synaptic HCN and T-type Ca 2+ channels. Nonetheless, our results show that functional HCN channels can be present presynaptically as well as on pyramid cell dendrites in the mature cortex. Thus there may be multiple means by which HCN channels can affect neural network excitability and ultimately processes such as learning and memory.

Methods

Slice preparation and electrophysiological experiments. HCN1 [START_REF] Nolan | The hyperpolarization-activated HCN1 channel is important for motor learning and neuronal integration by cerebellar Purkinje cells[END_REF] and Ca V 3.2 [START_REF] Chen | Abnormal coronary function in mice deficient in alpha1H Ttype Ca2+ channels[END_REF] heterozygote breeding pairs were used to generate HCN1 null (HCN1-/-), Ca V 3.2 null (Ca V 3.2-/-) and wildtype littermates as described in Huang et al. (2009) [START_REF] Huang | Loss of dendritic HCN1 subunits enhances cortical excitability and epileptogenesis[END_REF] . All electrophysiological experiments involving transgenic mice were done blindly.

Entorhinal-hippocampal slices were obtained from 6-9 week old HCN1-/-, Ca V 3.2-/and wildtype mice as described previously (see [START_REF] Huang | Loss of dendritic HCN1 subunits enhances cortical excitability and epileptogenesis[END_REF] ). For electrophysiological recordings, slices were placed in a chamber containing external solution of the following composition (mM): 125 NaCl, 2.5 KCl, 1.25 NaH 2 PO 4 , 25 NaHCO 3 , 2 CaCl 2 , 2 MgCl 2 , 10 glucose, 0.001 tetrodotoxin; bubbled with 95% O 2 /5% CO 2 (pH 7.2). Whole-cell voltage-clamp recordings were obtained from the soma and dendrites of EC neurons. The internal recording pipette solution for mEPSC recordings was composed of (in mM): 120 KMeSO 4 , 20 KCl, 10 HEPES, 2 MgCl 2 , 0.2 EGTA, 4 Na 2 -ATP, 0.3 Tris-GTP, 14 Tris-phosphocreatine, 0.015 ZD7288; pH was adjusted to 7.3 with KOH. In some experiments, CsMeSO 4 was used instead of KMeSO 4 . To record mIPSCs, KMeSO 4 was replaced with KCl. Pipettes containing any of these internal solutions had resistances of 5 -12 MΩ. In some recordings, biocytin (0.4% w/v; Vector Laboratories Ltd) was included in the intracellular pipette solution. Slices were fixed in 4% paraformaldehyde and stained with streptavidin Alexa Fluor 294 conjugate (Molecular Probes Ltd) 24 hr later as described previously [START_REF] Cossart | Dendritic but not somatic GABAergic inhibition is decreased in experimental epilepsy[END_REF] . For paired pulse recordings, tungsten electrodes (Biomedical Instruments, Germany) placed in EC layer I approx. 50 -100 μm from EC layer III distal dendrites were used to elicit single and 20 Hz pairs of synaptic potentials in interleaved sequences every 30 s. The recordings were obtained using a Axopatch 200B amplifier (Molecular Devices, UK), filtered at 1 kHz and sampled at 10 kHz. Series resistance was usually in the order of 10-30 MΩ and was approx. 70% compensated. Recordings were discarded if the series resistance increased by more than 20% during the course of the recordings. Data were acquired using pClamp 8.2 or pClamp 10.0 (Molecular Devices, UK).

Data Analysis

mEPSC and mIPSC recordings were analysed using Mini-analysis program (v6.07, Synpatosoft, USA). Events > 1.5 pA in amplitude (i.e. all events above baseline noise level) and with rise times of < 2 ms were detected and used for analysis. Decay times and amplitudes of these events were obtained by fitting the averaged EPSC or IPSC with a single exponential equation:

I(t) = A exp(-t/τ)
where I is the current amplitude at any given time (t), A is the peak amplitude of the EPSC or IPSC and τ is the decay time constant. visualized by the immunoperoxidase reaction, and Ca V 3.2 immunoreactivity was revealed with the silver-intensified immunogold reaction. Briefly, free-floating sections were obtained from 8-9 week old mice. Sections were then incubated for 48 h with either HCN1 antibody alone or a mixture of two HCN1 and Ca v 3.2 antibodies, at a final protein concentration of 1-2 μg/ml each. After primary antibody incubation, the sections were incubated at 4°C overnight with secondary antibodies: biotinylated goat anti-rabbit (diluted 1:100; Vector Laboratories) and goat anti-mouse (Fab fragment, diluted 1:100) coupled to 1.4 nm gold (Nanoprobes, Stony Brook, NY) antibodies. After washes in TBS, sections were washed in double-distilled water, followed by silver enhancement of the gold particles with an HQ Silver kit (Nanoprobes, Stony Brook, NY) for 8-10 min. Subsequently for double labelling, the sections were incubated for 4 hours in the ABC complex (Vector Laboratories) made up in TBS and then washed in TB. Peroxidase was visualized with DAB (0.05% in TB, pH 7.4) using 0.01% H 2 O 2 as substrate for 5-10 min. The sections were washed in PB and then post-fixed with OsO 4 (1% in 0.1 M PB), followed by block-staining with uranyl acetate, dehydration in graded series of ethanol and flat-embedding on glass slides in Durcupan (Fluka) resin. Regions of interest were cut at 70-90 nm thick sections using an ultramicrotome (Reichert Ultracut E, Leica, Austria). Ultrathin sections were mounted on 200-mesh nickel grids. Staining was performed on drops of 1% aqueous uranyl acetate followed by Reynolds's lead citrate. Ultrastructural analyses were performed in a Jeol-1010 electron microscope. Unless otherwise stated, electron microscopic samples were obtained from three different mice and three blocks of each animal were cut for electron microscopy. Electron micrographs were captured with CCD camera (Mega View III; Soft Imaging System, Germany).

  [START_REF] Huang | Loss of dendritic HCN1 subunits enhances cortical excitability and epileptogenesis[END_REF] ). Intriguingly, external application of ZD7288 enhanced wildtype somatic mEPSC frequency by 113.8 ± 19.1% (n=7, p < 0.01; Fig 1b). The outward holding current as well as mEPSC amplitude, rise-times and decay time constants were not further affected (Fig 1b). Further, 15 min external application of another I h blocker, zatebradine 27, 28 (10 μM) also enhanced mEPSC frequency in a comparable manner without affecting amplitude or kinetics (Supplementary Fig 1a).

  min bath-application of ZD7288 (Supplementary Fig 2a). Moreover, mIPSC frequency, amplitude and kinetics recorded from wildtype and HCN1-/-neurons did not differ (Supplementary Fig 2b). These findings suggest that in the EC, HCN1 channels selectively regulate glutamatergic synaptic release. We next asked whether HCN1 channels were present at excitatory synapses targeting other principal EC neurons, particularly layer II stellate (Fig 2a) and layer V pyramids (Fig 2c). Intriguingly, application of ZD7288 had no effect on wildtype mEPSC frequency, amplitudes or kinetics recorded from the soma of these neurons (Fig 2b, d). In addition, there were no differences between HCN1-/-and wildtype layer II and layer V mEPSCs (Fig 2). Since layer III pyramid dendrites are present in layer II and mEPSC frequency onto these is altered by pharmacological block of I h or HCN1 deletion (Fig 1c), these results suggest that HCN1 channels are predominantly located at glutamatergic synaptic terminals contacting EC layer III pyramids.

  ). Of these, 71% (92/129) were present in the active zone and 29%(37/129) in pre-synaptic terminal membranes. In contrast, only 16% (24/153 particles in 8 synapses) were located in the axonal plasma membranes establishing symmetrical synapses (Fig 3c). Interestingly, very few immunogold particles were observed in layer V (data not shown). No staining occurred if either the primary or secondary antibodies were omitted. Further, no signal was detected in HCN1-/-sections obtained from 4 mice under either light (Supplementary Fig 3) or electron microscopy (Fig 3d). Thus, these results together with the electrophysiological results suggest that HCN1 subunits are present at excitatory pre-synaptic terminals synapsing onto EC layer III neurons. These data are also consistent with our findings that ZD7288 only alters spontaneous excitatory synaptic transmission, and not inhibitory synaptic transmission, at layer III neurons (Fig 1, Supplementary Fig 1 and Supplementary Fig 2).

