Design of Ultrapotent Genetically Encoded Inhibitors of Kv4.2 for Gating Neural Plasticity.

The Kv4.2 potassium channel plays established roles in neuronal excitability, while also being implicated in plasticity. Current means to study the roles of Kv4.2 are limited, motivating us to design a genetically encoded membrane tethered Heteropodatoxin-2 (MetaPoda). We find that MetaPoda is an ultrapotent and selective gating-modifier of Kv4.2. We narrow its site of contact with the channel to two adjacent residues within the voltage sensitive domain (VSD) and, with docking simulations, suggest that the toxin binds the VSD from within the membrane. We also show that MetaPoda does not require an external linker of the channel for its activity. In neurons (obtained from female and male rat neonates), MetaPoda specifically, and potently, inhibits all Kv4 currents, leaving all other A-type currents unaffected. Inhibition of Kv4 in hippocampal neurons does not promote excessive excitability, as is expected from a simple potassium channel blocker. We do find that MetaPoda's prolonged expression (1 week) increases expression levels of the immediate early gene cFos and prevents potentiation. These findings argue for a major role of Kv4.2 in facilitating plasticity of hippocampal neurons. Lastly, we show that our engineering strategy is suitable for the swift engineering of another potent Kv4.2-selective membrane-tethered toxin, Phrixotoxin-1, denoted MetaPhix. Together, we provide two uniquely potent genetic tools to study Kv4.2 in neuronal excitability and plasticity.

Reconsidering editorial consideration: Changing editorial assessment could reduce subjectivity in the publication process.

Can rethinking editorial assessment reduce subjectivity and transform the way science is communicated?

Rescuing tri-heteromeric NMDA receptor function: the potential of pregnenolone-sulfate in loss-of-function GRIN2B variants.

N-methyl-D-aspartate receptors (NMDARs emerging from GRIN genes) are tetrameric receptors that form diverse channel compositions in neurons, typically consisting of two GluN1 subunits combined with two GluN2(A-D) subunits. During prenatal stages, the predominant channels are di-heteromers with two GluN1 and two GluN2B subunits due to the high abundance of GluN2B subunits. Postnatally, the expression of GluN2A subunits increases, giving rise to additional subtypes, including GluN2A-containing di-heteromers and tri-heteromers with GluN1, GluN2A, and GluN2B subunits. The latter  emerge as the major receptor subtype at mature synapses in the hippocampus. Despite extensive research on purely di-heteromeric receptors containing two identical GRIN variants, the impact of a single variant on the function of other channel forms, notably tri-heteromers, is lagging. In this study, we systematically investigated the effects of two de novo GRIN2B variants (G689C and G689S) in pure, mixed di- and tri-heteromers. Our findings reveal that incorporating a single variant in mixed di-heteromers or tri-heteromers exerts a dominant negative effect on glutamate potency, although 'mixed' channels show improved potency compared to pure variant-containing di-heteromers. We show that a single variant within a receptor complex does not impair the response of all receptor subtypes to the positive allosteric modulator pregnenolone-sulfate (PS), whereas spermine completely fails to potentiate tri-heteromers containing GluN2A and -2B-subunits. We examined PS on primary cultured hippocampal neurons transfected with the variants, and observed a positive impact over current amplitudes and synaptic activity. Together, our study supports previous observations showing that mixed di-heteromers exhibit improved glutamate potency and extend these findings towards the exploration of the effect of Loss-of-Function variants over tri-heteromers. Notably, we provide an initial and crucial demonstration of the beneficial effects of GRIN2B-relevant potentiators on tri-heteromers. Our results underscore the significance of studying how different variants affect distinct receptor subtypes, as these effects cannot be inferred solely from observations made on pure di-heteromers. Overall, this study contributes to ongoing efforts to understand the pathophysiology of GRINopathies and provides insights into potential treatment strategies.

