Tau mutants bind tubulin heterodimers with enhanced affinity

Tau is a microtubule binding protein that forms pathological aggregates in the brain in Alzheimer’s disease and other tauopathies. Disease etiology is thought to arise from loss of native interactions between tau and microtubules, as well as from gain of toxicity tied to tau aggregation, although neither mechanism is well understood. Here we investigate the link between function and disease using disease-associated and disease-motivated mutants of tau. We find that mutations to highly conserved proline residues in repeats 2 and 3 of the microtubule binding domain have differential effects on tau binding to tubulin and the capacity of tau to enhance tubulin polymerization. Notably, mutations to these residues result in an increased affinity for tubulin dimers while having a negligible effect on binding to stabilized microtubules. We measure conformational changes in tau on binding to tubulin that provide a structural framework for the observed altered affinity and function. Additionally, we find that these mutations do not necessarily enhance aggregation, which could have important implications for tau therapeutic strategies that focus solely on searching for tau aggregation inhibitors. We propose a model that describes tau binding to tubulin dimers and a mechanism by which disease-relevant alterations to tau impact its function. Together, these results draw attention to the interaction between tau and free tubulin as playing an important role in mechanisms of tau pathology.Tau is a microtubule (MT)-associated protein that plays a critical role in the pathology of several neurodegenerative disorders including Alzheimer’s disease, frontotemporal dementias, and traumatic brain injury (1). Normally, tau is found primarily in the axons of neurons where it regulates the dynamic instability of MTs (2, 3) and plays an important role in axonal transport (4, 5). Both in vitro and in vivo measurements find that tau increases the rate of MT polymerization, as well as decreasing rates of catastrophe (2, 6, 7). In disease, tau is found as aggregated, filamentous deposits that are the defining feature of a diverse class of neurodegenerative diseases, called tauopathies (1, 8). Pathological mutations to tau are thought to alter the native interactions of tau with MTs, in addition to increasing the propensity of tau to aggregate (9–11). Although the precise cause or mechanism by which tau contributes to toxicity in disease is unknown, both a loss of native function and a gain of toxic function are implicated (1, 12, 13).Tau consists of a C-terminal microtubule binding domain (MTBR) composed of imperfect repeats (R1–R4; Fig. 1A) (14), a flanking proline-rich region that enhances MT binding and assembly (3), and an N-terminal projection domain with putative roles in MT spacing (15) and membrane anchoring (16). Alternative splicing results in the expression of six isoforms of tau in the adult human brain, with zero, one, or two N-terminal inserts and three or four MT binding repeat units. The repeat units contain an 18-residue imperfect repeat sequence, which terminates with a highly conserved proline-glycine-glycine-glycine (PGGG), and are linked by a 13–14 residue interrepeat sequence (Fig. 1C). Early biochemical studies depicted the conserved regions as binding weakly to MTs, with the interrepeats acting as spacers between them (14, 17). More recently, it was shown that the interrepeats are also directly involved in binding and polymerization (18, 19), with the N-terminal proline-rich region playing a regulatory role (20). Tau binds MTs with a biphasic pattern indicative of two distinct binding sites on the MT lattice with unequal affinities (21, 22). Binding to MTs is negatively regulated by phosphorylation on sites in the MTBR and adjacent regions (23). Tau derived from aggregates in the brain is hyperphosphorylated (24), suggesting a role for aberrant phosphorylation in disease.Open in a separate windowFig. 