Dysregulation of ribosome-associated quality control elicits cognitive disorders via overaccumulation of TTC3.

Ribosome-associated quality control (RQC) pathway is responsible for degradation of nascent polypeptides in aberrantly stalled ribosomes, and its defects may lead to neurological diseases. However, the underlying molecular mechanism of how RQC dysfunction elicits neurological disorders remains poorly understood. Here we revealed that neurons with knockout (KO) of ubiquitin ligase LTN1, a key gene in the RQC pathway, show developmental defects in neurons via upregulation of TTC3 and UFMylation signaling proteins. The abnormally enhanced TTC3 protein in Ltn1 KO neurons reduced further accumulation of translationally arrested products by preventing translation initiation of selective genes. However, the overaccumulated TTC3 protein in turn caused dendritic abnormalities and reduced surface-localized GABAA receptors during neuronal development. Ltn1 KO mice showed behavioral deficits associated with cognitive disorders, a subset of which were restored by TTC3 knockdown in medial prefrontal cortex. Together, the overactivated cellular compensatory mechanism against defective RQC through TTC3 overaccumulation induced synaptic and cognitive deficits. More broadly, these findings represent a novel cellular mechanism underlying neuronal dysfunctions triggered by exaggerated cellular stress response to accumulated abnormal translation products in neurons.

Evidence of Orientation-Dependent Early States of Prion Protein Misfolded Structures from Single Molecule Force Spectroscopy.

Prion diseases are neurodegenerative disorders characterized by the presence of oligomers and amyloid fibrils. These are the result of protein aggregation processes of the cellular prion protein (PrPC) into amyloidal forms denoted as prions or PrPSc. We employed atomic force microscopy (AFM) for single molecule pulling (single molecule force spectroscopy, SMFS) experiments on the recombinant truncated murine prion protein (PrP) domain to characterize its conformations and potential initial oligomerization processes. Our AFM-SMFS results point to a complex scenario of structural heterogeneity of PrP at the monomeric and dimer level, like other amyloid proteins involved in similar pathologies. By applying this technique, we revealed that the PrP C-terminal domain unfolds in a two-state process. We used two dimeric constructs with different PrP reciprocal orientations: one construct with two sequential PrP in the N- to C-terminal orientation (N-C dimer) and a second one in the C- to C-terminal orientation (C-C dimer). The analysis revealed that the different behavior in terms of unfolding force, whereby the dimer placed C-C dimer unfolds at a higher force compared to the N-C orientation. We propose that the C-C dimer orientation may represent a building block of amyloid fibril formation.

How Staying Negative Is Good for the (Adult) Brain: Maintaining Chloride Homeostasis and the GABA-Shift in Neurological Disorders.

Excitatory-inhibitory (E-I) imbalance has been shown to contribute to the pathogenesis of a wide range of neurodevelopmental disorders including autism spectrum disorders, epilepsy, and schizophrenia. GABA neurotransmission, the principal inhibitory signal in the mature brain, is critically coupled to proper regulation of chloride homeostasis. During brain maturation, changes in the transport of chloride ions across neuronal cell membranes act to gradually change the majority of GABA signaling from excitatory to inhibitory for neuronal activation, and dysregulation of this GABA-shift likely contributes to multiple neurodevelopmental abnormalities that are associated with circuit dysfunction. Whilst traditionally viewed as a phenomenon which occurs during brain development, recent evidence suggests that this GABA-shift may also be involved in neuropsychiatric disorders due to the "dematuration" of affected neurons. In this review, we will discuss the cell signaling and regulatory mechanisms underlying the GABA-shift phenomenon in the context of the latest findings in the field, in particular the role of chloride cotransporters NKCC1 and KCC2, and furthermore how these regulatory processes are altered in neurodevelopmental and neuropsychiatric disorders. We will also explore the interactions between GABAergic interneurons and other cell types in the developing brain that may influence the GABA-shift. Finally, with a greater understanding of how the GABA-shift is altered in pathological conditions, we will briefly outline recent progress on targeting NKCC1 and KCC2 as a therapeutic strategy against neurodevelopmental and neuropsychiatric disorders associated with improper chloride homeostasis and GABA-shift abnormalities.

Regulation of sensorimotor gating via Disc1/Huntingtin-mediated Bdnf transport in the cortico-striatal circuit.

