VCP Protects Neurons from Proteopathic Seeding

Background: Neuronal uptake and subsequent spread of proteopathic seeds, such as αS (alpha-synuclein), tau, and TDP-43, contribute to neurodegeneration and disease progression. The cellular machinery necessary for this process is poorly understood. Methods: Cas9 expressing αS FRET biosensors were transduced with a whole-genome guide RNA (gRNA) library, seeded with αS fibrils, and flow-sorted. Candidate genes protective against αS seeding were identified following gRNA sequencing of FRET+ and FRET- cell populations. Secondary validation of the high probability candidate suppressor VCP, utilized VCP inhibitors or gene knockdown in αS biosensors and primary neurons. In vivo validation was performed in VCP disease mutation mice following intrastriatal injection of αS seeds. TDP-43 seeding was performed in primary neurons from control or VCP mutant mice.Results: We devised a genome-wide CRISPR-Cas9 screen to identify suppressors of αS seeding. This approach identified Valosin Containing Protein (VCP) as a suppressor of αS seeding. Dominant mutations in VCP cause multisystem proteinopathy (MSP) a phenotypically and pathologically variable neurodegeneraive disease characterized by myopathy, motor neuron disease and dementia with TDP-43, αS and tau inclusions. VCP inhibition or MSP disease mutations increased αS seeding in cells and primary cultured neurons. This was similar to treatment with the lysosomal damaging agent, LLoMe or knockdown of the endolysosomal damage response associated VCP cofactor, UBXD1. Intrastriatal injection of αS seeds into VCP disease mice demonstrated enhanced seeding efficiency as compared with controls. Finally, this phenomenon was not specific to αS since VCP disease mutant expression increased TDP-43 seeding in neurons.Conclusion: VCP surveillance of permeabilized late endosomes protects neurons against the proteopathic spread of pathogenic protein aggregates. The spread of distinct aggregate species may dictate the pleiotropic phenotypes and pathologies in VCP associated MSP.

Transcranial Focal Electrical Stimulation Modifies Biogenic Amines’ Alterations Induced by 6-Hydroxydopamine in Rat Brain

Transcranial focal stimulation (TFS) is a non-invasive neuromodulation strategy with neuroprotective effects. On the other hand, 6-hidroxidopamine (6-OHDA) induces neurodegeneration of the nigrostriatal system producing modifications in the dopaminergic, serotoninergic, and histaminergic systems. The present study was conducted to test whether repetitive application of TFS avoids the biogenic amines’ changes induced by the intrastriatal injection of 6-OHDA. Experiments were designed to determine the tissue content of dopamine, serotonin, and histamine in the brain of animals injected with 6-OHDA and then receiving daily TFS for 21 days. Tissue content of biogenic amines was evaluated in the cerebral cortex, hippocampus, amygdala, and striatum, ipsi- and contralateral to the side of 6-OHDA injection. Results obtained were compared to animals with 6-OHDA, TFS alone, and a Sham group. The present study revealed that TFS did not avoid the changes in the tissue content of dopamine in striatum. However, TFS was able to avoid several of the changes induced by 6-OHDA in the tissue content of dopamine, serotonin, and histamine in the different brain areas evaluated. Interestingly, TFS alone did not induce significant changes in the different brain areas evaluated. The present study showed that repetitive TFS avoids the biogenic amines’ changes induced by 6-OHDA. TFS can represent a new therapeutic strategy to avoid the neurotoxicity induced by 6-OHDA.

VCP protects neurons from proteopathic seeding

Uptake and spread of proteopathic seeds, such as αS, Tau, and TDP-43, contribute to neurodegeneration. The cellular machinery necessary for this process is poorly understood. Using a genome-wide CRISPR-Cas9 screen, we identified Valosin Containing Protein (VCP) as a suppressor of αS seeding. Dominant mutations in VCP cause multisystem proteinopathy (MSP) with muscle and neuronal degeneration. VCP inhibition or disease mutations increase αS seeding in cells and neurons. This is not associated with an increase in seed uptake and is similar to treatment with the lysosomal damaging agent, LLoME. Intrastriatal injection of αS seeds into VCP disease mice enhances seeding efficiency compared with controls. This is not specific to αS since VCP inhibition or disease mutations increased TDP-43 seeding in neurons. These data support that VCP protects against proteopathic spread of pathogenic aggregates. The spread of distinct aggregate species may dictate pleiotropic phenotypes and pathologies in VCP associated MSP.