SNAP-Tag-Targeted MRI-Fluorescent Multimodal Probes.

Magnetic resonance imaging (MRI) is a powerful imaging modality, widely employed in research and clinical settings. However, MRI images suffer from low signals and a lack of target specificity. We aimed to develop a multimodal imaging probe to detect targeted cells by MRI and fluorescence microscopy. We synthesized a trifunctional imaging probe consisting of a SNAP-tag substrate for irreversible and specific labelling of cells, cyanine dyes for bright fluorescence, and a chelated GdIII molecule for enhancing MRI contrast. Our probes exhibit specific and efficient labelling of genetically defined cells (expressing SNAP-tag at their membrane), bright fluorescence and MRI signal. Our synthetic approach provides a versatile platform for the production of multimodal imaging probes, particularly for light microscopy and MRI.

Daily Brief Heat Therapy Reduces Seizures in A350V IQSEC2 Mice and Is Associated with Correction of AMPA Receptor-Mediated Synaptic Dysfunction.

Purposeful induction of fever for healing, including the treatment of epilepsy, was used over 2000 years ago by Hippocrates. More recently, fever has been demonstrated to rescue behavioral abnormalities in children with autism. However, the mechanism of fever benefit has remained elusive due in large part to the lack of appropriate human disease models recapitulating the fever effect. Pathological mutations in the IQSEC2 gene are frequently seen in children presenting with intellectual disability, autism and epilepsy. We recently described a murine A350V IQSEC2 disease model, which recapitulates important aspects of the human A350V IQSEC2 disease phenotype and the favorable response to a prolonged and sustained rise in body core temperature in a child with the mutation. Our goal has been to use this system to understand the mechanism of fever benefit and then develop drugs that can mimic this effect and reduce IQSEC2-associated morbidity. In this study, we first demonstrate a reduction in seizures in the mouse model following brief periods of heat therapy, similar to what was observed in a child with the mutation. We then show that brief heat therapy is associated with the correction of synaptic dysfunction in neuronal cultures of A350V mice, likely mediated by Arf6-GTP.

A simplified Gibson assembly method for site directed mutagenesis by re-use of standard, and entirely complementary, mutagenesis primers.

BACKGROUND: Site-directed mutagenesis (SDM) is a key method in molecular biology; allowing to modify DNA sequences at single base pair resolution. Although many SDM methods have been developed, methods that increase efficiency and versatility of this process remain highly desired. METHOD: We present a versatile and simple method to efficiently introduce a variety of mutation schemes using Gibson-assembly but without the need to design uniquely designated Gibson primers. Instead, we explore the re-use of standard SDM primers (completely overlapping in sequence) in combination with regular primers (~ 25 bps long) for amplification of fragments flanking the site of mutagenesis. We further introduce a rapid amplification step of the Gibson-assembled product for analysis and quality control, as well as for ligation, or re-ligation at instances the process fails (avoiding expenditure of added Gibson reaction mixtures). RESULTS: We first demonstrate that standard SDM primers can be used with the Gibson assembly method and, despite the need for extensive digestion of the DNA past the entire primer sequence, the reaction is attainable within as short as 15 min. We also find that the amount of the assembled Gibson product is too low to be visualized on standard agarose gel. Our added amplification step (by use of the same short primers initially employed) remedies this limitation and allows to resolve whether the desired Gibson-assembled product has been obtained on agarose gel or by sequencing of amplicons. It also provides large amounts of amplicons for subsequent ligations, bypassing the need to re-employ Gibson mixtures. Lastly, we find that our method can easily accommodate SDM primers with degenerate sequences. CONCLUSION: We employ our alternative approach to delete, replace, insert, and degenerate sequences within target DNA sequences, specifically DNA sequences that proved very resistant to mutagenesis by multiple other SDM methods (standard and commercial). Importantly, our approach involves the re-use of SDM primers from our primer-inventory. Our scheme thereby reduces the need (and time and money) to design and order new custom Gibson-primers. Together, we provide a simple and versatile protocol that spans only 4 days (including the added amplification step), requires minimal primer sets and provides very high yields and success rates (> 98%).

