Excitoprotective effects of conditional tau reduction in excitatory neurons and in adulthood.

Tau reduction is a promising therapeutic strategy for Alzheimer's disease. In numerous models, tau reduction via genetic knockout is beneficial, at least in part due to protection against hyperexcitability and seizures, but the underlying mechanisms are unclear. Here we describe the generation and initial study of a new conditional Tau flox model to address these mechanisms. Given the protective effects of tau reduction against hyperexcitability, we compared the effects of selective tau reduction in excitatory or inhibitory neurons. Tau reduction in excitatory neurons mimicked the protective effects of global tau reduction, while tau reduction in inhibitory neurons had the opposite effect and increased seizure susceptibility. Since most prior studies used knockout mice lacking tau throughout development, we crossed Tau flox mice with inducible Cre mice and found beneficial effects of tau reduction in adulthood. Our findings support the effectiveness of tau reduction in adulthood and indicate that excitatory neurons may be a key site for its excitoprotective effects. SUMMARY: A new conditional tau knockout model was generated to study the protective effects of tau reduction against hyperexcitability. Conditional tau reduction in excitatory, but not inhibitory, neurons was excitoprotective, and induced tau reduction in adulthood was excitoprotective without adverse effects.

Chronic hyperactivation of midbrain dopamine neurons causes preferential dopamine neuron degeneration.

Parkinson's disease (PD) is characterized by the death of substantia nigra (SNc) dopamine (DA) neurons, but the pathophysiological mechanisms that precede and drive their death remain unknown. The activity of DA neurons is likely altered in PD, but we understand little about if or how chronic changes in activity may contribute to degeneration. To address this question, we developed a chemogenetic (DREADD) mouse model to chronically increase DA neuron activity, and confirmed this increase using ex vivo electrophysiology. Chronic hyperactivation of DA neurons resulted in prolonged increases in locomotor activity during the light cycle and decreases during the dark cycle, consistent with chronic changes in DA release and circadian disturbances. We also observed early, preferential degeneration of SNc projections, recapitulating the PD hallmarks of selective vulnerability of SNc axons and the comparative resilience of ventral tegmental area axons. This was followed by eventual loss of midbrain DA neurons. Continuous DREADD activation resulted in a sustained increase in baseline calcium levels, supporting an important role for increased calcium in the neurodegeneration process. Finally, spatial transcriptomics from DREADD mice examining midbrain DA neurons and striatal targets, and cross-validation with human patient samples, provided insights into potential mechanisms of hyperactivity-induced toxicity and PD. Our results thus reveal the preferential vulnerability of SNc DA neurons to increased neural activity, and support a potential role for increased neural activity in driving degeneration in PD.

Analysis of hemisphere-dependent effects of unilateral intrastriatal injection of α-synuclein pre-formed fibrils on mitochondrial protein levels, dynamics, and function.

Genetic and neuropathological evidence strongly implicates aberrant forms of α-synuclein in neurodegeneration. Antibodies specific for α-synuclein phosphorylated at serine 129 (pS129) are selective for the pathological protein aggregates that are characteristic of Parkinson's disease (PD) and other synucleinopathies, such as dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Although the etiology of most synucleinopathies remains uncertain, a large body of evidence points to mitochondrial dysfunction. The recent development of animal models based on intracranial injection of α-synuclein pre-formed fibrils (PFFs) has provided a valuable experimental system in which to study the spread and neurotoxicity of α-synuclein aggregates, yet the effects of PFF-induced protein aggregates on mitochondrial function and dynamics have not been rigorously examined in vivo. To help fill this knowledge gap, we injected the striatum of mice unilaterally with well-characterized small length (< 30 nm) PFFs or monomeric α-synuclein control and measured the distribution and extent of pS129 α-synuclein-immunoreactive aggregates, the loss of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra, the abundance of mitochondrial proteins, and the activity of mitochondrial respiratory chain components at 3 months and 6 months post injection. Intrastriatal injection of small length PFFs, but not monomeric α-synuclein control, induced robust pS129 α-synuclein immunoreactive inclusions in the cortex, ventral midbrain, and striatum, as well as in rarely reported brain regions, such as the hippocampus, as early as 3 months post injection. Significant loss of nigral tyrosine hydroxylase-immunoreactive neurons was observed in the PFF-injected hemisphere at 3 months and 6 months post injection. The unilateral striatal injection of small length PFFs also caused hemisphere-dependent and treatment-dependent changes in the cortical levels of mitochondrial proteins such as VDAC1, COX-IV, and DRP-1, as well as functional changes in mitochondrial complex I activity in the contralateral striatum. Together, these data demonstrate that intrastriatal injection of mice with small length PFFs induces extensive bilateral protein aggregates, significant unilateral nigral cell loss, and altered contralateral levels of mitochondrial proteins and respiratory chain activity. Our data suggest this animal model may be useful for studying the role of mitochondrial dysfunction in α-synucleinopathies, for studying the hemisphere-dependent effects of α-synuclein aggregates, and for testing neuroprotective therapies that target mitochondrial dysfunction and protein aggregation.