Alive Animal Model for Epilepsy by Intradorsal Striatum Injection of Colchicine

Background Epilepsy is a neural disorder with repeatable seizure attacks. We used the neurotoxin colchicine, which is derived from the plant Colchicum autumnale, to introduce a low cost but the more valuable alive animal model for epilepsy. Materials and Methods Wistar rats weighing 250 to 300 g after intraperitoneal injection of ketamine (100 mg/kg) and xylazine (20 mg/kg) were restrained in the stereotaxic apparatus; they were cannulated in the dorsal striatal area (AP: 0.5 mm; L: 3 mm; V: 3.6 mm). One week later, an injection cannula attached to a 5-µ Hamilton syringe by polyethylene tubing guided 0.05 to 25 μg/rat colchicine in the recovered healthy rats once daily for 4 consecutive days. The control group solely received the saline solution. The behavioral signs of all animals were daily recorded. Finally, the brains of rats under deep euthanasia were collected in 10% formalin and examined histopathologically. The dorsal striatal regions were cut coronally into 3 to 4 µm-thick slices, and then stained with hematoxylin-eosin. They were eventually examined under the light microscope to verify the injection placement or possibility of lesions. All data were analyzed by analysis of variance under α = 0.05. Results Behaviors were quantified based on Racine five-stage scoring and showed the significant epileptic generalized seizures in alive animal treated by intrastriatal injection of colchicine. However, tissue damage was invisible in the target brain area. Conclusion The colchicine, using injection successively into the dorsal striatal region of rat, can create recurring epileptic convulsions in the animal.

Cortical Circuit Dysfunction in a Mouse Model of Alpha-synucleinopathy in Vivo

Considerable fluctuations in cognitive performance and eventual dementia are an important characteristic of alpha-synucleinopathies, such as Parkinson’s disease (PD) and Lewy Body dementia (LBD) and are linked to cortical dysfunction. The presence of misfolded and aggregated alpha-synuclein (a-syn) in the cerebral cortex of patients has been suggested to play a crucial role in this process. However, the consequences of a-syn accumulation on the function of cortical networks at cellular resolution in vivo are largely unknown. Here we used the striatal seeding model in wildtype mice in order to induce robust a-synuclein pathology in the cerebral cortex. 9 months after a single intrastriatal injection of a-syn preformed fibrils, we performed in vivo two-photon calcium imaging in awake mice. We observed profound alterations of the function of layer 2/3 cortical neurons in somatosensory cortex (S1), as witnessed by an enhanced response to whisking and increased synchrony, accompanied by a decrease in baseline Ca2+ levels. Stereological analyses revealed a reduction in GAD67-positive inhibitory cells in S1 in PFF-injected brains. These findings point to a disturbed excitation/inhibition balance as an important driver of circuit dysfunction in alpha-synucleinopathies, which may underly cognitive changes in these diseases.

Pro-Inflammatory Role of AQP4 in Mice Subjected to Intrastriatal Injections of the Parkinsonogenic Toxin MPP+

Aquaporin-4 (AQP4) is critically involved in brain water and volume homeostasis and has been implicated in a wide range of pathological conditions. Notably, evidence has been accrued to suggest that AQP4 plays a proinflammatory role by promoting release of astrocytic cytokines that activate microglia and other astrocytes. Neuroinflammation is a hallmark of Parkinson’s disease (PD), and we have previously shown that astrocytes in substantia nigra (SN) are enriched in AQP4 relative to cortical astrocytes, and that their complement of AQP4 is further increased following treatment with the parkinsonogenic toxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). Here, we investigated the effect of Aqp4 deletion on microglial activation in mice subjected to unilateral intrastriatal injection of 1-methyl-4-phenylpyridinium (MPP+, the toxic metabolite of MPTP). Our results show that MPP+ injections lead to a pronounced increase in the expression level of microglial activating genes in the ventral mesencephalon of wild type (WT) mice, but not Aqp4−/− mice. We also show, in WT mice, that MPP+ injections cause an upregulation of nigral AQP4 and swelling of astrocytic endfeet. These findings are consistent with the idea that AQP4 plays a pro-inflammatory role in Parkinson’s disease, secondary to the dysregulation of astrocytic volume homeostasis.

Striatal seeding of protofibrillar alpha-synuclein causes cortical hyperreactivity in behaving mice

SummaryAlpha-synucleinopathies are characterized by self-aggregation of the protein alpha-synuclein (a-syn), causing alterations on the molecular and cellular level. To unravel the impact of transneuronal spreading and templated misfolding of a-syn on the microcircuitry of remotely connected brain areas, we investigated cortical neuron function in awake mice 9 months after a single intrastriatal injection of a-syn preformed fibrils (PFFs), using in vivo two-photon calcium imaging. We found altered function of layer 2/3 cortical neurons in somatosensory cortex (S1) of PFF-inoculated mice, as witnessed by an enhanced response to whisking and increased synchrony, accompanied by a decrease in baseline Ca2+ levels. Stereological analyses revealed a reduction in GAD67-positive inhibitory cells in S1 in PFF-injected brains. These findings point to a disturbed excitation/inhibition balance as an important pathomechanism in alpha-synucleinopathies and demonstrate a clear association between the spread of toxic proteins and the initiation of altered neuronal function in remotely connected areas.