Sensorimotor information processing underlies normal cognitive and behavioral traits and has classically been evaluated through prepulse inhibition (PPI) of a startle reflex. PPI is a behavioral dimension deregulated in several neurological and psychiatric disorders, yet the mechanisms underlying the cross-diagnostic nature of PPI deficits across these conditions remain to be understood. To identify circuitry mechanisms for PPI, we performed circuitry recording over the prefrontal cortex and striatum, two brain regions previously implicated in PPI, using wild-type (WT) mice compared to Disc1-locus-impairment (LI) mice, a model representing neuropsychiatric conditions. We demonstrated that the corticostriatal projection regulates neurophysiological responses during the PPI testing in WT, whereas these circuitry responses were disrupted in Disc1-LI mice. Because our biochemical analyses revealed attenuated brain-derived neurotrophic factor (Bdnf) transport along the corticostriatal circuit in Disc1-LI mice, we investigated the potential role of Bdnf in this circuitry for regulation of PPI. Virus-mediated delivery of Bdnf into the striatum rescued PPI deficits in Disc1-LI mice. Pharmacologically augmenting Bdnf transport by chronic lithium administration, partly via phosphorylation of Huntingtin (Htt) serine-421 and its integration into the motor machinery, restored striatal Bdnf levels and rescued PPI deficits in Disc1-LI mice. Furthermore, reducing the cortical Bdnf expression negated this rescuing effect of lithium, confirming the key role of Bdnf in lithium-mediated PPI rescuing. Collectively, the data suggest that striatal Bdnf supply, collaboratively regulated by Htt and Disc1 along the corticostriatal circuit, is involved in sensorimotor gating, highlighting the utility of dimensional approach in investigating pathophysiological mechanisms across neuropsychiatric disorders.

Amyloid conformation-dependent disaggregation in a reconstituted yeast prion system.

Disaggregation of amyloid fibrils is a fundamental biological process required for amyloid propagation. However, due to the lack of experimental systems, the molecular mechanism of how amyloid is disaggregated by cellular factors remains poorly understood. Here, we established a robust in vitro reconstituted system of yeast prion propagation and found that heat-shock protein 104 (Hsp104), Ssa1 and Sis1 chaperones are essential for efficient disaggregation of Sup35 amyloid. Real-time imaging of single-molecule fluorescence coupled with the reconstitution system revealed that amyloid disaggregation is achieved by ordered, timely binding of the chaperones to amyloid. Remarkably, we uncovered two distinct prion strain conformation-dependent modes of disaggregation, fragmentation and dissolution. We characterized distinct chaperone dynamics in each mode and found that transient, repeated binding of Hsp104 to the same site of amyloid results in fragmentation. These findings provide a physical foundation for otherwise puzzling in vivo observations and for therapeutic development for amyloid-associated neurodegenerative diseases.

A Perspective on the Potential Involvement of Impaired Proteostasis in Neuropsychiatric Disorders.

Recent genetic approaches have demonstrated that genetic factors contribute to the pathologic origins of neuropsychiatric disorders. Nevertheless, the exact pathophysiological mechanism for most cases remains unclear. Recent studies have demonstrated alterations in pathways of protein homeostasis (proteostasis) and identified several proteins that are misfolded and/or aggregated in the brains of patients with neuropsychiatric disorders, thus providing early evidence that disrupted proteostasis may be a contributing factor to their pathophysiology. Unlike neurodegenerative disorders in which massive neuronal and synaptic losses are observed, proteostasis impairments in neuropsychiatric disorders do not lead to robust neuronal death, but rather likely act via loss- and gain-of-function effects to disrupt neuronal and synaptic functions. Furthermore, abnormal activation of or overwhelmed endoplasmic reticulum and mitochondrial quality control pathways may exacerbate the pathophysiological changes initiated by impaired proteostasis, as these organelles are critical for proper neuronal functions and involved in the maintenance of proteostasis. This perspective article reviews recent findings implicating proteostasis impairments in the pathophysiology of neuropsychiatric disorders and explores how neuronal and synaptic functions may be impacted by disruptions in protein homeostasis. A greater understanding of the contributions by proteostasis impairment in neuropsychiatric disorders will help guide future studies to identify additional candidate proteins and new targets for therapeutic development.

Short disordered protein segment regulates cross-species transmission of a yeast prion.

