Harnessing diversity to study Alzheimer's disease: A new iPSC resource from the NIH CARD and ADNI.

The iDA Project (iPSCs to Study Diversity in Alzheimer's and Alzheimer's Disease-related Dementias) is generating 200 induced pluripotent stem cell lines from Alzheimer's Disease Neuroimaging Initiative participants. These lines are sex balanced, include common APOE genotypes, span disease stages, and are ancestrally diverse. Cell lines and characterization data will be shared openly.

Mis-spliced transcripts generate de novo proteins in TDP-43-related ALS/FTD.

Functional loss of TDP-43, an RNA binding protein genetically and pathologically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), leads to the inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote the degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. Here, we show that mRNA transcripts harboring cryptic exons generated de novo proteins in TDP-43-depleted human iPSC-derived neurons in vitro, and de novo peptides were found in cerebrospinal fluid (CSF) samples from patients with ALS or FTD. Using coordinated transcriptomic and proteomic studies of TDP-43-depleted human iPSC-derived neurons, we identified 65 peptides that mapped to 12 cryptic exons. Cryptic exons identified in TDP-43-depleted human iPSC-derived neurons were predictive of cryptic exons expressed in postmortem brain tissue from patients with TDP-43 proteinopathy. These cryptic exons produced transcript variants that generated de novo proteins. We found that the inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Last, we showed that 18 de novo peptides across 13 genes were present in CSF samples from patients with ALS/FTD spectrum disorders. The demonstration of cryptic exon translation suggests new mechanisms for ALS/FTD pathophysiology downstream of TDP-43 dysfunction and may provide a potential strategy to assay TDP-43 function in patient CSF.

Defining the causes of sporadic Parkinson's disease in the global Parkinson's genetics program (GP2).

The Global Parkinson's Genetics Program (GP2) will genotype over 150,000 participants from around the world, and integrate genetic and clinical data for use in large-scale analyses to dramatically expand our understanding of the genetic architecture of PD. This report details the workflow for cohort integration into the complex arm of GP2, and together with our outline of the monogenic hub in a companion paper, provides a generalizable blueprint for establishing large scale collaborative research consortia.

Mis-spliced transcripts generate de novo proteins in TDP-43-related ALS/FTD.

Functional loss of TDP-43, an RNA-binding protein genetically and pathologically linked to ALS and FTD, leads to inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. However, the possibility of de novo protein synthesis from cryptic exon transcripts has not been explored. Here, we show that mRNA transcripts harboring cryptic exons generate de novo proteins both in TDP-43 deficient cellular models and in disease. Using coordinated transcriptomic and proteomic studies of TDP-43 depleted iPSC-derived neurons, we identified numerous peptides that mapped to cryptic exons. Cryptic exons identified in iPSC models were highly predictive of cryptic exons expressed in brains of patients with TDP-43 proteinopathy, including cryptic transcripts that generated de novo proteins. We discovered that inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Finally, we showed that these de novo peptides were present in CSF from patients with ALS. The demonstration of cryptic exon translation suggests new mechanisms for ALS pathophysiology downstream of TDP-43 dysfunction and may provide a strategy for novel biomarker development.

Physiological Evidence for Delayed Age-related Hearing Loss in Two Long-lived Rodent Species (Peromyscus leucopus and P. californicus).

Deer mice (genus Peromyscus) are an emerging model for aging studies due to their longevity relative to rodents of similar size. Although Peromyscus species are well-represented in genetic, developmental, and behavioral studies, relatively few studies have investigated auditory sensitivity in this genus. Given the potential utility of Peromyscus for investigations of age-related changes to auditory function, we recorded auditory brainstem responses (ABRs) in two Peromyscus species, P. californicus, and P. leucopus, across the lifespan. We compared hearing sensitivity and ABR wave metrics measured in these species with measurements from Mus musculus (CBA/CaJ strain) to assess age-related effects on hearing across species. Recordings in young animals showed that all species had similar hearing ranges and thresholds with peak sensitivity ranging from 8 to 16 kHz; however, P. californicus and P. leucopus were more sensitive to frequencies below 8 kHz. Although M. musculus showed significant threshold shifts across a broad range of frequencies beginning at middle age and worsening among old individuals, older Peromyscus mice retained good sensitivity to sound across their lifespan. Middle-aged P. leucopus had comparable thresholds to young for frequencies below 24 kHz. P. leucopus also had notably large ABRs that were robust to age-related amplitude reductions, although response latencies increased with age. Old P. californicus were less sensitive to mid-range tones (8-16 kHz) than young individuals; however, there were no significant age-effects on ABR amplitudes or latencies in this species. These results indicate that longevity in Peromyscus mice may be correlated with delayed aging of the auditory system and highlight these species as promising candidates for longitudinal hearing research.

