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Truncating tau reveals different pathophysiological actions of oligomers in single neurons

Tau protein is involved in maintaining neuronal structure. In Alzheimer’s disease, small numbers of tau molecules can aggregate to form oligomers. However, how these oligomers produce changes in neuronal function remains unclear. Previously, oligomers made from full-length human tau were found to have multiple effects on neuronal properties. Here we have cut the tau molecule into two parts: the first 123 amino acids and the remaining 124-441 amino acids. These truncated tau molecules had specific effects on neuronal properties, allowing us to assign the actions of full-length tau to different regions of the molecule. We identified one key target for the effects of tau, the voltage gated sodium channel, which could account for the effects of tau on the action potential. By truncating the tau molecule, we have probed the mechanisms that underlie tau dysfunction, and this increased understanding of tau’s pathological actions will build towards developing future tau-targeting therapies.


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Tau is a microtubule-associated protein which can modify neuronal morphology, vesicle transport and

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To address these limitations, whole-cell patch-clamp recording has recently been used to introduce 62 oTau into single neurons and has enabled specific targeting to either pre-or post-synaptic cells 63 (11,18). This approach only requires small amounts of oligomers, each cell acts as its own control and 64 electrophysiological alterations can be observed in real time. Furthermore, direct comparisons can be effects on firing rate can occur independently of changes in input resistance ( Figure 2C-E). In contrast 111 to FL-oTau, which in agreement with (11), significantly increased action potential width and decreased 112 action potential height, introduction of C FRAG -oTau had no significant effects on the action potential 113 waveform ( Figure 2D-G). Thus, loss of the first 123 amino acids from tau removes the effects on input 114 resistance and action potential waveform. These effects could either be a direct effect of the N-115 terminal sequence or could result from a change in the structural conformation of the oligomers, which 116 could differ following truncation.

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A reduction in the rheobase current induced by C FRAG -oTau is consistent with its effects on 118 firing rate.

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To evaluate the effects on neuronal excitability in more detail, we examined the rheobase (the 120 minimal current required to elicit an action potential). The rheobase was measured by injecting a 121 current ramp (see methods). There was no significant difference in the rheobase in control (vehicle) 122 vs FL or C FRAG -oTau injected neurons at time 0 (Table 1, Figure 3). In control neurons, there was no 123 significant difference in the rheobase current over the duration of the recording. However, when either 124 FL or C FRAG -oTau were introduced, the rheobase was significantly reduced (Table 1, Figure 3). This 125 is consistent with the observed increased in firing rate in response to fluctuating noisy current injection 126 ( Figure 2F, G).

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We hypothesized that the decrease in rheobase current might be due to a smaller difference between 130 the resting membrane potential and spike initiation threshold (value of membrane potential that needs 131 to be surpassed to fire an action potential). Since there is no change in resting membrane potential, 132 this could only occur if there was a hyperpolarising shift in spike initiation threshold. The dynamic IV 133 method (25-27) provides a protocol to accurately parameterise neurons including spike initiation 134 threshold (see methods). In (11), it was not possible to map changes in neuronal parameters over 135 time using the dynamic IV method, as it is not very effective if there are significant changes in the 136 action potential waveform. However, as C FRAG -oTau has no effect on the action potential waveform, it 137 is possible to use this method to measure spike initiation threshold. There was no significant 138 difference between the spike initiation threshold in control neurons (vehicle) vs neurons receiving in 139 C FRAG -oTau at 0 mins. In control neurons, there was no significant difference in spike initiation 140 threshold after 40 minutes (recording duration), whereas for neurons where C FRAG -oTau was 141 introduced, spike initiation threshold was significantly hyperpolarised. Although the reduction in spike 142 initiation threshold was only ~4 mV, across a network this would cause a large shift in excitability. We 143 also noted an increase in spike onset sharpness, which is a measure of how narrow the voltage range 144 within which a spike starts to initiate, as well as how sharply the spike begins to rise (sharper DIV 145 curve). In control neurons, there was no significant difference in spike onset over the duration of the 146 recording, whereas for neurons where C FRAG -oTau was introduced, the onset was increased (steeper, 147 reflecting increased excitability). It can be noted that though the spike-onset range was broader at 40 148 rate (see Figure 4D).

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N FRAG -tau aggregates generate a rapid increase in input resistance and change action potential 152 waveform.

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As C FRAG -oTau did not replicate the changes to input resistance and action potential waveform

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To test the possibility that this rapid change to whole-cell resistance and capacitance could be due to  Figure 5C). We hypothesised that if N FRAG -tau was injected via pipette 1, there will 173 be an increase in the whole cell resistance, less current will leak out from the neuron and thus the 174 voltage response measured from both pipettes will be increased without a change in the ratio. If 175 however the N FRAG -tau aggregates in the soma and interferes with the current flow between the 176 pipettes then the ratio of the voltage response between the pipettes will be reduced as less current 177 will reach pipette 2.