Soluble prion proteins contingently encounter foreign prion aggregates, leading to cross-species prion transmission. However, how its efficiency is regulated by structural fluctuation of the host soluble prion protein remains unsolved. In the present study, through the use of two distantly related yeast prion Sup35 proteins, we found that a specific conformation of a short disordered segment governs interspecies prion transmissibility. Using a multidisciplinary approach including high-resolution NMR and molecular dynamics simulation, we identified critical residues within this segment that allow interspecies prion transmission in vitro and in vivo, by locally altering dynamics and conformation of soluble prion proteins. Remarkably, subtle conformational differences caused by a methylene group between asparagine and glutamine sufficed to change the short segment structure and substantially modulate the cross-seeding activity. Thus, our findings uncover how conformational dynamics of the short segment in the host prion protein impacts cross-species prion transmission. More broadly, our study provides mechanistic insights into cross-seeding between heterologous proteins.

Translation from the Ribosome to the Clinic: Implication in Neurological Disorders and New Perspectives from Recent Advances.

De novo protein synthesis by the ribosome and its multitude of co-factors must occur in a tightly regulated manner to ensure that the correct proteins are produced accurately at the right time and, in some cases, also in the proper location. With novel techniques such as ribosome profiling and cryogenic electron microscopy, our understanding of this basic biological process is better than ever and continues to grow. Concurrently, increasing attention is focused on how translational regulation in the brain may be disrupted during the progression of various neurological disorders. In fact, translational dysregulation is now recognized as the de facto pathogenic cause for some disorders. Novel mechanisms including ribosome stalling, ribosome-associated quality control, and liquid-liquid phase separation are closely linked to translational regulation, and may thus be involved in the pathogenic process. The relationships between translational dysregulation and neurological disorders, as well as the ways through which we may be able to reverse those detrimental effects, will be examined in this review.

Autophagy links MTOR and GABA signaling in the brain.

The disruption of MTOR-regulated macroautophagy/autophagy was previously shown to cause autistic-like abnormalities; however, the underlying molecular defects remained largely unresolved. In a recent study, we demonstrated that autophagy deficiency induced by conditional Atg7 deletion in either forebrain GABAergic inhibitory or excitatory neurons leads to a similar set of autistic-like behavioral abnormalities even when induced following the peak period of synaptic pruning during postnatal neurodevelopment. Our proteomic analysis and molecular dissection further revealed a mechanism in which the GABAA receptor trafficking function of GABARAP (gamma-aminobutyric acid receptor associated protein) family proteins was compromised as they became sequestered by SQSTM1/p62-positive aggregates formed due to autophagy deficiency. Our discovery of autophagy as a link between MTOR and GABA signaling may have implications not limited to neurodevelopmental and neuropsychiatric disorders, but could potentially be involved in other human pathologies such as cancer and diabetes in which both pathways are implicated.

GABARAPs dysfunction by autophagy deficiency in adolescent brain impairs GABAA receptor trafficking and social behavior.

Dysfunctional mTOR signaling is associated with the pathogenesis of neurodevelopmental and neuropsychiatric disorders. However, it is unclear what molecular mechanisms and pathogenic mediators are involved and whether mTOR-regulated autophagy continues to be crucial beyond neurodevelopment. Here, we selectively deleted Atg7 in forebrain GABAergic interneurons in adolescent mice and unexpectedly found that these mice showed a set of behavioral deficits similar to Atg7 deletion in forebrain excitatory neurons. By unbiased quantitative proteomic analysis, we identified γ-aminobutyric acid receptor-associated protein-like 2 (GABARAPL2) to differentially form high-molecular weight species in autophagy-deficient brains. Further functional analyses revealed a novel pathogenic mechanism involving the p62-dependent sequestration of GABARAP family proteins, leading to the reduction of surface GABAA receptor levels. Our work demonstrates a novel physiological role for autophagy in regulating GABA signaling beyond postnatal neurodevelopment, providing a potential mechanism for the reduced inhibitory inputs observed in neurodevelopmental and neuropsychiatric disorders with mTOR hyperactivation.

Distinct segregation patterns of yeast cell-peripheral proteins uncovered by a method for protein segregatome analysis.

Protein segregation contributes to various cellular processes such as polarization, differentiation, and aging. However, the difficulty in global determination of protein segregation hampers our understanding of its mechanisms and physiological roles. Here, by developing a quantitative proteomics technique, we globally monitored segregation of preexisting and newly synthesized proteins during cell division of budding yeast, and identified crucial domains that determine the segregation of cell-peripheral proteins. Remarkably, the proteomic and subsequent microscopic analyses demonstrated that the flow through the bud neck of the proteins that harbor both endoplasmic reticulum (ER) membrane-spanning and plasma membrane (PM)-binding domains is not restricted by the previously suggested ER membrane or PM diffusion barriers but by septin-mediated partitioning of the PM-associated ER (pmaER). Furthermore, the proteomic analysis revealed that although the PM-spanning t-SNARE Sso2 was retained in mother cells, its paralog Sso1 unexpectedly showed symmetric localization. We found that the transport of Sso1 to buds was required for enhancement of polarized cell growth and resistance to cell-wall stress. Taken together, these data resolve long-standing questions about septin-mediated compartmentalization of the cell periphery, and provide new mechanistic insights into the segregation of cell-periphery proteins and their cellular functions.