1.Schematic of tau constructs and an MTBR repeat. The functional domains of tau are indicated on the longest full-length isoform with alternatively spliced regions marked by dashed lines (A). The interrepeat regions that link the conserved repeat sequences are indicated by cross-hatching. On the fragments used in this study (B), K16 (residues 198–372) and K18 (residues 244–372), the residues mutated to cysteine for attachment of fluorophores (residues 244, 322, and 354), and proline to leucine/serine mutation sites (301 and 332) are indicated. A schematic of a repeat within the MTBR (C) illustrates interrepeat and repeats sequences, with the conserved residues shown.Attempts to obtain resolution structural information of tau have been hindered by the fact that it is intrinsically disordered under physiological conditions (25) and remains largely disordered even on binding to MTs. Contrasting models derived from cryo-EM suggest tau binds either along the outer protofilament ridge (26) or to the inner surface (27) of MTs. The MTBR carries a net positive charge, and binding of tau to the acidic carboxy termini of α and β tubulin has been observed both in the context of MTs (28) and for the isolated peptides corresponding to these tubulin sequences (29, 30). In addition, a secondary binding site has been mapped to an independent region within the C-terminal third of tubulin (30). Cleavage of the C-terminal tail of tubulin is sufficient to increase polymerization rates (30), and it has been suggested that tau may promote MT polymerization through a similar charge neutralization mechanism.The MTBR also plays a central role in tau pathology in that it forms the core of the aggregates found in disease (31) and contains the minimum sequence necessary to recapitulate relevant features of aggregation in vitro (32). Moreover, the majority of mutations to tau implicated in tauopathies are either point mutations within the MTBR or mutations that interfere with alternative splicing, altering the normal ∼1:1 ratio of 3R:4R tau (8, 9). Notable among the point mutants is P301L (tau_P301L) (Fig. 1B), implicated in frontotemporal dementia with Parkinsonism-17, which provided one of the first genetic links between tau and neurodegenerative disease (33). The P301L variant has emerged as a particularly reliable model for tau-based neurodegenerative disease, having successfully reproduced aspects of pathology in animal models of Alzheimer’s disease and other tauopathies (34).Although significant work has focused on tau interactions with MTs, very little is known about the mechanistic details of tau-mediated MT polymerization, including interactions of tau with tubulin dimers during the assembly process. Although many alterations to tau implicated in disease have been shown to affect MT polymerization both in vitro and in vivo, virtually nothing is known about the mechanism by which these changes occur. Tau_P301L exhibits impaired tubulin polymerization (35), as well as increased aggregation propensity relative to WT protein (tau_WT) (32) and thus serves as a model for both loss of function and gain of toxic function aspects of disease. This mutation is in the highly conserved PGGG sequence of R2 (Fig. 1C). To broaden our understanding of the impact of this point mutation on pathology, we designed a tau variant with the analogous proline to leucine substitution in the PGGG sequence of R3, tau_P332L, as well as a double mutant, tau_P301L/P332L. We use single molecule fluorescence to investigate structural and functional aspects of the interaction between tau and tubulin. In combination with ensemble polymerization and aggregation assays, this work provides insight into the relationship between functional and dysfunctional roles of tau.     