  Ca 2+ -dependent. Replacement of Ca 2+ in the extracellular solution with an equal concentration of Mg 2+ reversibly reduced wildtype mEPSC frequency by 56.3 ± 5.1% (n=9, p < 0.01) within 5 min (Fig 4a, b). Interestingly, substitution of Ca 2+ with Mg 2+ lowered HCN1-/-mEPSC frequency by 70.1 ± 3.6% (n=9, p < 0.01, Fig 4a) in a reversible manner (Fig 4a, b). This decrease in HCN1-/-mEPSC frequency was significantly (p = 0.04) greater than that in wildtypes. Neither the amplitudes nor the time constants of wildtype or HCN1-/-mEPSCs were affected by replacing external Ca 2+ with Mg 2+ . The outward holding current, though, was reduced significantly to a similar extent in wildtype and HCN1-/-neurones (average holding current in the presence of Ca 2+ in wildtypes and HCN1-/-cells = 30.0 ± 7.9 pA (n=9) and 39.4 ± 20.0 pA (n=9) respectively; holding current without Ca 2+ in wildtypes and HCN1-/pyramids = 5.0 ± 7.1 pA (n=9, p < 0.05) and 10.0 ± 20.3 (n=9, p < 0.05)). Given the larger Ca 2+ dependence of HCN1-/-mEPSCs, these results imply that pre-synaptic HCN1 channels regulate synaptic release by constraining Ca 2+ influx into terminals. Indeed, in the absence of extracellular Ca 2+ , bath application of ZD7288 no longer increased mEPSC frequency in wildtypes (Fig 4c).

  enhances excitatory synaptic transmission by altering T-type Ca 2+ channel activity. To investigate this further, we explored the effects of T-type Ca 2+ channel blockers on HCN1-/-mEPSC frequency. In contrast to the results obtained in wildtypes (Supplementary Fig 5), mibefradil (10 μM), TTA-A2 (500 nM) and TTA-P2 (1 μM) significantly reduced HCN1-/-somatic mEPSC frequency by 35.7 ± 4.8% (n=6, p < 0.05), 49.0 ± 4.8% (n=7, p < 0.05) and 50.4 ± 4.4% (n=6. P < 0.05) respectively (Fig 5b, d), whilst having no effect on amplitudes or time constants (Supplementary Table

Fig 8 ).

 8 Fig 8).No Ca V 3.2 signal was identified on sections obtained from 3 Ca V 3.2-/-mice

  (10 μM; Fig 7a).Interestingly, changing the external K + from 2.5 mM to 1.75 mM in HCN1-/-slices, where the RMP is expected to be approximately -80 mV[START_REF] Huang | Loss of dendritic HCN1 subunits enhances cortical excitability and epileptogenesis[END_REF] , had little effect on mEPSC frequency (% increase = -1.5 ± 3.6%, n=5, Fig 7b), presumably as T-type Ca 2+ channel activity was not further altered. The outward holding current, mEPSC amplitude or kinetics were not affected by reducing external K + concentration in wildtype or HCN1-/-neurons (Fig 7a, b). These results provide additional support for the notion that T-type Ca 2+ channels are present on pre-synaptic terminals and can contribute to synaptic release under certain conditions. HCN channels modulates evoked release onto EC layer III neurons. We also asked if HCN channels affect evoked release too. To determine this, we stimulated EC layer III distal dendrites to evoke pairs of EPSCs at 20 Hz in the absence and presence of 15 μM ZD7288 (see Methods). ZD7288 (15 μM) was included in the intracellular solution to block post-synaptic HCN channels. The amplitude of the first EPSC was adjusted between 50 to 100 pA (average amplitude = 71.3 ± 9.8 pA). Single EPSCs were also elicited in between pairs of EPSCs. Subtraction of these from the pairs of EPSCs was then used to elucidate the amplitudes of the first and second EPSCs within the pairs (Fig 8). Paired pulse ratio (PPR) was calculated as the amplitude of the second EPSC divided by the amplitude of the first. Under control conditions, the average PPR was 0.9 ± 0.1 (n=5, Fig 8). 15 min bath application of ZD7288 reduced PPR significantly to 0.7 ± 0.1 (n=5, p < 0.01, Fig 8) and increased the amplitude of the first evoked EPSC by 16.7 ± 5.8%

Fig 3 ).

 3 Spontaneous glutamatergic synaptic transmission was also augmented in HCN1-/-slices and was not further affected by the non-selective HCN channel blockers, ZD7288 (Fig 1) and zatebradine (Supplementary Fig 1). These agents, on the other hand, enhanced wildtype mEPSC frequency (Fig 1, Supplementary Fig 1). Further, ZD7288 reduced the wildtype EPSC paired pulse ratio (Fig 8). These findings indicate that pharmacological inhibition of HCN channels in normal tissue enhances glutamtergic synaptic transmission. Since ZD7288 has also been suggested to depress synaptic transmission independently of affecting HCN channels 26 , it is possible that the observed effects of ZD7288 might be underestimated (Fig 1, Fig 8). Further, the effects of zatebradine are dependent upon HCN channels being open 27, 28 and therefore, the increase in mEPSC frequency in the presence of this compound may also be undervalued (Supplementary Fig 1). This might account for the increase in spontaneous release in HCN1-/-slices being much greater than that obtained with application of pharmacological blockers on wildtype slices (Fig 1, Supplementary Fig 1).

(

  WT087363MA, MMS), a European Research Council Starter Independent Grant (ERC_2010_StG_20091118, MMS), the Spanish Ministry of Science and Innovation (BFU-2009-08404/BFI and CONSOLIDER-Ingenio CSD2008-00005, RL), the Junta de Comunidades de Castilla-La Mancha (PAI08-0174-6967, RL), an Epilepsy Research UK Grant (0803, ACD) and an MRC Project Grant (G0801756, ACD). The monoclonal antibodies HCN1 (Clone N70/28) and Ca V 3.2 (Clone N55/10) were developed by and obtained from the UC Davis/NIH NeuroMab Facility, supported by NIH grant U24NS050606 and maintained by the Department of Neurobiology, Physiology and Behaviour, College of Biological Sciences, University of California, Davis, CA 95616, USA. VNU and JJR are employees of Merck and Co., Inc. (USA) and potentially own stock and/or stock options in the company.

Figure 1

 1 Figure 1 Pharmacological block or deletion of HCN channels enhances mEPSC

Figure 2 :

 2 Figure 2: HCN channel block or deletion has no effect on mEPSCs onto EC layer

Figure 3 :

 3 Figure 3: HCN1 subunits are localized to active zones of synaptic terminals. a, b

Figure 4 :

 4 Figure 4: Ca 2+ dependence of EC layer III mEPSCs. a Traces from wildtype (Wt) and HCN1 null neurons respectively in the absence, presence and washout of Ca 2+ -

Figure 5 :

 5 Figure 5: T-type Ca 2+ channel blockers reduce the increase in mEPSC frequency

Figure 6 :

 6 Figure 6: Ca V 3.2 co-localise with HCN1 at EC synaptic terminals and cause the

Figure 7 :

 7 Figure 7: Hyperpolarization enhances mEPSC frequency by activating T-type Ca 2+ channels a and b Representative traces and their cumulative probability curves illustrating the effects of lowering the external K + concentration from 2.5 mM to 1.75

Figure 8 :

 8 Figure 8: Effects of pharmacologically blocking HCN channels on evoked

Acknowledgements

We thank Profs. D. A. Brown (University College, London, UK), D. Johnston (University of Texas, Austin, USA) and M. C. Walker (UCL Institute of Neurology, UK) and Dr. A. Constanti (School of Pharmacy, University of London, UK) for helpful discussions. We are also grateful to Mr. S. Martin (UCL Sequencing and Genotyping Facility, UK) for genotyping the transgenic mice, Ms. K. Venner (UCL Institute of Neurology, UK) for help with processing some samples for electron microscopy, Dr. D. McCarthy (School of Pharmacy, University of London, UK) for assistance with acquisition of some electron micrographs and Profs H. Beck (University of Bonn, Germany) and K. P. Campbell (University of Iowa, USA) for providing Ca V 3.2 heterozygote breeding pairs. This work was supported by an MRC New Investigator Award (G0700369, MMS), a Wellcome Trust project grant