TAR DNA-Binding Protein 43 and Disrupted in Schizophrenia 1 Coaggregation Disrupts Dendritic Local Translation and Mental Function in Frontotemporal Lobar Degeneration.

BACKGROUND: Neurodegenerative diseases involving protein aggregation often accompany psychiatric symptoms. Frontotemporal lobar degeneration (FTLD) associated with TAR DNA-binding protein 43 (TDP-43) aggregation is characterized by progressive neuronal atrophy in frontal and temporal lobes of cerebral cortex. Furthermore, patients with FTLD display mental dysfunction in multiple behavioral dimensions. Nevertheless, their molecular origin for psychiatric symptoms remains unclear. METHODS: In FTLD neurons and mouse models with TDP-43 aggregates, we examined coaggregation between TDP-43 and disrupted in schizophrenia 1 (DISC1), a key player in the pathology of mental conditions and its effects on local translation in dendrites and psychiatric behaviors. The protein coaggregation and the expression level of synaptic proteins were also investigated with postmortem brains from patients with FTLD (n = 6). RESULTS: We found cytosolic TDP-43/DISC1 coaggregates in brains of both FTLD mouse model and patients with FTLD. At the mechanistic levels, the TDP-43/DISC1 coaggregates disrupted the activity-dependent dendritic local translation through impairment of translation initiation and, in turn, reduced synaptic protein expression. Behavioral deficits detected in FTLD model mice were ameliorated by exogenous DISC1 expression. CONCLUSIONS: Our findings reveal a novel role of the aggregate-prone TDP-43/DISC1 protein complex in regulating local translation, which affects aberrant behaviors relevant to multiple psychiatric dimensions.

Genome-wide Translation Profiling by Ribosome-Bound tRNA Capture.

In the ribosome complex, tRNA is a critical element of mRNA translation. A rich repertoire of cell regulation is hypothesized to occur during the recruitment of specific tRNAs in polypeptide formation. However, this basic question in nascent chain biology remains unaddressed due to the lack of technologies to report the complete tRNA complement inside ribosomes during active translation. Here, we characterize a technique for profiling ribosome-embedded tRNA and their modifications. With this method, we generated a comprehensive survey of the quantity and quality of intra-ribosomal tRNAs. In cells under environmental stress, we show that methionine tRNA inside ribosomes is a robust biomarker for the impairment of translation initiation or elongation steps. Concurrent tRNA/mRNA ribosome profiling revealed a stress-dependent incorporation of damaged and uncharged tRNAs into ribosomes causing translation arrest. Thus, tRNA ribosome profiling can provide insights on translation control mechanisms in diverse biological contexts.

Molecular basis for diversification of yeast prion strain conformation.

Self-propagating ß-sheet-rich fibrillar protein aggregates, amyloid fibers, are often associated with cellular dysfunction and disease. Distinct amyloid conformations dictate different physiological consequences, such as cellular toxicity. However, the origin of the diversity of amyloid conformation remains unknown. Here, we suggest that altered conformational equilibrium in natively disordered monomeric proteins leads to the adaptation of alternate amyloid conformations that have different phenotypic effects. We performed a comprehensive high-resolution structural analysis of Sup35NM, an N-terminal fragment of the Sup35 yeast prion protein, and found that monomeric Sup35NM harbored latent local compact structures despite its overall disordered conformation. When the hidden local microstructures were relaxed by genetic mutations or solvent conditions, Sup35NM adopted a strikingly different amyloid conformation, which redirected chaperone-mediated fiber fragmentation and modulated prion strain phenotypes. Thus, dynamic conformational fluctuations in natively disordered monomeric proteins represent a posttranslational mechanism for diversification of aggregate structures and cellular phenotypes.

Aggregation of scaffolding protein DISC1 dysregulates phosphodiesterase 4 in Huntington's disease.