Effects of Noise Exposure and Aging on Behavioral Tone Detection in Quiet and Noise by Mice.

Aging leads to degeneration of the peripheral and central auditory systems, hearing loss, and difficulty understanding sounds in noise. Aging is also associated with changes in susceptibility to or recovery from damaging noise exposures, although the effects of the interaction between acute noise exposure and age on the perception of sounds are not well studied. We tested these effects in the CBA/CaJ mouse model of age-related hearing loss using operant conditioning procedures before and after noise exposure and longitudinally measured changes in their sensitivity for detecting tones in quiet or noise backgrounds. Cochleae from a subset of the behaviorally tested mice were immunolabeled to examine organ of Corti damage relative to what is expected based on aging alone. Mice tested in both quiet and noise background conditions experienced worse behavioral sensitivity immediately after noise exposure, but mice exposed at older ages generally showed greater threshold shifts and reduced recovery over time. Surprisingly, day-to-day stability in thresholds was markedly higher for mice detecting signals in the presence of a noise masker compared with detection in quiet conditions. Cochlear analysis revealed decreases in the total number of outer hair cells (OHCs) and the number of ribbons per inner cell in high-frequency regions in aged, noise-exposed mice relative to aging alone. Our findings build on previous work showing interactions between age and noise exposure and add that background noise can increase the stability of behavioral hearing sensitivity after noise damage.

Deafness in an auditory specialist, the big brown bat (Eptesicus fuscus).

Bats are long-lived animals that show presumed resistance to noise-induced and age-related hearing loss, which has been attributed to their dependence on sound processing for survival. Echolocation and basic auditory functions have been studied extensively in the big brown bat (Eptesicus fuscus), an insectivorous microchiropteran species. We conducted hearing tests and analysis of cochlear sensory cells in a group of big brown bats that exhibited anomalies in behavioral sonar tracking experiments and/or lacked neural responses to acoustic stimulation in subcortical auditory nuclei. We show for the first time the presence of profound deafness and extensive cochlear damage in an echolocating bat species. Auditory brainstem responses were abnormal or absent in these bats, and histological analyses of their cochleae revealed extensive loss of hair cells, supporting cells, and spiral ganglion neurons. The underlying cause of deafness is unknown.

Linking anatomical and physiological markers of auditory system degeneration with behavioral hearing assessments in a mouse (Mus musculus) model of age-related hearing loss.

Age-related hearing loss is a very common sensory disability, affecting one in three older adults. Establishing a link between anatomical, physiological, and behavioral markers of presbycusis in a mouse model can improve the understanding of this disorder in humans. We measured age-related hearing loss for a variety of acoustic signals in quiet and noisy environments using an operant conditioning procedure and investigated the status of peripheral structures in CBA/CaJ mice. Mice showed the greatest degree of hearing loss in the last third of their lifespan, with higher thresholds in noisy than in quiet conditions. Changes in auditory brainstem response thresholds and waveform morphology preceded behavioral hearing loss onset. Loss of hair cells, auditory nerve fibers, and signs of stria vascularis degeneration were observed in old mice. The present work underscores the difficulty in ascribing the primary cause of age-related hearing loss to any particular type of cellular degeneration. Revealing these complex structure-function relationships is critical for establishing successful intervention strategies to restore hearing or prevent presbycusis.

Functional, Morphological, and Evolutionary Characterization of Hearing in Subterranean, Eusocial African Mole-Rats.