Basal Synaptic Transmission and Long-Term Plasticity at CA3-CA1 Synapses Are Unaffected in Young Adult PINK1-Deficient Rats.

Loss of function mutations in PARK6, the gene that encodes the protein PTEN-induced kinase 1 (PINK1), cause autosomal recessive familial Parkinson's disease (PD). While PD is clinically diagnosed by its motor symptoms, recent studies point to the impact of non-motor symptoms, including cognitive dysfunction in the early pre-motor stages of the disease (Aarsland et al., 2004; Chaudhuri and Schapira, 2009). As the hippocampus is a key structure for learning and memory, this study aimed to determine whether synaptic transmission is affected at CA3-CA1 excitatory synapses in PINK1 knockout rats at an age when we recently reported a gain of function at excitatory synapses onto spiny projection neurons in the dorsal striatum (Creed et al., 2020) and when motor symptoms are beginning to appear (Dave et al., 2014). Using extracellular dendritic field excitatory postsynaptic potential recordings at CA3-CA1 synapses in dorsal hippocampus 4-to 5- month old PINK1 KO rats and wild-type littermate controls, we observed no detectable differences in the strength of basal synaptic transmission, paired-pulse facilitation, or long-term potentiation. Our results suggest that loss of PINK1 protein does not cause a general dysfunction of excitatory transmission throughout the brain at this young adult age when excitatory transmission is abnormal in the striatum.

Increased glutamate transmission onto dorsal striatum spiny projection neurons in Pink1 knockout rats.

Loss-of-function PTEN Induced Kinase 1 (PINK1) mutations cause early-onset familial Parkinson's disease (PD) with similar clinical and neuropathological characteristics as idiopathic PD. While Pink1 knockout (KO) rats have mitochondrial dysfunction, locomotor deficits, and α-synuclein aggregates in several brain regions such as cerebral cortex, dorsal striatum, and substantia nigra, the functional ramifications on synaptic circuits are unknown. Using whole cell patch clamp recordings, we found a significant increase in the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) onto striatal spiny projection neurons (SPNs) in Pink1 KO rats at ages 4 and 6 months compared to wild-type (WT) littermates, suggesting increased excitability of presynaptic neurons. While sEPSC amplitudes were also increased at 2 and 4 months, no changes were observed in AMPAR/NMDAR ratio or receptor expression. Further analysis revealed increased glutamate release probability and decreased recovery of the synaptic vesicle pool following a train of stimulation in Pink1 KO rats. Ultrastructural analysis revealed increased excitatory and inhibitory synapse number and increased levels of presynaptic α-synuclein, while the number and structure of striatal mitochondria appeared normal. Lastly, we found that Pink1 KO rats have altered striatal dopamine tone, which together with the abnormal α- synuclein distribution and dysfunctional mitochondria, could contribute to the increase in excitatory transmission. Together, these studies show that PINK1 is necessary for normal glutamatergic transmission onto striatal SPNs and reveal possible mechanisms underlying striatal circuit dysfunction in PD.

Enhanced Susceptibility of PINK1 Knockout Rats to α-Synuclein Fibrils.

