Background
The ability to maintain balance is an evolutionarily‐conserved behavior that is frequently disrupted found in patients with neurodegenerative diseases. One of the most prominent balance ...disorders is found in patients with progressive supranuclear palsy (PSP), a primary tauopathy pathologically characterized by tau over‐representation in the brainstem vestibulospinal (VS) nucleus, where they frequently exhibit accidental‐backward falls starting from the early stage of the disease. Although pathological features of PSP correlate well with its clinical phenotype, how tau aggregation affects neuronal and circuit functions, which eventually leads to behavioral deficits, remains unclear.
Method
To dissect disease mechanisms across molecular, cellular, circuitry, and behavioral levels, we generated tau fish by expressing human 0N/4R‐Tau in zebrafish VS nucleus. Tau expression and phosphorylation were validated using immunohistochemistry staining with PHF‐1 antibody. To examine the effect of tau on balance behavior, we measured postural control of free‐swimming tau fish and compared to that of tau‐negative siblings. Moreover, we tested response of VS neurons to nose‐down and nose‐up tilt stimulus using 2‐photon calcium imaging.
Result
Ttau‐expressing zebrafish exhibit impaired balance control while maintaining normal locomotor ability. Interestingly, we did not observe any neuronal death in the VS nucleus. Functional imaging of the VS nucleus shows impaired directional tuning in tau‐expressing neurons in response to tilt stimulus. We also found ectopic accumulation of acidic organelles in the cell bodies of tau‐positive neurons, suggesting abnormal lysosomal function.
Conclusion
Our results demonstrate how molecular abnormalities disrupt specific behavior in tauopathies before neurodegeneration appeared.
Optogenetic approaches in transparent zebrafish models have provided numerous insights into vertebrate neurobiology. The purpose of this study was to develop methods to activate light-sensitive ...transgene products simultaneously throughout an entire larval zebrafish.
We developed a LED illumination stand and microcontroller unit to expose zebrafish larvae reproducibly to full field illumination at defined wavelength, power, and energy.
The LED stand generated a sufficiently flat illumination field to expose multiple larval zebrafish to high power light stimuli uniformly, while avoiding sample bath warming. The controller unit allowed precise automated delivery of predetermined amounts of light energy at calibrated power. We demonstrated the utility of the approach by driving photoconversion of Kaede (398 nm), photodimerization of GAVPO (450 nm), and photoactivation of dL5**/MG2I (661 nm) in neurons throughout the CNS of larval zebrafish. Observed outcomes were influenced by both total light energy and its rate of delivery, highlighting the importance of controlling these variables to obtain reproducible results.
Our approach employs inexpensive LED chip arrays to deliver narrow-waveband light with a sufficiently flat illumination field to span multiple larval zebrafish simultaneously. Calibration of light power and energy are built into the workflow.
The LED illuminator and controller can be constructed from widely available materials using the drawings, instructions, and software provided. This approach will be useful for multiple optogenetic applications in zebrafish and other models.
Multiple convergent lines of evidence implicate both α-synuclein (encoded by SCNA) and mitochondrial dysfunction in the pathogenesis of sporadic Parkinson's disease (PD). Occupational exposure to the ...mitochondrial complex I inhibitor rotenone increases PD risk; rotenone-exposed rats show systemic mitochondrial defects but develop specific neuropathology, including α-synuclein aggregation and degeneration of substantia nigra dopaminergic neurons. Here, we inhibited expression of endogenous α-synuclein in the adult rat substantia nigra by adeno-associated virus-mediated delivery of a short hairpin RNA (shRNA) targeting the endogenous rat Snca transcript. Knockdown of α-synuclein by ~35% did not affect motor function or cause degeneration of nigral dopaminergic neurons in control rats. However, in rotenone-exposed rats, progressive motor deficits were substantially attenuated contralateral to α-synuclein knockdown. Correspondingly, rotenone-induced degeneration of nigral dopaminergic neurons, their dendrites, and their striatal terminals was decreased ipsilateral to α-synuclein knockdown. These data show that α-synuclein knockdown is neuroprotective in the rotenone model of PD and indicate that endogenous α-synuclein contributes to the specific vulnerability of dopaminergic neurons to systemic mitochondrial inhibition. Our findings are consistent with a model in which genetic variants influencing α-synuclein expression modulate cellular susceptibility to environmental exposures in PD patients. shRNA targeting the SNCA transcript should be further evaluated as a possible neuroprotective therapy in PD.