We next asked if Ca 2+ entry through specific VGCCs contributes to generation of spontaneous mEPSCs and whether pre-synaptic HCN1 channels limit the activity of particular VGCCs. To investigate this, we first examined the effects of various pharmacological inhibitors of VGCCs on wildtype mEPSC frequency. The P/Q Ca 2+ channel blocker, ω -agatoxin IVA [START_REF] Catterall | Structure and function of neuronal Ca 2+ channels and their role in neurotransmitter release[END_REF][START_REF] Tsien | Molecular diversity of voltagedependent Ca2+ channels[END_REF] (100 nM) and the N-type Ca 2+ channel inhibitor, ω-conotoxin GVIA [START_REF] Catterall | Structure and function of neuronal Ca 2+ channels and their role in neurotransmitter release[END_REF][START_REF] Tsien | Molecular diversity of voltagedependent Ca2+ channels[END_REF] 2). The effects occurred within 10 min of bath application. ω -agatoxin IVA and ωconotoxin GVIA co-application decreased mEPSC frequency by 48.6 ± 5.5% (n=6, Supplementary Fig 5). In contrast, neither the L-type Ca 2+ channel inhibitor, nifedipine [START_REF] Catterall | Structure and function of neuronal Ca 2+ channels and their role in neurotransmitter release[END_REF][START_REF] Tsien | Molecular diversity of voltagedependent Ca2+ channels[END_REF] (2 μM), the R-type Ca 2+ channel antagonist, SNX-482 [START_REF] Bourinet | Interaction of SNX482 with domains III and IV inhibits activation gating of alpha(1E) (Ca(V)2.3) calcium channels[END_REF] (200 nM) nor the selective T-type Ca 2+ channel blockers, mibefradil 36 (10 μM), TTA-P2 (1 μM) [START_REF] Dreyfus | Selective T-type calcium channel block in thalamic neurons reveals channel redundancy and physiological impact of I T window[END_REF] , TTA-A2 [START_REF] Shipe | Design, synthesis, and evaluation of a novel 4aminomethyl-4-fluoropiperidine as a T-type Ca 2+ channel antagonist[END_REF][START_REF] Uebele | T-type calcium channels regulate cortical plasticity invivo. [corrected][END_REF] (500 nM) had any effect on wildtype mEPSCs (Supplementary Fig 5 ,   Supplementary Table 2). Since wildtype mEPSC frequency inhibition in the presence of ω -agatoxin IVA and ω-conotoxin GVIA together is comparable to that resulting from removal of extracellular Ca 2+ (Fig 4a,b), it suggests that in these neurones, Ca 2+ -influx via P/Q-and N-type generates Ca 2+ -dependent mEPSCs.

Next we tested whether the ZD7288 induced enhancement in wildtype mEPSC immunoelectron microscopy experiments before and after embedding were described previously [START_REF] Lujan | Localization of metabotropic GABA receptor subunits GABAB1 and GABAB2 relative to synaptic sites in the rat developing cerebellum[END_REF] . Three antibodies were used: a monoclonal anti-HCN1 antibody was obtained from NeuroMab (USA); a polyclonal anti-HCN1 antibody was obtained from Alomone Labs (Israel); and a monoclonal Ca V 3.2 antibody was obtained from Neuromab. To examine the precise subcellular localization of HCN1 and Ca V 3.2 channels in the mouse entorhinal cortex, single and double labelling technique were used, as described previously [START_REF] Lujan | Preferential localization of the hyperpolarization-activated cyclic nucleotide-gated cation channel subunit HCN1 in basket cell terminals of the rat cerebellum[END_REF] .

When a single primary antibody was used, it was visualized by the silver-intensified immunogold reaction. In co-labeling experiments, HCN1 immunoreactivity was Digitized electron images were then modified for brightness and contrast by using Adobe PhotoShop CS1 (Mountain View, CA) to optimize them for printing.

Quantitative analysis. To establish the relative abundance of HCN1 in EC layer III, immunolabeling quantification was performed from 60 μm coronal slices as previously described [START_REF] Lujan | Perisynaptic location of metabotropic glutamate receptors mGluR1 and mGluR5 on dendrites and dendritic spines in the rat hippocampus[END_REF] . Randomly selected areas were captured at a final magnification of 45,000X, and measurements covered a total section area of ~5000 mm 2 . Dendritic shafts, dendritic spines and axon terminals were assessed for the presence of immunoparticles. The percentage of immunoparticles for HCN1 at postand presynaptic sites was calculated.

Estimation of number of synapses 6-9 week old mice were anaesthetised and perfused intracardially with a 3% glutaraldehyde in 0.05M sodium cacodylate buffer (pH 7.4).

The brains were removed and incubated in 1% OsO 4 for 3 hr at 4 o C. The tissue was then washed with distilled water followed by 70% ethanol, 90% ethanol and 100%

ethanol. The region of interest was then embedded in a beam capsule using a neat araldite CY212 resin mixture and polymerized for 3 days at 60 o C. Ultrathin sections were cut on a Reichert UCT at 85nm using a Diatome Ultra diamond knife, and collected on 400 mesh copper grids. Sections were subsequently stained was with 25% Uranyl acetate in methanol and Reynolds lead citrate and imaged using a Phillips CM 10 TEM. Electron micrographs were captured using a high sensitivity digital camera and synapses counted from digital images.

Western blot analysis EC samples from 6-8 week old wildtype and HCN1-/-or Ca V 3.2-/-mice were homogenized using a syringe and sonicated for 10 s in 50 mM Tris pH 7.4, 50 mM NaCl, 2 mM EDTA, 25 mM N-ethyl maleimide, with complete protease inhibitors (Roche). The buffer was then supplemented with Igepal and SDS to final concentrations of 1 % and 0.2 %, respectively. Samples were kept on ice for 29 30 -45 min and centrifuged for 30 min at 60000 x g. The pellet was discarded and the supernatant kept for SDS-PAGE analysis. The protein concentration of each sample was determined using a Bradford-based assay (Biorad). The amount corresponding to 50 µg of total protein was supplemented with gel loading buffer (50 mM Tris pH7.4, 100 mM DTT, 2 % SDS, 0.02% bromophenol blue, 2% glycerol), loaded onto a 3 -8 % Tris -acetate gel and subjected to SDS-PAGE following the manufacturer's instructions (Invitrogen). The transfer was performed on a PVDF membrane in a semi-dry chamber (Biorad). A slice of the membrane containing proteins larger than 150 kD was incubated with anti-Ca V 3.2 antibody (Neuromab, dilution 1:250), regions of same membrane containing proteins of lower molecular weight was incubated with either anti-HCN1 (Neuromab, dilution 1:3000) or anti-GAPDH antibody (Sigma, dilution 1:25,000), followed by the appropriate secondary antibodies conjugated to horseradish peroxidise, and enhanced chemiluminescence detection, using a Typhoon 9410 Variable Mode Imager (APB), set in chemiluminescence mode. The protein bands were background-subtracted and the signal quantified using Imagequant v5.2.

The same amount of total protein was loaded for all samples on each gel, for accurate comparison between lanes, and the data were normalized to the corresponding GAPDH signal.


James Robert Tollervey

Tomaž Curk

Boris Rogelj

Michael Briese

Matteo Cereda

Melis Kayikci

Tibor Hortobagyi

Agnes L Nishimura

Vera Zupunski

Rickie Patani

Tibor Hortobágyi

Vera Župunski

Siddharthan Chandran

Gregor Rot

Blaž Zupan

Christopher E Shaw

Jernej Ule

Characterising the RNA targets and position-dependent

TAR DNA binding protein (TDP-43) is a predominantly nuclear protein reported to regulate transcription, alternative splicing and RNA stability [START_REF] Buratti | Multiple roles of TDP-43 in gene expression, splicing regulation, and human disease[END_REF][START_REF] Kuo | Structural insights into TDP-43 in nucleic-acid binding and domain interactions[END_REF] . TDP-43 has two RNArecognition motif (RRM) domains, which bind UG repeat sequences with high affinity [START_REF] Buratti | Characterization and functional implications of the RNA binding properties of nuclear factor TDP-43, a novel splicing regulator of CFTR exon 9[END_REF] . TDP-43 mislocalisation and aggregation is implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD-TDP). Mutations in the gene encoding TDP-43 are associated with 1-4% of familial and sporadic ALS and rare FTLD cases, and confer toxicity [START_REF] Sreedharan | TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis[END_REF][START_REF] Lagier-Tourenne | TDP-43 and FUS/TLS: emerging roles in RNA processing and neurodegeneration[END_REF] . The pathology of ~60% of FTLD and ~90% of ALS cases are characterised by the formation of cytoplasmic inclusion bodies in neurons and astroglia that are rich in ubiquitinated, hyperphosphorylated C-terminal fragments of TDP-43 [START_REF] Lagier-Tourenne | TDP-43 and FUS/TLS: emerging roles in RNA processing and neurodegeneration[END_REF][START_REF] Neumann | Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis[END_REF] . Neurons containing cytoplasmic TDP-43 inclusions often have dramatically reduced TDP-43 staining in the nucleus [START_REF] Lagier-Tourenne | TDP-43 and FUS/TLS: emerging roles in RNA processing and neurodegeneration[END_REF][START_REF] Neumann | Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis[END_REF] . Thus, TDP-43 inclusions might have a toxic gain of function in the cytoplasm and/or sequester nuclear TDP-43, disrupting its role in RNA processing. TDP-43 cleavage products found in the cytoplasmic inclusions have been shown to alter splicing of a TDP-43 regulated exon in minigene studies, indicating that they are capable of interfering with wild-type TDP-43 function [START_REF] Igaz | Expression of TDP-43 C-terminal Fragments in Vitro Recapitulates Pathological Features of TDP-43 Proteinopathies[END_REF] . The toxicity of TDP-43 overexpression is dependent on retaining its RNA binding function in yeast [START_REF] Johnson | A yeast TDP-43 proteinopathy model: Exploring the molecular determinants of TDP-43 aggregation and cellular toxicity[END_REF] , drosophila and chick [START_REF] Voigt | TDP-43-mediated neuron loss in vivo requires RNA-binding activity[END_REF] .