Interneuron precursor transplants in adult hippocampus reverse psychosis-relevant features in a mouse model of hippocampal disinhibition

GABAergic interneuron hypofunction is hypothesized to underlie hippocampal dysfunction in schizophrenia. Here, we use the cyclin D2 knockout (Ccnd2^−/−) mouse model to test potential links between hippocampal interneuron deficits and psychosis-relevant neurobehavioral phenotypes. Ccnd2^−/− mice show cortical PV⁺ interneuron reductions, prominently in hippocampus, associated with deficits in synaptic inhibition, increased in vivo spike activity of projection neurons, and increased in vivo basal metabolic activity (assessed with fMRI) in hippocampus. Ccnd2^−/− mice show several neurophysiological and behavioral phenotypes that would be predicted to be produced by hippocampal disinhibition, including increased ventral tegmental area dopamine neuron population activity, behavioral hyperresponsiveness to amphetamine, and impairments in hippocampus-dependent cognition. Remarkably, transplantation of cells from the embryonic medial ganglionic eminence (the major origin of cerebral cortical interneurons) into the adult Ccnd2^−/− caudoventral hippocampus reverses these psychosis-relevant phenotypes. Surviving neurons from these transplants are 97% GABAergic and widely distributed within the hippocampus. Up to 6 mo after the transplants, in vivo hippocampal metabolic activity is lowered, context-dependent learning and memory is improved, and dopamine neuron activity and the behavioral response to amphetamine are normalized. These findings establish functional links between hippocampal GABA interneuron deficits and psychosis-relevant dopaminergic and cognitive phenotypes, and support a rationale for targeting limbic cortical interneuron function in the prevention and treatment of schizophrenia.Precursors of most γ-aminobutyric acid (GABA)-releasing interneurons of the cerebral cortex and the hippocampus originate in the embryonic medial ganglionic eminence (MGE) (1–3). A subpopulation of MGE-derived cells differentiates into fast-spiking, parvalbumin-expressing (PV⁺) interneurons that tightly regulate the activity and synchronization of cortical projection neurons (2, 4). Structural and functional deficits in PV⁺ interneurons are hypothesized as a pathophysiological mechanism in schizophrenia and psychotic disorders (4–6).Although psychotic disorders are clearly heterogeneous in etiology, disinhibition within temporolimbic cortical circuits is postulated as a core pathophysiology underlying positive symptoms (e.g., delusions and hallucinations) and a subset of cognitive disturbances that manifest with psychosis (4, 5, 7). Postmortem studies of brains from individuals with psychotic disorders show reduced molecular markers of the number and/or function of PV⁺ interneurons in the hippocampus (6, 8). Consistent with these observations, basal metabolic activity in the hippocampus, as measured with functional magnetic resonance imaging (fMRI), is increased in schizophrenia, a phenotype that predicts psychosis and positive symptom severity (5, 7). This abnormal resting activity is postulated to underlie abnormal recruitment of hippocampal circuits during cognitive performance (5, 9). Striatal dopamine (DA) release capacity is also increased and correlated with positive symptoms in schizophrenia and its risk states (10, 11). Importantly, hippocampal hyperactivity may contribute to DA dysregulation (12), because rodent studies show that caudoventral hippocampal (in the primate, anterior hippocampal) efferents regulate the activity of DA neurons and medial striatal DA release (13, 14).Thus, converging evidence implicates hippocampal disinhibition in the abnormal striatal DA transmission and cognitive impairment in schizophrenia. However, the role of hippocampal inhibitory interneurons in psychosis-relevant circuitry remains to be established. To this end, we used the cyclin D2 (Ccnd2) knockout mouse model (15), which displays a relatively selective deficit in cortical PV⁺ interneurons, and transplantation of interneuron precursors from the MGE to elucidate relationships between reduced hippocampal GABA interneuron function and multiple psychosis-relevant phenotypes, and to explore a novel treatment strategy for psychosis.     

Quantifying a Rare Disease in Administrative Data: The Example of Calciphylaxis

BACKGROUND

Calciphylaxis, a rare disease seen in chronic dialysis patients, is associated with significant morbidity and mortality. As is the case with other rare diseases, the precise epidemiology of calciphylaxis remains unknown. Absence of a unique International Classification of Diseases (ICD) code impedes its identification in large administrative databases such as the United States Renal Data System (USRDS) and hinders patient-oriented research. This study was designed to develop an algorithm to accurately identify cases of calciphylaxis and to examine its incidence and mortality.

DESIGN, PARTICIPANTS, AND MAIN MEASURES

Along with many other diagnoses, calciphylaxis is included in ICD-9 code 275.49, Other Disorders of Calcium Metabolism. Since calciphylaxis is the only disorder listed under this code that requires a skin biopsy for diagnosis, we theorized that simultaneous application of code 275.49 and skin biopsy procedure codes would accurately identify calciphylaxis cases. This novel algorithm was developed using the Partners Research Patient Data Registry (RPDR) (n?=?11,451 chronic hemodialysis patients over study period January 2002 to December 2011) using natural language processing and review of medical and pathology records (the gold-standard strategy). We then applied this algorithm to the USRDS to investigate calciphylaxis incidence and mortality.

KEY RESULTS

Comparison of our novel research strategy against the gold standard yielded: sensitivity 89.2 %, specificity 99.9 %, positive likelihood ratio 3,382.3, negative likelihood ratio 0.11, and area under the curve 0.96. Application of the algorithm to the USRDS identified 649 incident calciphylaxis cases over the study period. Although calciphylaxis is rare, its incidence has been increasing, with a major inflection point during 2006–2007, which corresponded with specific addition of calciphylaxis under code 275.49 in October 2006. Calciphylaxis incidence continued to rise even after limiting the study period to 2007 onwards (from 3.7 to 5.7 per 10,000 chronic hemodialysis patients; r?=?0.91, p?=?0.02). Mortality rates among calciphylaxis patients were noted to be 2.5–3 times higher than average mortality rates for chronic hemodialysis patients.

CONCLUSIONS

By developing and successfully applying a novel algorithm, we observed a significant increase in calciphylaxis incidence. Because calciphylaxis is associated with extremely high mortality, our study provides valuable information for future patient-oriented calciphylaxis research, and also serves as a template for investigating other rare diseases.