The NMDA receptor activation by d-serine and glycine is controlled by an astrocytic Phgdh-dependent serine shuttle.

Astrocytes express the 3-phosphoglycerate dehydrogenase (Phgdh) enzyme required for the synthesis of l-serine from glucose. Astrocytic l-serine was proposed to regulate NMDAR activity by shuttling to neurons to sustain d-serine production, but this hypothesis remains untested. We now report that inhibition of astrocytic Phgdh suppressed the de novo synthesis of l-and d-serine and reduced the NMDAR synaptic potentials and long-term potentiation (LTP) at the Schaffer collaterals-CA1 synapse. Likewise, enzymatic removal of extracellular l-serine impaired LTP, supporting an l-serine shuttle mechanism between glia and neurons in generating the NMDAR coagonist d-serine. Moreover, deletion of serine racemase (SR) in glutamatergic neurons abrogated d-serine synthesis to the same extent as Phgdh inhibition, suggesting that neurons are the predominant source of the newly synthesized d-serine. We also found that the synaptic NMDAR activation in adult SR-knockout (KO) mice requires Phgdh-derived glycine, despite the sharp decline in the postnatal glycine levels as a result of the emergence of the glycine cleavage system. Unexpectedly, we also discovered that glycine regulates d-serine metabolism by a dual mechanism. The first consists of tonic inhibition of SR by intracellular glycine observed in vitro, primary cultures, and in vivo microdialysis. The second involves a transient glycine-induce d-serine release through the Asc-1 transporter, an effect abolished in Asc-1 KO mice and diminished by deleting SR in glutamatergic neurons. Our observations suggest that glycine is a multifaceted regulator of d-serine metabolism and implicate both d-serine and glycine in mediating NMDAR synaptic activation at the mature hippocampus through a Phgdh-dependent shuttle mechanism.

Synapses in the spotlight with synthetic optogenetics.

Membrane receptors and ion channels respond to various stimuli and relay that information across the plasma membrane by triggering specific and timed processes. These include activation of second messengers, allowing ion permeation, and changing cellular excitability, to name a few. Gaining control over equivalent processes is essential to understand neuronal physiology and pathophysiology. Recently, new optical techniques have emerged proffering new remote means to control various functions of defined neuronal populations by light, dubbed optogenetics. Still, optogenetic tools do not typically address the activity of receptors and channels native to neurons (or of neuronal origin), nor gain access to their signaling mechanisms. A related method-synthetic optogenetics-bridges this gap by endowing light sensitivity to endogenous neuronal receptors and channels by the appending of synthetic, light-receptive molecules, or photoswitches. This provides the means to photoregulate neuronal receptors and channels and tap into their native signaling mechanisms in select regions of the neurons, such as the synapse. This review discusses the development of synthetic optogenetics as a means to study neuronal receptors and channels remotely, in their natural environment, with unprecedented spatial and temporal precision, and provides an overview of tool design, mode of action, potential clinical applications and insights and achievements gained.

Photoactivatable genetically encoded calcium indicators for targeted neuronal imaging.

Circuit mapping requires knowledge of both structural and functional connectivity between cells. Although optical tools have been made to assess either the morphology and projections of neurons or their activity and functional connections, few probes integrate this information. We have generated a family of photoactivatable genetically encoded Ca(2+) indicators that combines attributes of high-contrast photolabeling with high-sensitivity Ca(2+) detection in a single-color protein sensor. We demonstrated in cultured neurons and in fruit fly and zebrafish larvae how single cells could be selected out of dense populations for visualization of morphology and high signal-to-noise measurements of activity, synaptic transmission and connectivity. Our design strategy is transferrable to other sensors based on circularly permutated GFP (cpGFP).