To date, only a handful of RNAs regulated by TDP-43 have been identified. In order to understand the function of TDP-43, and its dysfunction in disease, we have comprehensively identified the RNA sites interacting with TDP-43 in human brain and characterised changes in RNA following reduced TDP-43 expression. In order to identify RNA targets on a global scale, we used individual-nucleotide resolution CLIP (iCLIP), which captures truncated cDNAs and quantifies them using a random barcode contained within the primer used for reverse transcription [START_REF] König | iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution[END_REF] . Duplicate sequences sharing the same random barcode were removed from analysis in order to avoid any apparent binding-site bias due to PCR artefacts.

In this study, we sought to identify the RNAs bound by TDP-43 in the healthy and FTLD brain tissue containing TDP-43 inclusions (FTLD-TDP). Although this was the first study of protein-RNA interactions in post-mortem human brain, we identified a high number of RNA binding sites using these samples. Most binding mapped to introns, long ncRNAs and intergenic transcripts, which also had the greatest enrichment of UG-rich motifs. We characterised the changes in alternative splicing following TDP-43 knockdown in neuroblastoma cells and identified splicing changes in 158 alternative cassette exons. Analysis of TDP-43 iCLIP crosslink sites in the vicinity of these exons provided insights into the mechanisms of TDP-43 splicing regulation. The exons regulated by TDP-43 are enriched in genes involved in neuronal development and those implicated in a range of neurodegenerative diseases.

RESULTS

Comparison of TDP-43 binding between healthy and FTLD-TDP brains.

TDP-43 in complex with its direct RNA targets was purified from UV-crosslinked cells and analysed on SDS-PAGE gel. The size of the dominant TDP-43-RNA complex present in the low and high RNase conditions corresponded to a single TDP-43 molecule bound to the RNA, however a complex corresponding to two TDP-43 molecules bound to the RNA was also present, particularly in the low RNase experiment (Fig. 1a). This result agreed with the finding of a past study that TDP-43 binds RNA as a homodimer [START_REF] Kuo | Structural insights into TDP-43 in nucleic-acid binding and domain interactions[END_REF] . iCLIP experiments were performed with human cortical tissue from human post-mortem brain samples of three cognitively normal (healthy) and three sporadic FTLD-TDP patients, as well as from embryonic stem cells (hES) and SH-SY5Y neuroblastoma cells (Supplementary Table 1). In total, 17 million sequence reads aligned as single hits to the human genome by allowing a maximum of one nucleotide mismatch. After eliminating PCR amplification artefacts by removing sequences that truncated at the same nucleotide in the genome and shared the same random barcode, 3,7 million reads representing unique cDNA molecules were identified (Supplementary Table 2).

Genomic annotation of the cDNA sequences showed that the majority of TDP-43 binding occurred in introns, 74% in the cell lines and 58% in brain samples (Fig. 1b). The decreased proportion in brain samples, and corresponding increase in exonic sequences, may be a result of a greater sensitivity of intronic RNA to degradation in postmortem samples. Approximately 5% of iCLIP cDNAs mapped to long ncRNAs in all samples (Fig. 1c). Increased binding of TDP-43 to snoRNAs, snRNAs, rRNAs and telomeric ncRNAs was seen in FTLD-TDP (Fig. 1c). In total, 2139 ncRNA genes contained a crosslink site in control and 2702 in FTLD-TDP experiments.

The high proportion of intronic cDNA sequences indicated that the iCLIP data mainly represent nuclear RNA targets of TDP-43. To further evaluate this, we quantified the crosslinked complexes that were obtained using the iCLIP procedure from nuclear and cytoplasmic fractions of healthy and FTLD-TDP brain tissue. Only 6.5% of the total TDP-43-RNA complexes were obtained from cytoplasmic fractions of brain tissue, with insignificant difference between healthy and FTLD-TDP tissues (Supplementary Fig. 1b). iCLIP analysis of the RNAs bound by TDP-43 in the two fractions in SH-SY5Y cells showed 34% 3'UTR binding in the cytoplasm, 3.2% in the nuclear fraction and 3.8% in total cell extract (Supplementary Fig. 1d, Fig. 1b). As a control, we analysed crosslink complexes with TIAL1, a protein that is known to shuttle between nucleus and cytoplasm. 31.5% of total TIAL1-RNA crosslinked complexes were obtained from cytoplasmic fractions of healthy and FTLD-TDP brain tissue (Supplementary Fig. 1b). Thus, even though the TIAL1 analysis demonstrates that iCLIP can efficiently purify proteins bound to cytoplasmic RNAs, our analysis indicates that over 93% of TDP-43 iCLIP data presented in this study represents interactions with nuclear RNAs both in healthy and FTLD-TDP brain tissue.

To study the changes in TDP-43 binding to individual transcripts in FTLD-TDP, we analysed the proportion of cDNAs mapping to individual co-expressed genomic regions in the different brain samples. A significant (p value < 0.05 by Student's t-test, one-tailed, unequal variance) and greater than 0.025% change in proportion of cDNAs between control and FTLD-TDP brain was found in four ncRNAs, 3' UTRs of seven transcripts and introns of 48 transcripts (Supplementary Table 3). The fact that introns had the greatest number of changes indicates that the changes detected by iCLIP mainly reflect changes in RNA interactions that occur in cell nuclei.

Four transcripts had a significant and greater than 0.1% change in proportion of cDNAs (p value < 0.05, Fig. 1d). Analysis of the abundance of these transcripts by RNA-seq and real-time PCR showed that the change in TDP-43 binding agreed with a similar extent of change in transcript expression (Fig. 1e, Supplementary Fig. 2). The most significant increase in TDP-43 binding in FTLD-TDP was observed in the nuclear paraspeckle assembly transcript 1 (NEAT1) and the metastasis associated lung adenocarcinoma transcript 1 (MALAT1, also known as NEAT2), which are transcribed from proximal loci on chromosome 11. Whereas NEAT1 was previously identified as a 4 kb long ncRNA [START_REF] Clemson | An architectural role for a nuclear noncoding RNA: NEAT1 RNA is essential for the structure of paraspeckles[END_REF] , we detected TDP-43 binding along the length of a 18kb ncRNA, with the peak binding in a tandem UG-repeat sequence close to the 3' end (Fig. 1d,f). The primary binding sites in these two ncRNAs contained long UG repeats, and were identified in a highly reproducible manner (Fig. 1f, Supplementary Fig. 1f). The neurexin 3 (NRXN3) transcript and the glial excitatory amino acid transporter-2 (EAAT2, also SLC1A2) had significantly decreased cDNA proportion in FTLD-TDP (Fig. 1d,g). EEAT2 is necessary for glutamate clearance at the synapse, a process that prevents glutamate excitotoxicity [START_REF] Cleveland | From Charcot to Lou Gehrig: deciphering selective motor neuron death in ALS[END_REF] .

TDP-43 binds to clusters of UG-rich sequences

In order to compare the RNA sequence specificity of TDP-43 in the different iCLIP experiments, we assessed the enrichment of all possible pentamers within 30 nt on either side of all crosslink sites. In all iCLIP data sets, the most significantly enriched pentamers were GUGUG and UGUGU (Fig. 2a, Supplementary Fig. 3a-c). The pentamer enrichment scores were correlated between healthy and FTLD-TDP brain (r=0.91, Fig. 2a).

We analysed the relationship between TDP-43 binding and UG repeats length by monitoring the frequency of crosslinking in the last ten nucleotides of UG tandem repeats compared to randomised positions. An example of a UG tandem repeat can be seen in the NEAT1 ncRNA (Fig. 1f). We found that enrichment of TDP-43 crosslinking to UG repeats increased up to a repeat length of 15 nt, in a manner that was similar in healthy and FTLD-TDP brain (Fig. 2b,c). For comparison, we performed iCLIP with CELF2, which also binds UG-rich motifs in the human brain (Supplementary Fig. 3c). In contrast to TDP-43, binding of CELF2 to UG repeats increased only up to a length of 10 nt, and dropped once the repeats became longer than 15 nt (Fig. 2b). This indicates that TDP-43 has a stronger preference for tandem UG repeats compared to CELF2.