This article describes a method to quantify the movements of larval zebrafish in multiwell plates, using the open-source MATLAB applications LSRtrack and LSRanalyze. The protocol comprises four ...stages: generation of high-quality, flatly illuminated video recordings with exposure settings that facilitate object recognition; analysis of the resulting recordings using tools provided in LSRtrack to optimize tracking accuracy and motion detection; analysis of tracking data using LSRanalyze or custom MATLAB scripts; and implementation of validation controls. The method is reliable, automated and flexible, requires <1 h of hands-on work for completion once optimized and shows excellent signal:noise characteristics. The resulting data can be analyzed to determine the following: positional preference; displacement, velocity and acceleration; and duration and frequency of movement events and rest periods. This approach is widely applicable to the analysis of spontaneous or stimulus-evoked zebrafish larval neurobehavioral phenotypes resulting from a broad array of genetic and environmental manipulations, in a multiwell plate format suitable for high-throughput applications.
Human spinal cord injury (SCI) is characterized by permanent loss of damaged axons, resulting in chronic disability. In contrast, zebrafish can regenerate axonal projections following central nervous ...system injury and re‐establish synaptic contacts with distant targets; elucidation of the underlying molecular events is an important goal with translational potential for improving outcomes in SCI patients. We generated transgenic zebrafish with GFP‐labeled axons and transected their spinal cords at 10 days post‐fertilization. Intravital confocal microscopy revealed robust axonal regeneration following the procedure, with abundant axons bridging the transection site by 48 h post‐injury. In order to analyze neurological function in this model, we developed and validated new open‐source software to measure zebrafish lateral trunk curvature during propulsive and turning movements at high temporal resolution. Immediately following spinal cord transection, axial movements were dramatically decreased caudal to the lesion site, but preserved rostral to the injury, suggesting the induction of motor paralysis below the transection level. Over the subsequent 96 h, the magnitude of movements caudal to the lesion recovered to baseline, but the rate of change of truncal curvature did not fully recover, suggesting incomplete restoration of caudal strength over this time course. Quantification of both morphological and functional recovery following SCI will be important for the analysis of axonal regeneration and downstream events necessary for restoration of motor function. An extensive array of genetic and pharmacological interventions can be deployed in the larval zebrafish model to investigate the underlying molecular mechanisms.
New open‐source software allowed measurement of trunk curvature during dark‐evoked larval zebrafish movements following spinal cord transection. Motor deficits caudal to the lesion recovered incompletely, even after regeneration of spinal axons, suggesting that evaluation of function will be critical to understand restoration of circuitry in this central nervous system injury model.
Bi-allelic mutations in the glucocerebrosidase gene (GBA1) cause Gaucher's disease, the most common human lysosomal storage disease. We previously reported a marked increase in miR-155 transcript ...levels and early microglial activation in a zebrafish model of Gaucher's disease (gba1−/−). miR-155 is a master regulator of inflammation and has been implicated in a wide range of different neurodegenerative disorders. The observed miR-155 upregulation preceded the subsequent development of widespread pathology with marked neuroinflammation, closely resembling human Gaucher's disease pathology. We now report similar increases of miR-155 expression in mammalian models of GD, confirming that miR-155 upregulation is a shared feature in glucocerebrosidase (GCase) deficiency across different species. Using CRISPR/Cas9 mutagenesis we then generated a miR-155 mutant zebrafish line (miR-155−/−) with completely abolished miR-155 expression. Unexpectedly, loss of miR-155 did not mitigate either the reduced lifespan or the robust inflammatory phenotypes of gba1−/− mutant zebrafish. Our data demonstrate that neither neuroinflammation nor disease progression in GCase deficiency are dependent on miR-155 and suggest that miR-155 inhibition would not be a promising therapeutic target in Gaucher's disease.
•miR-155 is a master regulator of inflammation.•miR-155 is upregulated in different neurodegenerative disorders.•miR-155 is increased in a zebrafish model of Gaucher disease (GD).•We now show that miR-155 is also upregulated in mammalian models of GD.•However, miR-155 inactivation does not rescue the GD phenotype.