Two-photon brightness of azobenzene photoswitches designed for glutamate receptor optogenetics.

Mammalian neurotransmitter-gated receptors can be conjugated to photoswitchable tethered ligands (PTLs) to enable photoactivation, or photoantagonism, while preserving normal function at neuronal synapses. "MAG" PTLs for ionotropic and metabotropic glutamate receptors (GluRs) are based on an azobenzene photoswitch that is optimally switched into the liganding state by blue or near-UV light, wavelengths that penetrate poorly into the brain. To facilitate deep-tissue photoactivation with near-infrared light, we measured the efficacy of two-photon (2P) excitation for two MAG molecules using nonlinear spectroscopy. Based on quantitative characterization, we find a recently designed second generation PTL, L-MAG0460, to have a favorable 2P absorbance peak at 850 nm, enabling efficient 2P activation of the GluK2 kainate receptor, LiGluR. We also achieve 2P photoactivation of a metabotropic receptor, LimGluR3, with a new mGluR-specific PTL, D-MAG0460. 2P photoswitching is efficiently achieved using digital holography to shape illumination over single somata of cultured neurons. Simultaneous Ca(2+)-imaging reports on 2P photoswitching in multiple cells with high temporal resolution. The combination of electrophysiology or Ca(2+) imaging with 2P activation by optical wavefront shaping should make second generation PTL-controlled receptors suitable for studies of intact neural circuits.

A Quantitative Model of the GIRK1/2 Channel Reveals That Its Basal and Evoked Activities Are Controlled by Unequal Stoichiometry of Gα and Gßγ.

G protein-gated K+ channels (GIRK; Kir3), activated by Gßγ subunits derived from Gi/o proteins, regulate heartbeat and neuronal excitability and plasticity. Both neurotransmitter-evoked (Ievoked) and neurotransmitter-independent basal (Ibasal) GIRK activities are physiologically important, but mechanisms of Ibasal and its relation to Ievoked are unclear. We have previously shown for heterologously expressed neuronal GIRK1/2, and now show for native GIRK in hippocampal neurons, that Ibasal and Ievoked are interrelated: the extent of activation by neurotransmitter (activation index, Ra) is inversely related to Ibasal. To unveil the underlying mechanisms, we have developed a quantitative model of GIRK1/2 function. We characterized single-channel and macroscopic GIRK1/2 currents, and surface densities of GIRK1/2 and Gßγ expressed in Xenopus oocytes. Based on experimental results, we constructed a mathematical model of GIRK1/2 activity under steady-state conditions before and after activation by neurotransmitter. Our model accurately recapitulates Ibasal and Ievoked in Xenopus oocytes, HEK293 cells and hippocampal neurons; correctly predicts the dose-dependent activation of GIRK1/2 by coexpressed Gßγ and fully accounts for the inverse Ibasal-Ra correlation. Modeling indicates that, under all conditions and at different channel expression levels, between 3 and 4 Gßγ dimers are available for each GIRK1/2 channel. In contrast, available Gαi/o decreases from ~2 to less than one Gα per channel as GIRK1/2's density increases. The persistent Gßγ/channel (but not Gα/channel) ratio support a strong association of GIRK1/2 with Gßγ, consistent with recruitment to the cell surface of Gßγ, but not Gα, by GIRK1/2. Our analysis suggests a maximal stoichiometry of 4 Gßγ but only 2 Gαi/o per one GIRK1/2 channel. The unique, unequal association of GIRK1/2 with G protein subunits, and the cooperative nature of GIRK gating by Gßγ, underlie the complex pattern of basal and agonist-evoked activities and allow GIRK1/2 to act as a sensitive bidirectional detector of both Gßγ and Gα.

Tracking Ca2+-dependent and Ca2+-independent conformational transitions in syntaxin 1A during exocytosis in neuroendocrine cells.