Proteins interacting with multiple identical sequences on the RNA often allow spacing between different binding sites; for instance, NOVA proteins bind clusters of a minimum of three YCAY tetramers, but can tolerate variable spacing between these [START_REF] Ule | CLIP identifies Nova-regulated RNA networks in the brain[END_REF] . To analyse if TDP-43 can similarly bind to more dispersed UG-rich motifs, we monitored the frequency of UGUGU motifs over a 200 nt region surrounding the TDP-43 crosslink sites compared to randomised positions. UGUGU was seven-fold enriched at the TDP-43 crosslink sites, and remained two-fold enriched even at a distance of 100 nts away from the crosslink sites (Fig. 2d,e). One cause for this phenomenon may be a tendency of TDP-43 to bind to long UG-rich RNA regions, such as the 90 nucleotide region in the 3' UTRs of TARDBP mRNA, where TDP-43 binds to autoregulate its own mRNA levels (Fig. 2f) [START_REF] Ayala | TDP-43 regulates its mRNA levels through a negative feedback loop[END_REF][START_REF] Polymenidou | Disrupted processing of long pre-mRNAs and widespread RNA missplicing are components of neuronal vulnerability from loss of nuclear TDP-43[END_REF] . In contrast, even though UGUGU is four-fold enriched at the CELF2 crosslink sites, no such enrichment was seen at a distance of 100 nts (Fig. 2d). This indicates that TDP-43 has a unique capacity to recognise dispersed clusters of UG-rich motifs, or to spread its RNA binding to positions proximal to the UG-rich motifs.

To specifically analyse the high-affinity RNA binding sites of TDP-43, we grouped all data and searched for crosslink sites clustered with a maximum spacing of 15 nt that contained a significant cDNA count when compared to randomised positions (FDR < 0.05). 111,691 such crosslink clusters were identified. The reproducibility of crosslinking within these clusters between different samples increased with the number of iCLIP cDNA sequences that mapped to a cluster (Supplementary Fig. 3 d-g). For instance, 76% of clusters with summed cDNA counts of five or more in healthy brain were similarly bound in FTLD (Supplementary Fig. 3d). Clustered crosslink sites were highly enriched in UG tandem repeats, with increased enrichment at repeat lengths up to 30 nts (Fig. 2c). UGUGU was 18-fold enriched at the TDP-43 crosslink sites, and remained five-fold enriched even at a distance of 100 nts away from the crosslink sites (Fig. 2e). The increased incidence of UG-rich motifs at the crosslink clusters confirms that these clusters are the likely high-affinity binding sites of TDP-43.

To compare TDP-43 binding to different types of RNAs, we analysed the enrichment of UGUGU motif at the crosslink sites. The rRNAs, snRNAs, tRNAs and snoRNAs had the lowest UGUGU enrichment, indicating that crosslinking in these RNAs mainly reflected low-affinity, transient interactions (Supplementary Fig. 4). Three-fold enrichment was seen in 5'UTRs and ORFs, and four-fold UG enrichment was seen in 3' UTRs, miRNA precursors and telomeric transcripts (Supplementary Fig. 4). Similar UG enrichment was seen in the 3' UTRs when isolated with nuclear or cytoplasmic mRNA, indicating that TDP-43 binds to RNA in sequence-specific manner both in nucleus and the cytoplasm (Supplementary Fig. 1e). Seven-fold enrichment of UGUGU pentamers was seen in introns, long ncRNAs and intergenic RNAs, which together contain most TDP-43 binding (90% in cell lines and 76% in brain samples, Fig 1b,c). The high UG enrichment in intergenic RNAs is particularly interesting, indicating that high-affinity TDP-43 interactions occur with a large number of as yet non-annotated transcripts. Taken together, the iCLIP results indicate that TDP-43 binds pre-mRNAs and several types of ncRNAs in a sequence-specific manner.

The TDP-43 RNA splicing map

As the majority of iCLIP cDNAs mapped to introns, we reasoned that TDP-43 might have an important role in splicing regulation. Therefore, we used Affymetrix highresolution splice-junction microarrays to evaluate splicing changes in TDP-43 knockdown SH-SY5Y cells. Analysis of splicing isoform reciprocity using the ASPIRE3 software identified splicing changes in 158 alternative cassette exons and 71 other types of splicing changes (⏐∆Irank⏐≥1) out of 30154 alternative exons that were detected by the microarray. 40 of the cassette exons were further assessed using RT-PCR and capillary electrophoresis. For 32 splicing events, primer pairs generated both isoforms, and 28 of these reproduced the direction of the observed splicing change, resulting in a 87% (28/32) validation rate of the microarray data (Supplementary Table 4).

To study how TDP-43 binds pre-mRNAs to regulate splicing, we visualised the positions of crosslink clusters in the form of an RNA splicing map, where the crosslink sites are plotted within 500 nt of the silenced (blue clusters) or enhanced (red clusters) cassette exons and the flanking exons (Fig. 3). The crosslink clusters identified three regions on the RNA splicing map where TDP-43 binding was mainly restricted to silenced or enhanced exons (Fig. 3). Binding in the region 1, 0-150 nt upstream or within the alternative exons, was present at 11 silenced and 1 enhanced exon (group S1). Binding in the region 2, 150-500 nt downstream of the alternative exons, was present at 7 silenced and 1 enhanced exon (group S2). Five of the silenced exons contained multiple crosslink clusters that spanned a sequence of 100 nt or longer in this region. This indicates that 'deep' intronic binding involving multiple dispersed binding sites might contribute to the ability of TDP-43 to silence exon inclusion. Finally, binding in the region 3, 0-150 nt downstream of the alternative exons (but not region 1 or 2), was present at four enhanced exons (group E).

To evaluate the proportion of TDP-43 iCLIP targets that changed splicing after knockdown, we analysed splicing of exons with crosslink clusters in the three regions defined by the RNA splicing map. We used RT-PCR to evaluate splicing of 41 putative alternative exons that had highest number of cDNAs mapping to these regions. 29% (12/41) of these exons showed no evidence of alternative splicing in SH-SY5Y cells (single band detected by RT-PCR). 79% (23/29) of the remaining exons changed splicing after knockdown, indicating that iCLIP data alone has high predictive value (Supplementary Table 4, Supplementary Fig. 5b). We also assayed splicing of 12 alternative exons that had UG-rich regions within 200 nt of the alternative exons, but were not identified either by microarray or iCLIP. A splicing change in 5 of these exons was validated by RT-PCR (Supplementary Table 4). Similar to the iCLIP binding, UGrich motifs upstream of the exon or dispersed motifs downstream of the exon were associated with silencing, whereas proximal UG-rich motifs downstream of the exon were associated with enhancing function of TDP-43 (Supplementary Fig. 5c).

TDP-43 regulates splicing of mRNAs involved in neuronal development

TDP-43 crosslink clusters were also present in introns of 7,499 protein-coding genes and 3' UTRs of 1,172 genes. 67% of the genes with 3' UTR clusters (785/1,172) also had intronic clusters. For instance, two crosslink clusters were present in the intron downstream of the alternative exon 11 and three clusters in the 3' UTR of myocyte enhancer factor 2D (MEF2D) mRNA (Fig. 4a). We identified a 29% decrease in inclusion of the MEF2D exon 11 in TDP-43 knockdown SH-SY5Y cells (Fig. 4a). The additional binding sites in the 3' UTR indicate that TDP-43 can remain associated with the MEF2D mRNA after splicing is finished and thereby regulate additional aspects of RNA processing.

Due to the strong sequence-specificity in binding to long ncRNAs (Supplementary Fig. 4), we also speculated that TDP-43 might regulate processing of such ncRNAs. iCLIP identified an alternative exon in the long ncRNA myocardial infarction associated transcript (MIAT). Eleven crosslink clusters are dispersed over the 1 kb region of both introns that flank this exon (Fig. 4b). We identified a 43% decrease in inclusion of the MIAT exon 10 in TDP-43 knockdown SH-SY5Y cells (Fig. 4b, Supplementary Fig. 5b).

We also identified a crosslink cluster in the CFTR pre-mRNA, which was the first identified RNA target of TDP-43 [START_REF] Buratti | Characterization and functional implications of the RNA binding properties of nuclear factor TDP-43, a novel splicing regulator of CFTR exon 9[END_REF] . Importantly, the crosslink overlapped with the UG-rich element upstream of exon 9, which is required for TDP-43-dependent splicing regulation of this exon (Supplementary Fig. 6). Past studies identified several candidate RNA targets of TDP-43, for which the direct in vivo interaction with TDP-43 has not been validated. Here, we identified TDP-43 binding sites in the FUS pre-mRNA [START_REF] Sephton | Identification of neuronal RNA targets of TDP-43-containing Ribonucleoprotein complexes[END_REF] , hNFL 3' UTR [START_REF] Strong | TDP43 is a human low molecular weight neurofilament (hNFL) mRNA-binding protein[END_REF] , the CDK6 intron and 3' UTR [START_REF] Ayala | TDP-43 regulates retinoblastoma protein phosphorylation through the repression of cyclin-dependent kinase 6 expression[END_REF] , and the intronic region overlapping with the mir558 gene [START_REF] Buratti | Nuclear factor TDP-43 can affect selected microRNA levels[END_REF] (Supplementary Fig. 6). Also of particular interest, we observed crosslink clusters in the mRNAs encoding HDAC6 and Casein kinase 1 and 2, which were reported to regulate phosphorylation and expression of TDP-43, respectively [START_REF] Kametani | Identification of casein kinase-1 phosphorylation sites on TDP-43[END_REF][START_REF] Fiesel | Knockdown of transactive response DNA-binding protein (TDP-43) downregulates histone deacetylase 6[END_REF] (Supplementary Fig. 6).