Naked mole-rats are highly vocal, eusocial, subterranean rodents with, counterintuitively, poor hearing. The causes underlying their altered hearing are unknown. Moreover, whether altered hearing is degenerate or adaptive to their unique lifestyles is controversial. We used various methods to identify the factors contributing to altered hearing in naked and the related Damaraland mole-rats and to examine whether these alterations result from relaxed or adaptive selection. Remarkably, we found that cochlear amplification was absent from both species despite normal prestin function in outer hair cells isolated from naked mole-rats. Instead, loss of cochlear amplification appears to result from abnormal hair bundle morphologies observed in both species. By exploiting a well-curated deafness phenotype-genotype database, we identified amino acid substitutions consistent with abnormal hair bundle morphology and reduced hearing sensitivity. Amino acid substitutions were found in unique groups of six hair bundle link proteins. Molecular evolutionary analyses revealed shifts in selection pressure at both the gene and the codon level for five of these six hair bundle link proteins. Substitutions in three of these proteins are associated exclusively with altered hearing. Altogether, our findings identify the likely mechanism of altered hearing in African mole-rats, making them the only identified mammals naturally lacking cochlear amplification. Moreover, our findings suggest that altered hearing in African mole-rats is adaptive, perhaps tailoring hearing to eusocial and subterranean lifestyles. Finally, our work reveals multiple, unique evolutionary trajectories in African mole-rat hearing and establishes species members as naturally occurring disease models to investigate human hearing loss.

Perception of Ultrasonic Vocalizations by Socially Housed and Isolated Mice.

It is currently unclear whether mice use their ultrasonic vocalizations (USVs) for communication purposes. It is also unknown whether mice require previous experience with USVs to understand conspecifics. There is some evidence that experience changes the perception of juvenile USVs; however, it is unclear whether similar plasticity also occurs for adult USVs. To examine whether social exposure or deprivation throughout development leads to changes in USV perception, eleven female CBA/CaJ mice were trained to discriminate between 18 USVs of three different categories using operant conditioning procedures. Mice were group housed with four females or housed individually from weaning for the duration of the experiment. Socially housed and isolated mice differed in initial training times on pure tones, suggesting isolated mice had a more difficult time learning the task. Both groups completed USV discrimination conditions quicker at the end of the testing phases relative to the beginning. The overall discrimination of USVs did not differ between the two housing conditions, but a multidimensional scaling analysis revealed that socially experienced and isolated mice perceive some USVs differently, illustrated by differences in locations of USVs on the scaling maps from the two groups. Finally, a negative correlation was found between spectrotemporal similarity and percent discrimination, and analyses support the idea that mice may show categorical perception of at least two of the three USV categories. Thus, experience with USVs changes USV perception.

Social isolation produces no effect on ultrasonic vocalization production in adult female CBA/CaJ mice.

Mice produce ultrasonic vocalizations (USVs) in a wide variety of social contexts, including courtship, investigation, and territorial defense. Despite the belief that mouse USVs are innate, social experience may be necessary for mice to learn the appropriate situation to emit USVs. Mouse USVs have been divided into categories based on their spectrotemporal parameters, but it is currently unclear if social experience changes these parameters (e.g., frequency and duration) or the proportion of calls from each category produced. Social isolation has been found to influence USV production in male mice. To investigate the influence of social isolation on vocal behavior in female mice, recordings were made of USVs emitted to unfamiliar male and female mice by subjects with one of three types of social experience. Twenty-four adult female CBA/CaJ mice either lived alone, lived with other females only, or lived with other females and had limited access to a male. Mice were recorded while in isolation, ensuring all recorded USVs were from the female of interest. Vocalizations were separated into nine categories and peak frequency, duration, and bandwidth were measured for every call. Socially isolated mice did not produce significantly more USVs or USV types than socially experienced mice. Social isolation did not have a significant effect on the features of USVs, suggesting production of USVs may not be learned in female mice.

Behavioral measurements of auditory streaming and build-up by budgerigars (Melopsittacus undulatus).