A key issue for understanding exocytosis is elucidating the various protein interactions and the associated conformational transitions underlying soluble N-ethylmeleimide-sensitive factor attachment protein receptor (SNARE) protein assembly. To monitor dynamic changes in syntaxin 1A (Syx) conformation along exocytosis, we constructed a novel fluorescent Syx-based probe that can be efficiently incorporated within endogenous SNARE complexes, support exocytosis, and report shifts in Syx between 'closed' and 'open' conformations by fluorescence resonance energy transfer analysis. Using this probe we resolve two distinct Syx conformational transitions during membrane depolarization-induced exocytosis in PC12 cells: a partial 'opening' in the absence of Ca(2+) entry and an additional 'opening' upon Ca(2+) entry. The Ca(2+)-dependent transition is abolished upon neutralization of the basic charges in the juxtamembrane regions of Syx, which also impairs exocytosis. These novel findings provide evidence of two conformational transitions in Syx during exocytosis, which have not been reported before: one transition directly induced by depolarization and an additional transition that involves the juxtamembrane region of Syx. The superior sensitivity of our probe also enabled detection of subtle Syx conformational changes upon interaction with VAMP2, which were absolutely dependent on the basic charges of the juxtamembrane region. Hence, our results further suggest that the Ca(2+)-dependent transition in Syx involves zippering between the membrane-proximal juxtamembrane regions of Syx and VAMP2 and support the recently implied existence of this zippering in the final phase of SNARE assembly to catalyze exocytosis.

Recruitment of Gßγ controls the basal activity of G-protein coupled inwardly rectifying potassium (GIRK) channels: crucial role of distal C terminus of GIRK1.

The G-protein coupled inwardly rectifying potassium (GIRK, or Kir3) channels are important mediators of inhibitory neurotransmission via activation of G-protein coupled receptors (GPCRs). GIRK channels are tetramers comprising combinations of subunits (GIRK1-4), activated by direct binding of the Gßγ subunit of Gi/o proteins. Heterologously expressed GIRK1/2 exhibit high, Gßγ-dependent basal currents (Ibasal) and a modest activation by GPCR or coexpressed Gßγ. Inversely, the GIRK2 homotetramers exhibit low Ibasal and strong activation by Gßγ. The high Ibasal of GIRK1 seems to be associated with its unique distal C terminus (G1-dCT), which is not present in the other subunits. We investigated the role of G1-dCT using electrophysiological and fluorescence assays in Xenopus laevis oocytes and protein interaction assays. We show that expression of GIRK1/2 increases the plasma membrane level of coexpressed Gßγ (a phenomenon we term 'Gßγ recruitment') but not of coexpressed Gαi3. All GIRK1-containing channels, but not GIRK2 homomers, recruited Gßγ to the plasma membrane. In biochemical assays, truncation of G1-dCT reduces the binding between the cytosolic parts of GIRK1 and Gßγ, but not Gαi3. Nevertheless, the truncation of G1-dCT does not impair activation by Gßγ. In fluorescently labelled homotetrameric GIRK1 channels and in the heterotetrameric GIRK1/2 channel, the truncation of G1-dCT abolishes Gßγ recruitment and decreases Ibasal. Thus, we conclude that G1-dCT carries an essential role in Gßγ recruitment by GIRK1 and, consequently, in determining its high basal activity. Our results indicate that G1-dCT is a crucial part of a Gßγ anchoring site of GIRK1-containing channels, spatially and functionally distinct from the site of channel activation by Gßγ.

Datasets assessing lipid-content in optically cleared brains.