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A 200 pA (1s) hyperpolarising step was only injected via pipette (1.) and voltage responses were 180 measured from both pipettes ( Figure 5C, pipettes 1. and 2.). As the current needs to travel from 181 pipette 1 to pipette 2, the voltage response was always slightly smaller in pipette 2 than in pipette 1. In 182 cells where both pipettes contained vehicle, there was no change in the relative amplitude of the 183 voltage responses between 0 mins (whole-cell breakthrough) and 5 mins ( Figure 5D). However, when 184 N FRAG -oTau was introduced via pipette 1, there was a large increase in the voltage response 185 measured by pipette 1 as a result of the increase in cell resistance ( Figure 5E) . However, this was result is consistent with N FRAG -tau accumulating in the soma, impeding the flow of current between 189 the two pipettes, resulting in an 'increase in the apparent input resistance and a decrease in the 190 observed capacitance due to the cell appearing electronically smaller.  in acute brain slices is challenging due to problems with space clamp, given the size of the neurons 221 and the large amplitude and speed of the currents. One way to effectively clamp the currents, is to 222 electronically isolate the soma. We followed the protocol outlined in (28), using a short depolarising 223 pre-pulse to inactivate the axonal sodium channels, leaving only the somatic channels to be activated 224 (see methods). It was then possible to achieve reasonable voltage clamp ( Figure 7A). Normalised

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We then implemented a simplified model of the neuronal action potential with general applicability

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(29) to evaluate whether the conductance changes mediated by FL-oTau could feasibly underly the 239 changes we observed in action potential waveform. Our experimental voltage gated sodium current 240 recordings showed that FL-oTau mediates a reduction of ḡ to by two thirds of the value at 0 241 minutes after 20 minutes (7F-I). Therefore, we first simulated an action potential with ḡ = 242 120 / (28, Figure 7J, red) and then again with ḡ = 40 / ( Figure 7J, red trace). We

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found that this reduction in maximal conductance matches the experimental phenotype well (in 244 particular the changes in action potential amplitude and speed of rise to threshold; Figure 7K).

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In Alzheimer's disease (AD) and in other tauopathies, tau aggregates into small soluble toxic

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It is conceivable that some of the changes in electrophysiological properties that we have observed 262 could result from tau oligomers blocking the tip of the patch pipette and increasing series resistance.

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In particular the changes in action potential amplitude and waveform kinetics. In previous studies . These oligomers are much larger than the tau oligomers but had no effect on the action potential 276 amplitude or waveform kinetics (19).

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The N-terminal truncation (C FRAG ) at position 123, has been identified as a major N-terminal cleavage 279 site in tau found in human CSF (21) and a truncated tau construct at a nearby position has been used 280 to study effects on synaptic transmission (31). We found that C FRAG -oTau did not replicate the effects 281 on action potential kinetics or input resistance that were observed with FL-oTau but did still increase 282 firing rate. This firing rate increase is mediated by a reduction in the minimum current required to elicit 283 an action potential. Using the dynamic IV protocol we found that C FRAG -oTau reduces spike threshold

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We then investigated the tau fragment that was removed to generate C FRAG (aa 1-123; N FRAG ). This

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Through measurements of the cell time constant we found that the apparent capacitance was 302 significantly decreased. This led us to hypothesise that N FRAG was inferring with current flow, making 303 the neuron electrotonically smaller. By making simultaneous dual patch clamp recordings from the 304 soma of a single neuron, we found that although input resistance was significantly increased, the 305 current flow between the electrodes had significantly decreased. This is consistent with N FRAG tau 306 aggregating in the soma and impeding current flow across the cell.

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increasing input resistance or changing cell capacitance. Thus, the changes in action potential 310 waveform were not simply due to increased filtering. Surprisingly, N FRAG monomers produced 311 comparable changes to APs. Thus, the effects of N FRAG are unrelated to its quaternary structure and 312 instead maybe associated with a specific sequence within N FRAG and FL-oTau. Given the nature of the 313 changes in action potential waveform we hypothesised that tau might be interacting with voltage-314 gated sodium channels and altering their function.