We performed GO term analysis of genes regulated by TDP-43 at the level of alternative splicing, as determined by analysis of SH-SY5Y knockdown cells. This identified several terms related to development, including neural tube formation, organ morphogenesis and chordate embryonic development (Table 1). 15 of the exons regulated by TDP-43 are within genes encoding proteins with well-characterised functions in neuronal survival or development (Table 2). TDP-43 promotes inclusion of alternative exon 3 in Bcl-2 interacting mediator of cell death (BIM, also BCL2l11), and thereby suppresses production of the most cytotoxic BIM isoform (BIM S ) [START_REF] O'connor | Bim: a novel member of the Bcl-2 family that promotes apoptosis[END_REF] . We have evaluated splicing of BIM in FTLD-TDP, and have found that the BIM S isoform increases in FTLD-TDP tissue to a similar extent as in TDP-43 knockdown cells (Fig. 4c). This result indicates that aberrant splicing might contribute to the neurodegenerative process in human brain.

DISCUSSION

Our results show that TDP-43 interacts with a diverse spectrum of RNAs with important functions in the brain. We identified binding of TDP-43 to non-coding RNAs, introns and 3' UTRs of mRNAs, indicating a role for TDP-43 in coupling different aspects of gene expression. Even though only a minor portion of the protein was detected in the cytoplasm, we found a ten-fold increase of TDP-43 binding to 3' UTR elements when comparing RNAs bound to cytoplasmic and nuclear protein. We also identified the changes in alternative splicing associated with reduced level of TDP-43 protein. Analysis of crosslink clusters in the regulated pre-mRNAs indicates that the splicing function of TDP-43 depends on the position where it interacts with pre-mRNAs.

We found that TDP-43 preferentially binds to UG tandem repeats or long clusters of UGrich motifs. These results agree with the past in vitro studies which showed that RNA binding of TDP-43 increases with the length of the UG repeat [START_REF] Kuo | Structural insights into TDP-43 in nucleic-acid binding and domain interactions[END_REF][START_REF] Buratti | Characterization and functional implications of the RNA binding properties of nuclear factor TDP-43, a novel splicing regulator of CFTR exon 9[END_REF] . iCLIP results also indicate that the increased affinity for long binding sites differentiates the RNA specificity of TDP-43 from CELF2, which recognises shorter clusters of UG-rich motifs. Since a single RRM domain generally recognises four nucleotides [START_REF] Clery | RNA recognition motifs: boring? Not quite[END_REF] , this RNA specificity for long binding sites indicates that TDP-43 can cooperatively recognise multiple RNA binding sites and thereby achieve high-affinity RNA binding. The apparent RNA binding of TDP-43 in a homodimer state might contribute to its ability to recognise binding sites composed of multiple dispersed UG-rich motifs (Fig. 1a).

TDP-43 crosslink clusters were located upstream and within the silenced exons and immediately downstream of enhanced exons, which is also a characteristic of several other RBPs, including the Nova proteins [START_REF] Witten | Understanding splicing regulation through RNA splicing maps[END_REF] . In addition, dispersed clusters of TDP-43 crosslinking further downstream of exons were associated with splicing silencing. Similar mode of splicing silencing, involving multiple binding sites flanking an alternative exon, has also been reported for Sxl, hnRNP A1, PTB, hnRNP L and hnRNP C [START_REF] König | iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution[END_REF][START_REF] Black | Mechanisms of alternative pre-messenger RNA splicing[END_REF][START_REF] Hung | Diverse roles of hnRNP L in mammalian mRNA processing: a combined microarray and RNAi analysis[END_REF] . The glycine-rich C-terminal domain of Sxl and hnRNP A1 is required for the cooperative assembly of these proteins on multiple proximal binding sites and the associated splicing silencing [START_REF] Black | Mechanisms of alternative pre-messenger RNA splicing[END_REF] . Interestingly, TDP-43 also contains a glycine-rich C-terminus. Mutations in the TARDBP gene that were identified in ALS or FTLD patients are concentrated in the region encoding the glycine-rich C-terminus [START_REF] Lagier-Tourenne | TDP-43 and FUS/TLS: emerging roles in RNA processing and neurodegeneration[END_REF] , indicating that the ability of TDP-43 for cooperative assembly on long RNA binding sites plays a role in the disease mechanisms.

The tandem UG repeat sequences recognised by TDP-43 correspond to the most common microsatellite (AC) in the human genome. Variation in the length of microsatellites can introduce heritable phenotypic variation [START_REF] Rockman | Abundant raw material for cis-regulatory evolution in humans[END_REF] or lead to changes in transcription and alternative splicing [START_REF] Kashi | Simple sequence repeats as advantageous mutators in evolution[END_REF] . Genes involved in organ morphogenesis and neurogenesis are enriched in the variable tandem repeats [START_REF] Legendre | Sequence-based estimation of minisatellite and microsatellite repeat variability[END_REF] . Similarly, the genes containing the exons regulated by TDP-43 are enriched for neural tube formation and organ morphogenesis. This is consistent with the finding that TDP-43 knockout is lethal for mice from embryonic day 3.5 [START_REF] Wu | TDP-43, a neuro-pathosignature factor, is essential for early mouse embryogenesis[END_REF] . It could be speculated that the ability of TDP-43 to regulate splicing by binding the UG repeats in the human genome might contribute to variation in splicing regulation of genes involved in development.

TDP-43 regulates splicing of several transcripts encoding proteins involved in neuronal survival, as well as seven mRNAs encoding proteins with relevance for neurodegenerative diseases (Table 2). Delta-catenin (CTNND1) plays a role in the development and maintenance of dendritic spines [START_REF] Matter | Delta-catenin is required for the maintenance of neural structure and function in mature cortex in vivo[END_REF][START_REF] Ishiyama | Dynamic and static interactions between p120 catenin and Ecadherin regulate the stability of cell-cell adhesion[END_REF] . Furthermore, CTNND1 interacts with presenilin-1 to inhibit production of Aβ peptide, the main constituent of amyloid plaques in the brains of Alzheimer's disease patients [START_REF] Kouchi | p120 catenin recruits cadherins to gamma-secretase and inhibits production of Abeta peptide[END_REF] . MEF2D is a transcription factor that was reported to play a role in neuronal survival in Parkinson's disease [START_REF] Yang | Regulation of neuronal survival factor MEF2D by chaperonemediated autophagy[END_REF] . BIM is a core factor in the neuron-specific JNK-mediated apoptotic pathway, and loss of BIM protects mice from ischemia-induced damage [START_REF] Ness | Selective involvement of BH3-only Bcl-2 family members Bim and Bad in neonatal hypoxia-ischemia[END_REF][START_REF] Becker | Pin1 in neuronal apoptosis[END_REF] . The BIM splice isoforms regulated by TDP-43 have been functionally characterised; BIM S antagonizes the pro-survival Bcl-2 family members more effectively than BIM L , while BIM EL is the least potent [START_REF] O'connor | Bim: a novel member of the Bcl-2 family that promotes apoptosis[END_REF] . We found that production of the most cytotoxic BIM isoform (BIM S ) was increased in TDP-43 knockdown cells and in FTLD-TDP. However, analysis of other exons regulated by TDP-43 did not indicate a significant correlation with the splicing changes present in FTLD-TDP (unpublished observation).

We also found that TDP-43 binds to long ncRNAs in highly sequence-specific manner both in control and FTLD-TDP samples. The greatest increase in TDP-43 binding in FTLD-TDP is seen in the NEAT1 ncRNA. Expression of NEAT1 and MALAT1 significantly increases in FTLD-TDP, which might be the primary cause of the increased association of TDP-43 with these RNAs. NEAT1 functions in paraspeckle assembly [START_REF] Clemson | An architectural role for a nuclear noncoding RNA: NEAT1 RNA is essential for the structure of paraspeckles[END_REF] , and the MALAT1 ncRNA, which recruits splicing factors to nuclear speckles and affects phosphorylation of SR proteins [START_REF] Tripathi | The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation[END_REF] . In conclusion, we have characterised the splicing function of TDP-43 and quantified the changes in its RNA binding in FTLD-TDP. Analysis of these RNAs in model organisms and in neurons containing TDP-43 inclusions will be required for full understanding of the mechanisms of TDP-43 loss-or gain-of-function effects in the neurodegenerative process.