Multi-modal imaging, by light-microscopy (LM) and Magnetic Resonance Imaging (MRI), holds promise for examining the brain across various resolutions and scales. While MRI acquires images in three dimensions, acquisition of intact whole-brain by LM requires a process of tissue clearing that renders the brain transparent. Removal of lipids (delipidation) is a critical step in the tissue clearing process, and was previsouly suggested to be the cause for absence of MRI contrast in cleared brains. Yet, the association between MRI contrast, delipidation and the different clearing techniques is debatable. Here, we provide datasets concerning lipid-content in cleared brain tissues obtained by various approaches. Fixed mouse and rat brains were cleared by CLARITY, Scale, uDISCO and ECi clearing techniques. Lipid-content was assessed at various intermediate steps of the different clearing methods, as well as at the end of the processes. Methods employed included whole brain MRI acquisition, Oil Red O (ORO)- and carbocyanine DiI-staining of cryosections, and DiI-washout assay from brain slices. MRI contrast-to-noise ratio, staining intensities and integrity of tissue were systematically analyzed. We demonstrate that lipid electrophoresis, an essential step of the CLARITY approach, engenders progressive reduction in MRI contrast in non-cleared (PFA-fixed) control brains, as well as strongly reduces contrast from uDISCO and ECi-cleared brains. ORO minimally stained CLARITY-cleared brains, however efficiently labelled uDISCO and ECi-cleared brains. Conversely, and in contrast to ORO-staining, DiI equally stained control, CLARITY, ECi and uDISCO-cleared brains. Both ORO- and DiI-staining demonstrated impairment in brain tissue integrity following CLARITY, but less so in uDISCO and ECi brains. DiI-washout assay demonstrated that each of the solvents employed along the process of uDISCO and ECi are highly delipidating, as well as the SDS-electrophoresis employed during CLARITY clearing. However, Scale treatment preserved most of the DiI dye. These data emphasize the variability in lipid assessment of cleared tissues by common techniques, and may help to resolve the contribution of lipids in brain MRI contrast.

Revealing the MRI-Contrast in Optically Cleared Brains.

The current consensus holds that optically-cleared specimens are unsuitable for Magnetic Resonance Imaging (MRI); exhibiting absence of contrast. Prior studies combined MRI with tissue-clearing techniques relying on the latter's ability to eliminate lipids, thereby fostering the assumption that lipids constitute the primary source of ex vivo MRI-contrast. Nevertheless, these findings contradict an extensive body of literature that underscores the contribution of other features to contrast. Furthermore, it remains unknown whether non-delipidating clearing methods can produce MRI-compatible specimens or whether MRI-contrast can be re-established. These limitations hinder the development of multimodal MRI-light-microscopy (LM) imaging approaches. This study assesses the relation between MRI-contrast, and delipidation in optically-cleared whole brains following different tissue-clearing approaches. It is demonstrated that uDISCO and ECi-brains are MRI-compatible upon tissue rehydration, despite both methods' substantial delipidating-nature. It is also demonstrated that, whereas Scale-clearing preserves most lipids, Scale-cleared brain lack MRI-contrast. Furthermore, MRI-contrast is restored to lipid-free CLARITY-brains without introducing lipids. Our results thereby dissociate between the essentiality of lipids to MRI-contrast. A tight association is found between tissue expansion, hyperhydration and loss of MRI-contrast. These findings then enabled us to develop a multimodal MRI-LM-imaging approach, opening new avenues to bridge between the micro- and mesoscale for biomedical research and clinical applications.

A custom-made AAV1 variant (AAV1-T593K) enables efficient transduction of Japanese quail neurons in vitro and in vivo.

The widespread use of rodents in neuroscience has prompted the development of optimized viral variants for transduction of brain cells, in vivo. However, many of the viruses developed are less efficient in other model organisms, with birds being among the most resistant to transduction by current viral tools. Resultantly, the use of genetically-encoded tools and methods in avian species is markedly lower than in rodents; likely holding the field back. We sought to bridge this gap by developing custom viruses towards the transduction of brain cells of the Japanese quail. We first develop a protocol for culturing primary neurons and glia from quail embryos, followed by characterization of cultures via immunostaining, single cell mRNA sequencing, patch clamp electrophysiology and calcium imaging. We then leveraged the cultures for the rapid screening of various viruses, only to find that all yielded poor to no infection of cells in vitro. However, few infected neurons were obtained by AAV1 and AAV2. Scrutiny of the sequence of the AAV receptor found in quails led us to rationally design a custom-made AAV variant (AAV1-T593K; AAV1*) that exhibits improved transduction efficiencies in vitro and in vivo (14- and five-fold, respectively). Together, we present unique culturing method, transcriptomic profiles of quail's brain cells and a custom-tailored AAV1 for transduction of quail neurons in vitro and in vivo.