Mitochondrial dysfunction is implicated in the pathogenesis of multiple neurological diseases, but elucidation of underlying mechanisms is limited experimentally by the inability to damage specific ...mitochondria in defined neuronal groups. We developed a precision chemoptogenetic approach to target neuronal mitochondria in the intact nervous system in vivo. MG2I, a chemical fluorogen, produces singlet oxygen when bound to the fluorogen-activating protein dL5** and exposed to far-red light. Transgenic zebrafish expressing dL5** within neuronal mitochondria showed dramatic MG2I- and light-dependent neurobehavioral deficits, caused by neuronal bioenergetic crisis and acute neuronal depolarization. These abnormalities resulted from loss of neuronal respiration, associated with mitochondrial fragmentation, swelling and elimination of cristae. Remaining cellular ultrastructure was preserved initially, but cellular pathology downstream of mitochondrial damage eventually culminated in neuronal death. Our work provides powerful new chemoptogenetic tools for investigating mitochondrial homeostasis and pathophysiology and shows a direct relationship between mitochondrial function, neuronal biogenetics and whole-animal behavior.
Traumatic brain injury (TBI) is a prominent risk factor for dementias including tauopathies like chronic traumatic encephalopathy (CTE). The mechanisms that promote prion-like spreading of Tau ...aggregates after TBI are not fully understood, in part due to lack of tractable animal models. Here, we test the putative role of seizures in promoting the spread of tauopathy. We introduce 'tauopathy reporter' zebrafish expressing a genetically encoded fluorescent Tau biosensor that reliably reports accumulation of human Tau species when seeded via intraventricular brain injections. Subjecting zebrafish larvae to a novel TBI paradigm produced various TBI features including cell death, post-traumatic seizures, and Tau inclusions. Bath application of dynamin inhibitors or anticonvulsant drugs rescued TBI-induced tauopathy and cell death. These data suggest a role for seizure activity in the prion-like seeding and spreading of tauopathy following TBI. Further work is warranted regarding anti-convulsants that dampen post-traumatic seizures as a route to moderating subsequent tauopathy.
Optokinetic reflex (OKR) responses provide a convenient means to evaluate visual, integrative and oculomotor function in larval zebrafish. We measured multiple aspects of the OKR response in ...zebrafish exposed systemically to compounds altering signaling at GABA
receptors in order to derive quantitative concentration-response relationships. The GABA
antagonist picrotoxin caused concentration-dependent decreases in reflex gain, saccade velocity, saccade amplitude, interocular concordance and interocular gain. Conversely, the GABA
agonist gaboxadol provoked increases in reflex gain, saccade velocity, saccade amplitude and ocular range at low concentrations, and decreases in some of these parameters at higher concentrations. These data show that GABA
signaling influences multiple aspects of the OKR (including gain, generation of saccades, and coordination between the two eyes) and provide proof of concept that quantitative OKR analysis can be used as a tool for chemical biology and neuropharmacology applications.
•We evaluated GABAA-modulating agents in the zebrafish optokinetic reflex (OKR).•Picrotoxin decreased reflex gain, and saccade velocity and amplitude.•Gaboxadol increased reflex gain, and saccade ...velocity and amplitude.•Quantitative changes were robust and concentration-dependent.•The OKR may be useful as a chemical biology and neuropharmacology tool.
Optokinetic reflex (OKR) responses provide a convenient means to evaluate visual, integrative and oculomotor function in larval zebrafish. We measured multiple aspects of the OKR response in zebrafish exposed systemically to compounds altering signaling at GABAA receptors in order to derive quantitative concentration-response relationships. The GABAA antagonist picrotoxin caused concentration-dependent decreases in reflex gain, saccade velocity, saccade amplitude, interocular concordance and interocular gain. Conversely, the GABAA agonist gaboxadol provoked increases in reflex gain, saccade velocity, saccade amplitude and ocular range at low concentrations, and decreases in some of these parameters at higher concentrations. These data show that GABAA signaling influences multiple aspects of the OKR (including gain, generation of saccades, and coordination between the two eyes) and provide proof of concept that quantitative OKR analysis can be used as a tool for chemical biology and neuropharmacology applications.
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