Huntington's disease (HD) is a polyglutamine (polyQ) disease caused by aberrant expansion of the polyQ tract in Huntingtin (HTT). While motor impairment mediated by polyQ-expanded HTT has been intensively studied, molecular mechanisms for nonmotor symptoms in HD, such as psychiatric manifestations, remain elusive. Here we have demonstrated that HTT forms a ternary protein complex with the scaffolding protein DISC1 and cAMP-degrading phosphodiesterase 4 (PDE4) to regulate PDE4 activity. We observed pathological cross-seeding between DISC1 and mutant HTT aggregates in the brains of HD patients as well as in a murine model that recapitulates the polyQ pathology of HD (R6/2 mice). In R6/2 mice, consequent reductions in soluble DISC1 led to dysregulation of DISC1-PDE4 complexes, aberrantly increasing the activity of PDE4. Importantly, exogenous expression of a modified DISC1, which binds to PDE4 but not mutant HTT, normalized PDE4 activity and ameliorated anhedonia in the R6/2 mice. We propose that cross-seeding of mutant HTT and DISC1 and the resultant changes in PDE4 activity may underlie the pathology of a specific subset of mental manifestations of HD, which may provide an insight into molecular signaling in mental illness in general.

[KIL-d] Protein Element Confers Antiviral Activity via Catastrophic Viral Mutagenesis.

Eukaryotic cells are targeted by pathogenic viruses and have developed cell defense mechanisms against viral infection. In yeast, the cellular extrachromosomal genetic element [KIL-d] alters killer activity of M double-stranded RNA killer virus and confers cell resistance against the killer virus. However, its underlying mechanism and the molecular nature of [KIL-d] are unknown. Here, we demonstrate that [KIL-d] is a proteinaceous prion-like aggregate with non-Mendelian cytoplasmic transmission. Deep sequencing analyses revealed that [KIL-d] selectively increases the rate of de novo mutation in the killer toxin gene of the viral genome, producing yeast harboring a defective mutant killer virus with a selective growth advantage over those with WT killer virus. These results suggest that a prion-like [KIL-d] element reprograms the viral replication machinery to induce mutagenesis and genomic inactivation via the long-hypothesized mechanism of "error catastrophe." The findings also support a role for prion-like protein aggregates in cellular defense and adaptation.

Autophagy-related protein 7 deficiency in amyloid ß (Aß) precursor protein transgenic mice decreases Aß in the multivesicular bodies and induces Aß accumulation in the Golgi.

Alzheimer disease (AD) is biochemically characterized by increased levels of amyloid ß (Aß) peptide, which aggregates into extracellular Aß plaques in AD brains. Before plaque formation, Aß accumulates intracellularly in both AD brains and in the brains of AD model mice, which may contribute to disease progression. Autophagy, which is impaired in AD, clears cellular protein aggregates and participates in Aß metabolism. In addition to a degradative role of autophagy in Aß metabolism we recently showed that Aß secretion is inhibited in mice lacking autophagy-related gene 7 (Atg7) in excitatory neurons in the mouse forebrain. This inhibition of Aß secretion leads to intracellular accumulation of Aß. Here, we used fluorescence and immunoelectron microscopy to elucidate the subcellular localization of the intracellular Aß accumulation which accumulates in Aß precursor protein mice lacking Atg7. Autophagy deficiency causes accumulation of p62(+) aggregates, but these aggregates do not contain Aß. However, knockdown of Atg7 induced Aß accumulation in the Golgi and a concomitant reduction of Aß in the multivesicular bodies. This indicates that Atg7 influences the transport of Aß possibly derived from Golgi to multivesicular bodies.

Self-propagating amyloid as a critical regulator for diverse cellular functions.

Amyloids are ß-sheet-rich fibrillar protein aggregates characterized by structural properties of self-propagation and strong resistance to detergent and proteinase. Although a number of causative proteins for neurodegenerative disorders are known to undergo amyloid formation, recent studies have revealed that amyloids may also play beneficial roles in cells. Cellular processes that could be regulated by amyloids are diverse and include translational regulation, programmed cell death and protein storage. Yeast prions of Mod5 and Mot3, non-Mendelian extra-chromosomal factors, also show amyloid-like biophysical properties and have recently been shown to confer host cells resistant to environmental stressors. Furthermore, yeast cells actively respond to environmental stress for fitness adaptation to environmental changes by converting soluble yeast prion proteins into their amyloid forms, allowing cells to survive under stress conditions. Therefore, amyloids are not simply the terminal end-products of protein misfolding but a growing body of evidence suggests that they may possess physiological roles by using their self-propagating properties. Here, we present an overview on recent progress of the research on such functional amyloids.