Two distinct aspects of coupling between Gα(i) protein and G protein-activated K+ channel (GIRK) revealed by fluorescently labeled Gα(i3) protein subunits.

G protein-activated K(+) channels (Kir3 or GIRK) are activated by direct interaction with Gßγ. Gα is essential for specific signaling and regulates basal activity of GIRK (I(basal)) and kinetics of the response elicited by activation by G protein-coupled receptors (I(evoked)). These regulations are believed to occur within a GIRK-Gα-Gßγ signaling complex. Fluorescent energy resonance transfer (FRET) studies showed strong GIRK-Gßγ interactions but yielded controversial results regarding the GIRK-Gα(i/o) interaction. We investigated the mechanisms of regulation of GIRK by Gα(i/o) using wild-type Gα(i3) (Gα(i3)WT) and Gα(i3) labeled at three different positions with fluorescent proteins, CFP or YFP (xFP). Gα(i3)xFP proteins bound the cytosolic domain of GIRK1 and interacted with Gßγ in a guanine nucleotide-dependent manner. However, only an N-terminally labeled, myristoylated Gα(i3)xFP (Gα(i3)NT) closely mimicked all aspects of Gα(i3)WT regulation except for a weaker regulation of I(basal). Gα(i3) labeled with YFP within the Gα helical domain preserved regulation of I(basal) but failed to restore fast I(evoked). Titrated expression of Gα(i3)NT and Gα(i3)WT confirmed that regulation of I(basal) and of the kinetics of I(evoked) of GIRK1/2 are independent functions of Gα(i). FRET and direct biochemical measurements indicated much stronger interaction between GIRK1 and Gßγ than between GIRK1 and Gα(i3). Thus, Gα(i/o)ßγ heterotrimer may be attached to GIRK primarily via Gßγ within the signaling complex. Our findings support the notion that Gα(i/o) actively regulates GIRK. Although regulation of I(basal) is a function of Gα(i)(GDP), our new findings indicate that regulation of kinetics of I(evoked) is mediated by Gα(i)(GTP).

Differential roles for DNAJ isoforms in HTT-polyQ and FUS aggregation modulation revealed by chaperone screens.

Protein aggregation is a hallmark of neurodegeneration. Here, we find that Huntington's disease-related HTT-polyQ aggregation induces a cellular proteotoxic stress response, while ALS-related mutant FUS (mutFUS) aggregation leads to deteriorated proteostasis. Further exploring chaperone function as potential modifiers of pathological aggregation in these contexts, we reveal divergent effects of naturally-occurring chaperone isoforms on different aggregate types. We identify a complex of the full-length (FL) DNAJB14 and DNAJB12, that substantially protects from mutFUS aggregation, in an HSP70-dependent manner. Their naturally-occurring short isoforms, however, do not form a complex, and lose their ability to preclude mutFUS aggregation. In contrast, DNAJB12-short alleviates, while DNAJB12-FL aggravates, HTT-polyQ aggregation. DNAJB14-FL expression increases the mobility of mutFUS aggregates, and restores the deteriorated proteostasis in mutFUS aggregate-containing cells and primary neurons. Our results highlight a maladaptive cellular response to pathological aggregation, and reveal a layer of chaperone network complexity conferred by DNAJ isoforms, in regulation of different aggregate types.