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Changes in action potential kinetics and amplitude can readily be measured using whole cell current

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There are four subtypes of voltage-gated sodium channel present in the mammalian central nervous

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From unnormalized conductance plots, we noticed that the rate of conductance increase appeared 356 faster for FL-oTau despite ḡ being reduced ( Figure 7I). We therefore modelled the initial rise of the 357 sodium current using the same approach (33) used to derive the original model: Where 1 is 1 nS and V1 is the voltage where the conductance is 1 nS. For FL-oTau introduced cells,

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Δ was calculated to be stable ~2.65 mV for 0 mins and 20 mins but 1 varied over time. At 0 mins, 363 1 = -32 mV and by 20 mins, 1 = -36 mV, demonstrating that a sodium conductance of 1 nS is 364 achieved at a much lower voltage, thus the cell is more excitable (despite the peak sodium 365 conductance being lower overall, Figure 7I). In the EIF model, the sodium current is modelled as: Empirically, the first term V 1 on the right-hand-side is dependent on parameters that change between 373 0 mins and 40 mins, whereas the second term does not change significantly between these time 374 steps. Hence the modelling predicts that the change in the action-potential threshold parameter V T is 375 largely due to changes in V 1 which is the voltage at which the sodium current is 1nS. The 376 experiments predict that the difference in VT at 0 and 40 mins is 4 mV, which fits exactly with the 377 excitability phenotype modelled using C FRAG . Despite the differences in temperature for these 378 recordings, these two independent methodologies further support that the tau-mediated shift in 379 excitability comes from a lowering of the action-potential threshold (rather than input resistance

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oTau is dependent on voltage, at low voltages the conductance is bigger at 20 mins in FL-oTau but at 388 high voltages it is lower at 20 mins in o-Tau, thus, the effect is non-monotonic. Globally it appears that 389 there is a lower maximal Na conductance at 20 mins (and therefore action potential height is 390 reduced), but there is a small region at lower voltages where the conductance is higher for FL-oTau 391 which means the model is more excitable. This means the profile of the activation has been 392 changed by oTau; it is not just an issue of different maximal conductance magnitudes.

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The simplest explanation for these changes to sodium conductance is that a specific N FRAGtau 395 sequence binds to the sodium channel. The mammalian sodium channel is a molecular complex of an 396 ∼2000 amino acid α-subunit, which contains the pore and drug interaction sites, and smaller β-

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This work has shown that by modifying the tau molecule it is possible to dissect apart the multiple 409 effects it has on neuronal function (Table 2) and identify the molecular targets that tau might bind to.

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This not only provides information about the mechanisms of action of tau but also raises interesting 411 possibilities. Since the truncations used in this study have been found in the CSF of AD patients, then 412 the effects of tau will depend on the mixture of these molecules inside neurons. Thus, if tau is 413 primarily present as the C FRAG then it will affect excitability and not change action potential waveform.

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However, if the N FRAG accumulates in the soma it will have large effects on neuronal integration and 415 action potential waveform. It woluld also be interesting to identify whether these effects are specific to 416 specific sodium channel isoforms, and therefore could effect some neurons more than others 417 depending on the isofroms present.

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The different fragments were PCR amplified using custom designed primers representing the 5' and    when bubbled with 95% O2 and 5% CO 2 , 300 mOsm). Following preparation, slices were left to 483 recover in recording aCSF (as above but with 1 mM MgCl 2 and 2 mM CaCl 2 ) at 34°C. Slices were 484 used within 1-8 hours after preparation.

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to align with the findings of this study and of (11).

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Series resistance (R s ) was measured throughout the recording and were typically in the range of 6-12 567 MΩ. Cells with R s over 15 MΩ or those that differed by more than 20 % over the period of recording 568 were discarded. R s was not compensated, but the liquid junction potential of ~ 8 mV was 569 corrected. Leak currents were subtracted using the P/n protocol (1/4) and the resulting data 570 displayed.

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The stimulation protocol was adapted from (28) to allow fully clamped somatic Na + channel currents to 572 be recorded. Neurons were held at -80 mV. A depolarising pre-pulse was given (5 ms, -40 mV) to then voltage steps (from -60 to 60 mV, 5 ms duration) were used to elicit controlled Na + channel 575 current responses. The inter-sweep interval was 2 s. From the current responses, plots of current vs 576 voltage or conductance vs voltage were calculated.

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Conductance is given by the following equation:

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The membrane current is given by: Where is the total membrane current and is the membrane capacitance, , and are the 595 potassium, sodium and leak conductance's per unit area, and , and are reversal potentials.

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The sodium conductance can be given by: Where ḡ Na is the maximal sodium conductance and m and h are quantities between 0 and 1 that are 599 associated with sodium channel activation and inactivation, respectively. V m is the membrane 600 potential and E Na is the reversal potential for sodium.

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Statistics and reproducibility 607 illustrated the induced-changes to neuronal properties in the manuscript using normalised data the tau constructs will not have had time to diffuse into the recorded cell, this acts as an internal 612 control. Therefore normalising the data to the values at whole-cell breathrough makes it easier to 613 observe any changes that occur over time and compare this between conditions.

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Due to small sample sizes (n < 15), statistical analysis was performed using non-parametric methods

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The authors declare no competing interests.

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In the same recordings that are illustrated in Figure