Methods iCLIP analysis

The iCLIP protocol was performed as described previously [START_REF] König | iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution[END_REF] , with the following modifications. SH-SY5Y neuroblastoma or H9 human embryonic stem cells were irradiate once with 150 mJ/cm 2 in a Stratlinker 2400 at 254 nm, and brain tissue was dissociated in cold PBS and the suspension was crosslinked four times with 100 mJ/cm 2 . TDP-43 was immunoprecipitated with protein A Dynabeads (Invitrogen) conjugated to rabbit-anti TDP-43 (Proteintech, 10782-2-AP). For iCLIP of CELF2, protein G Dynabeads conjugated to mouse anti-CELF2 (Sigma, C9367) were used. In both cases, the region corresponding to 55-100kDa complexes was excised from the membrane to isolate the RNA. High-throughput sequencing using Illumina GA2 was done using 54 or 72 cycles (Supplementary Table S1). The barcode sequences corresponding to the individual experiment were as described (Supplementary table 1). The random barcodes were registered and the barcodes were removed before mapping the sequences to the human genome sequence (version GRCh37/hg19) allowing one mismatch using Bowtie version 0.10.1 (command line: -a -m 1 -v 1).

Nucleocytoplasmic fractionation

50mg of crosslinked SH-SY5Y cells or brain tissue was resuspended in cold 1ml of cytoplasmic lysis buffer (50 mM Tris-HCl, pH 7.4, 10mM NaCl, 0.5% NP-40, 0.25% Triton X-100, 1mM EDTA, 0.5% RNAsin). After rotation in cold room for 5 minutes, homogeniser was used to complete the lysis. After centrifugation at 4°C, 3000 x g for 5 minutes, supernatant was collected and centrifugation was repeated at 4°C, 10000 x g for 10 minutes. Supernatant was collected, and 200ul of 0.5% SDS, 0.25% sodium deoxycholate, 0.5M NaCl was added; this was used as cytoplasmic fraction for CLIP or western blot analyses. The pellet (after 3000 x g spin) was resuspended in 1ml of CLIP Lysis Buffer 10 , sonicated and used as nuclear fraction for CLIP or western blot analyses. The radioactive CLIP protein-RNA complexes on nitrocellulose membrane were quantified using phosphoimager.

Sequence analyses

Analysis of reproducibility of crosslink sites was done as described previously [START_REF] König | iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution[END_REF] . Identification of the significant iCLIP crosslink clusters and z-score analysis of enriched pentamers was done as described previously [START_REF] Wang | iCLIP predicts the dual splicing effects of TIA-RNA interactions[END_REF] . For analysis of crosslinking to UG repeats of different lengths, the number of UG repeats in transcribed genome with a crosslink site in at least one of the last ten nucleotides is determined for each data set. Enrichment represents the ratio in the number of UG repeats with a crosslink site in real data compared to randomized data. The pentamer with highest z-scores in TDP-43 iCLIP (UGUGU) was used to assess the positions at which the TDP-43 binding sites were enriched relative to the position of crosslink sites. The genomic sequences at positions within 100 nts of the iCLIP crosslink site were filtered to retain only the sequence containing the pentamer. The sequences were aligned at each iCLIP crosslink site, and the number of sequences containing the pentamer was determined at each nucleotide position between -100 to +100 nts. Enrichment represents the ratio in the number of UG repeats with a crosslink site in real data compared to randomized data.

siRNA knockdown

Acell Smartpool oligos against TARDBP (E-012394-00-0005, Dharmacon) were transfected according to the manufacturer's instructions (Acell, Dharmacon). Accell nontargeting pool oligos (D-001910-10-05, Dharmacon) were used as a control.

A-012394-14, TARDBP: 5'-GGCUCAAGCAUGGAUUCUA -3' A-012394-15, TARDBP: 5'-GUCUCAAGUCAAAUGGAUU -3' A-012394-16, TARDBP: 5'-GUGUUAAGUGAAAUGAUAC -3' A-012394-17, TARDBP: 5'-GGGUGAUGUUCUAUUUACA -3'

Western blot

SH-SY5Y cells were lysed in iCLIP lysis buffer and sonicated. To digest DNA, 10 μl of DNase (Ambion) was added per 1 ml of lysate, and incubated at 37 C for 3 minutes. The samples were centrifuged and the supernatants were collected for Western analysis. The protein concentration was determined using Lowry's Assay (Bio-RAD). Equal amounts of protein were loaded on 4-12% Bis-Tris gels (Invitrogen) and transferred to a nitrocellulose membrane. The membrane was incubated with rabbit anti-TDP-43 antibody (Proteintech, 10782-2-AP) (1:1000 dilution). Rabbit anti-GAPDH (Cell Signalling) (1:5000 dilution) or anti-actin antibodies were used as a loading control.

Splice-junction microarray

A total of 6 samples were used, 3 from the siRNA control and 3 from KD. The highresolution AltSplice splice-junction microarrays were produced by Affymetrix, the cDNA samples were prepared using the GeneChip WT cDNA Synthesis and Amplification Kit (Affymetrix) and data was analysed with the version 3 of ASPIRE (Analysis of SPlicing Isoform REciprocity) [START_REF] König | iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution[END_REF] .

RT-PCR

Total RNA was extracted using RNasy Kit (Qiagen) and 200 ng of RNA was used for reverse transcription using Superscript III (Invitrogen) according to the manufacturer's instructions. For analysis of transcript levels, real-time PCR was performed using SYBR Green Fast PCR master mix (Applied Biosystems). For analysis of splicing, PCR was performed using Immomix (Bioline) using primers listed in Supplementary Table 4. The PCR products were visualised using QIAxcel capillary electrophoresis system (Qiagen). To calculate exon inclusion (I), the percentage of the peak representing exon inclusion was divided by the total percentage of peaks representing exon inclusion and skipping. Splicing change was calculated by subtracting the exon inclusion in the knockdown cells from the inclusion in wild-type cells (thus, a positive ∆I represents exons enhanced by TDP-43, and negative those silenced by TDP-43).

RNA splicing map

In order to analyze the impact of TDP-43 positioning on splicing regulation, we assessed the positioning of crosslink clusters in proximity of the exon/intron boundaries of alternative exons and flanking exons, including 50 nt of exonic and 500 nt of intronic sequence. All cassette exons with identified splicing change in knockdown SH-SY5Y cells and at least one significant crosslink cluster in the analysed regions are shown in the RNA splicing map.

Figure 1 :

 1 Figure 1: Comparison of TDP-43 RNA binding in healthy and FTLD-TDP brain. (a)To validate specificity of TDP-43 antibody for iCLIP, the[START_REF] Ishiyama | Dynamic and static interactions between p120 catenin and Ecadherin regulate the stability of cell-cell adhesion[END_REF] P-labelled RNA bound to TDP-43 gel was isolated from control (Ctr) or knockdown (KD) HeLa cells in the presence or absence of UV crosslinking or anti-TDP-43 antibody. High and low RNase concentrations were used to confirm the presence of RNA bound to TDP-43. The arrows mark the positions on the gel corresponding to the size of TDP-43 monomer or dimer. TDP-43 Western analysis of input extracts confirmed TDP-43 knockdown, and GAPDH was used as a loading control. The full image is shown in Supplementary Fig.1a (b) The proportion of cDNAs (out of all cDNAs that mapped to human genome) from the TDP-43 iCLIP experiments in the four types of samples that mapped to different RNA regions. (c) The proportion of cDNAs that mapped to different types of ncRNAs. (d) The proportion of cDNAs that mapped to individual RNAs with at least 10 cDNAs in any experiment. The RNAs with the largest significant change between control and FTLD-TDP brain (difference in proportion of cDNAs > 0.1% and p value < 0.05 by Student's ttest, one-tailed, unequal variance) are marked in red. The long ncRNA MEG3 (maternally expressed 3) with the largest decrease in TDP-43 binding in FTLD-TDP is also marked. (e) Real-time PCR analysis of transcripts with largest TDP-43 iCLIP changes in total RNA isolated from control and FTLD-TDP brain samples. (f) iCLIP cDNA counts for TDP-43 crosslink positions in the NEAT1 gene in experiments from SH-SY5Y and hES cell lines, and healthy and FTLD-TDP tissue from different individuals. The blue bars represent sequences on the sense strand of the genome, and the height of the bars corresponds to the cDNA count. The positions of significant crosslink clusters (XL cluster) are shown on top, and the RNA sequence underlying the two man clusters is shown below, with UG repeats in pink. The graph shows the average proportion of cDNAs that map to NEAT1 transcript in each experiment (out of all cDNAs maping to the human genome), as well as standard deviation between experiments from different individuals. (g) iCLIP cDNA counts for TDP-43 crosslink positions in the SLC1A2 gene. The orange bars represent sequences on the antisense strand of the genome. The graph shows the average proportion of cDNAs that map to SLC1A2 3' UTR in different experiments, as well as standard deviation between experiments from different individuals. The RNA sequence underlying the peak crosslinking sites is shown below, with UG repeats in pink.