The perception of the build-up of auditory streaming has been widely investigated in humans, while it is unknown whether animals experience a similar perception when hearing high (H) and low (L) tonal pattern sequences. The paradigm previously used in European starlings (Sturnus vulgaris) was adopted in two experiments to address the build-up of auditory streaming in budgerigars (Melopsittacus undulatus). In experiment 1, different numbers of repetitions of low-high-low triplets were used in five conditions to study the build-up process. In experiment 2, 5 and 15 repetitions of high-low-high triplets were used to investigate the effects of repetition rate, frequency separation, and frequency range of the two tones on the birds' streaming perception. Similar to humans, budgerigars subjectively experienced the build-up process in auditory streaming; faster repetition rates and larger frequency separations enhanced the streaming perception, and these results were consistent across the two frequency ranges. Response latency analysis indicated that the budgerigars needed a longer amount of time to respond to stimuli that elicited a salient streaming perception. These results indicate, for the first time using a behavioral paradigm, that budgerigars experience a build-up of auditory streaming in a manner similar to humans.

Preference in female laboratory mice is influenced by social experience.

Mice must perceive and interpret the signals they are presented with to evaluate potential mates, and should show a preference for high quality conspecifics. The present experiment was designed to investigate preference for acoustic, olfactory, and acoustic + olfactory signals in laboratory mice. Eighteen female CBA/CaJ mice, socially housed or isolated, were tested to evaluate preference for signals produced by males. Social experience influenced female preference for male USV playback and soiled bedding. Female mice who had experience with males showed a stronger preference for olfactory signals, whereas female mice with experience only with other females preferred USV playback. Isolated mice showed a greater preference for signals of different modalities presented together than socially experienced mice. This preference may have occurred because of increased motivation to make social contact. The findings of this experiment illustrate that social experience could play a role in mate choice.

CBA/CaJ mouse ultrasonic vocalizations depend on prior social experience.

Mouse ultrasonic vocalizations (USVs) have variable spectrotemporal features, which researchers use to parse them into different categories. USVs may be important for communication, but it is unclear whether the categories that researchers have developed are relevant to the mice. Instead, other properties such as the number, rate, peak frequency, or bandwidth of the vocalizations may be important cues that the mice are using to interpret the nature of the social interaction. To investigate this, a comprehensive catalog of the USVs that mice are producing across different social contexts must be created. Forty male and female adult CBA/CaJ mice were recorded in isolation for five minutes following either a one-hour period of isolation or an exposure to a same- or opposite-sex mouse. Vocalizations were separated into nine categories based on the frequency composition of each USV. Additionally, USVs were quantified based on the bandwidth, duration, peak frequency, total number, and proportion of vocalizations produced. Results indicate that mice differentially produce their vocalizations across social encounters. There were significant differences in the number of USVs that mice produce across exposure conditions, the proportional probability of producing the different categories of USVs across sex and conditions, and the features of the USVs across conditions. In sum, there are sex-specific differences in production of USVs by laboratory mice, and prior social experiences matter for vocalization production. Furthermore, this study provides critical evidence that female mice probably produce vocalizations in opposite-sex interactions, which is important because this is an often overlooked variable in mouse communication research.

Discrimination of frequency modulated sweeps by mice.

Mice often produce ultrasonic vocalizations (USVs) that sweep upwards in frequency from around 60 to around 80 kHz and downwards in frequency from 80 to 60 kHz. Whether or not these USVs are used for communication purposes is still unknown. Here, mice were trained and tested using operant conditioning procedures and positive reinforcement to discriminate between synthetic upsweeps and downsweeps. The stimuli varied in bandwidth, duration, and direction of sweep. The mice performed significantly worse when discriminating between background and test stimuli when the stimuli all occupied the same bandwidths. Further, the mice's discrimination performance became much worse for stimuli that had durations similar to those natural vocalizations of the mice. Sweeps composed of different frequency ranges and longer durations had improved discrimination. These results collected using artificial stimuli created to mimic natural USVs indicate that the bandwidth of the vocalizations may be much more important for communication than the frequency contours of the vocalizations.