Figure 2 :

 2 Figure 2: TDP-43 binding motif analysis. (a) z-scores of penamer occurrence within the 61 nt sequence surrounding all crosslink sites (-30 nt to +30 nt) are shown for healthy and FTLD-TDP brain iCLIP. The sequences of the two most enriched pentamers and the Pearson correlation coefficient (r) between the two samples are given. (b) Enrichment of crosslinking compared to randomised data in UG repeats of different lengths in TDP-43 iCLIP experiments from healthy brains (blue) and FTLD-TDP brains (red), and CELF2 experiments in healthy brain (green). (d) Enrichment of crosslinking compared to

Figure 3 :

 3 Figure 3: The RNA splicing map of TDP-43. The map of crosslink clusters at positions within 500 nt of alternative exons and flanking exons. The cassette exons with ∆Irank > 1 (enhanced exons, red clusters) or ∆Irank < -1 (silenced exons, blue clusters) with at least one crosslink cluster in these regions are shown. The exons are grouped by sequential analysis of crosslink cluster positions in three regions: group S1 is identified by clusters in region 1, 150nt upstream till the exon and within the exon; group S2 by clusters in region 2, 150-500nt downstream of the exon; and group E by clusters present 0-150nt downstream of the exon.

Figure 4 :

 4 Figure 4: TDP-43 regulates splicing of non-coding and protein-coding RNAs. (a) TDP-43 crosslinking in MIAT ncRNA. UG repeat lengths are shown in pink bars and crosslink clusters are shown as black bars. Analysis of MIAT exon 10 inclusion (gel electropherogram, left, and quantification, right) in control and TDP-43 knockdown SH-SY5Y cells is shown below. (b) TDP-43 crosslinking in MEF2D protein-coding transcript. UG repeat lengths are shown in pink bars and crosslink clusters are shown as black bars. Analysis of MEF2D exon 11 inclusion (gel electropherogram, left, and quantification, right) in control and TDP-43 knockdown SH-SY5Y cells is shown below. (c) Analysis of BIM exon 3 splicing (gel electropherogram, left, and quantification, right) in control and TDP-43 knockdown SH-SY5Y cells, and in healthy (C23, C25, C30) and FTLD-TDP (F19, F20, F21) brain samples.

Figure 1 a

 1 Figure 1 a

  Figure 2

Table 1 : Significant GO terms of the exons regulated by TDP-43.Table 2 : Neuronal functions of proteins encoded by the alternative mRNA isoforms regulated by TDP-43.

 12

Table 1 : Significant GO terms of the exons regulated by TDP-43.

 1

	GO term	p-value	e exons referenc		TDP-43 regulated exons
					BCL2L11_E5, COL4A3BP_E12, CTNND1_E30,
	organ morphogenesis	<0.001	811	13	DLC1_E2, FGFRL1_E4, FZD6_E2, MACF1_E68, MEF2D_E11, PLEKHA1_E21, PLEKHA1_E23, TFAP2A_E2, TLE1_E10, TPM1_E7
	neural tube closure	0.001	45	3	DLC1_E2, FZD6_E2, TFAP2A_E2
	homeostasis of number of cells	0.01	187	4 BCL2L11_E5, RPS24_E11, RPS24_E9, SOX6_E9
	mitotic cell cycle	0.016	856	9	CLIP1_E33, HORMAD1_E4, KIF2A_E19, MYO16_E8, PMF1_E2, PSMD6_E4, RBM38_E3, SIRT7_E9, STAG2_E36
	cell surface receptor linked signaling pathway	0.018	2005	16	ANXA1_E2, BRD8_E24, CNTFR_E2, CTNND1_E30 FZD6_E2, GRM4_E7, HOMER2_E5, IRAK3_E2, MACF1_E68, MADD_E40, PILRB_E25, PLEKHA1_E21, PLEKHA1_E23, PTPRD_E78, TLE1_E10, TNIK_E15
	chordate embryonic development	0.021	475	6	BCL2L11_E5, COL4A3BP_E12, DLC1_E2, FZD6_E2, SOX6_E9, TFAP2A_E2
	lipid transport	0.037	276	4	ANXA1_E2, ATP11C_E36, COL4A3BP_E12, STARD4_E5

Table 2 : Neuronal functions of proteins encoded by the alternative mRNA isoforms regulated by TDP-43.

 2

	Gene symbol	Gene name	neurologic disease association	neuronal function	Pubmed PMID
	AP2 (TFAP2A)	transcription factor AP-2 alpha		cranial neural-tube closure	11137286
	BIM (BCL2L11)	bcl-2 interacting mediator of cell death	ischemia-induced neuronal death	apoptotic signalling cascade	16780816 17568190
	CNTFR	ciliary neurotrophic factor receptor	Amyotropic lateral sclerosis	neuronal survival	19386761
	MADD	MAP-kinase activating death domain	Alzheimer's disease	neuronal survival conditions under stress	9482930
	MEF2D	myocyte enhancer factor 2D	Parkinson's disease	neuronal survival	19119233
		CDK5 regulatory	Primary autosomal-	maintainance of the
	CDK5RAP2	subunit associated	recessive	neural progenitor	20471352
		protein 2	microcephaly	pool
	CTNND1	delta catenin	mental retardation syndrome Cri-du-Chat	maintenance of dendritic spines	19914181
	DLC1	deleted in liver cancer 1		neural tube development	15710412
	FZ3 (FZD3) frizzled homolog 3		axonal guidance, neural tube closure	16495441
	KIF2A	kinesin family member 2A		axonal branching	12887924
	KIF1B	kinesin family member 1B	Charcot-Marie-Tooth neuropathy	axonal vesicle transport	11389829
	SOX6	transcription factor SOX-6		cortical interneuron development	19657336
	TLE1	transducin-like enhancer of split 1		cortical neuron differentiation	16314515
	TNIK	TRAF2 and NCK interacting kinase		dendrite formation	20159449
	UNC5C	unc-5 homolog C (netrin receptor)		spinal accessory motor neuron development

Acknowledgement

The authors wish to thank Kathi Zarnack and Julian König for their advice and comments on the manuscript, Francisco Baralle, Yuna Ayalla and Andrea D.Ambrogio for providing HeLa knockdown RNA, and James Hadfield and the genomic team at CRI for Illumina sequencing. This work was supported by the European Research Council grant (206726-CLIP), the Medical Research Council (MRC), Slovenian Research Agency (P2-0209, J2-2197, L2-1112, Z7-3665), Wellcome Trust and Medical Research Council Strategic Grant Award (089701/Z/09/Z), Motor Neuron Disease Association, Heaton-Ellis Trust and Psychiatry Research Trust.

Author contributions

J.R.T. carried out TDP-43 iCLIP, microarray and PCR experiments, M.B. carried out CELF2 iCLIP, T.C. and G.R. mapped the iCLIP sequence reads to genome, evaluated random barcodes, determined crosslink clusters and annotated the data, T.C. analyzed the reproducibility, sequence and positioning of TDP-43 crosslink sites and performed gene ontology analysis, B.R., A. L.N. and V.Ž. prepared RNA from knockdown cells and brain tissue, T.H. collected the brain tissue, M.C. and M.K. analyzed splice-junction microarray data and generated the RNA splicing map, R.P. prepared the embryonic stem cells, S.C, S.E.S, B.Z. and JU supervised the project, J.R.T., T.C., B.R., C.E.S and J.U. prepared the manuscript.

Supplementary Figure Legends

Supplementary Figure 1 Exon/intron structure of the transcript is indicated below. In the first panel, the alternative exon 9 is marked by a box and the RNA sequence underlying the TDP-43 binding cluster is given below (UG and GU motifs in pink).

Supplementary Table S1. Experimental annotation and barcoding.

Supplementary Table S2. Mapping information for iCLIP data. S3. RNA regions with a significant, greater than 0.025% change in proportion of cDNAs between control and FTLD-TDP brain (p value < 0.05 by Student's t-test, one-tailed, unequal variance). Supplementary Table S4. RT-PCR primers for validation of splicing events identified by the microarray, iCLIP crosslink clusters or UG repeats.

Supplementary Table