The main neuropathological hallmarks of Parkinson's disease (PD) are loss of dopaminergic neurons in the substantia nigra and intraneuronal protein aggregates immunoreactive for α-synuclein phosphorylated at serine 129 (pS129). Most cases of PD are idiopathic; however, genetic mutations have been identified in several genes linked to familial PD. Mutations in the gene encoding α-synuclein are causally linked to dominantly inherited forms of PD and mutations in the PTEN-induced kinase-1 (PINK1) gene are linked to recessively inherited forms of PD. Because abnormal α-synuclein protein aggregates appear spontaneously in PINK1 knockout (KO) rats, we hypothesize that PINK1-deficiency causes endogenous α-synuclein to be more prone to aggregation. α-Synuclein aggregation does not normally occur in mice or rats, however, it can be induced by intracranial injection of α-synuclein pre-formed fibrils (PFFs), which also induces loss of dopaminergic nigral neurons 3-6 months post-injection. Because PINK1-deficiency is linked to early-onset PD, we further hypothesize that PINK1 KO rats will show earlier PFF-induced neurodegeneration compared to wild-type (WT) rats. Herein, we report that intracranial injection of α-synuclein PFFs into the dorsal striatum induced more abundant pS129 α-synuclein in PINK1 KO rat brains compared to WT littermate controls. Moreover, the synuclein extracted from the brains of PFF-injected PINK1 KO rats was more insoluble compared to PFF-injected WT littermates, suggesting greater progression of α-synuclein pathology in PINK1 KO rats. Four weeks post-injection, PFFs caused significant loss of dopaminergic neurons in the substantia nigra of PINK1 KO rats, but not WT controls. Together, our results indicate that PINK1 deficiency increases vulnerability to α-synuclein aggregation and dopaminergic neurodegeneration in vivo.

PINK1 phosphorylates ubiquitin predominantly in astrocytes.

Loss-of-function mutations in PINK1 are causally linked to recessively inherited Parkinson's disease (PD), with marked loss of dopaminergic neurons in the substantia nigra that are required for normal movement. PINK1 is a nuclear-encoded mitochondrial-targeted kinase that phosphorylates a conserved serine at amino acid 65 (pS65) in ubiquitin as well as Parkin, another gene with loss-of-function mutations linked to recessive parkinsonism. The steady-state levels of PINK1 protein are very low, even in cells that express PINK1, because PINK1 is normally targeted for degradation after mitochondrial import by a process that is dependent upon mitochondrial membrane potential. Dissipation of the mitochondrial membrane potential with ionophores, such as CCCP and valinomycin, causes the accumulation of PINK1 on the outer mitochondrial membrane, a marked increase of pS65-ubiquitin and the recruitment of Parkin, which targets dysfunctional mitochondria for degradation by autophagy. While the high penetrance of PINK1 mutations establish its critical function for maintaining neurons, the activity of PINK1 in primary neurons has been difficult to detect. Mounting evidence implicates non-neuronal cells, including astrocytes and microglia, in the pathogenesis of both idiopathic and inherited PD. Herein we used both western analysis and immunofluorescence of pS65-ubiquitin to directly compare the activity of PINK1 in primary neurons, astrocytes, microglia, and oligodendrocyte progenitor cells cultured from the brains of wild-type (WT) and PINK1 knockout (KO) rat pups. Our findings that PINK1-dependent ubiquitin phosphorylation is predominantly in astrocytes supports increased priority for research on the function of PINK1 in astrocytes and the contribution of astrocyte dysfunction to PD pathogenesis.

Basal and Evoked Neurotransmitter Levels in Parkin, DJ-1, PINK1 and LRRK2 Knockout Rat Striatum.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder and is characterized by the loss of neurons in the substantia nigra that project to the striatum and release dopamine (DA), which is required for normal movement. Common non-motor symptoms likely involve abnormalities with other neurotransmitters, such as serotonin, norepinephrine, acetylcholine, glycine, glutamate and gamma-aminobutyric acid (GABA). As part of a broad effort to provide better PD research tools, the Michael J. Fox Foundation for Parkinson's Research funded the generation and characterization of knockout (KO) rats for genes with PD-linked mutations, including PINK1, Parkin, DJ-1 and LRRK2. Here we extend the phenotypic characterization of these lines of KO rats to include in vivo microdialysis to measure both basal and potassium-induced release of the above neurotransmitters and their metabolites in the striatum of awake and freely moving rats at ages 4, 8 and 12 months compared to wild-type (WT) rats. We found age-dependent abnormalities in basal DA, glutamate and acetylcholine in PINK1 KO rats and age-dependent abnormalities in basal DA metabolites in Parkin and LRRK2 KO rats. Parkin KO rats had increased glycine release while DJ-1 KO rats had decreased glutamate release and increased acetylcholine release compared to WT rats. All lines except DJ-1 KO rats showed age-dependent changes in release of one or more neurotransmitters. Our data suggest these rats may be useful for studies of PD-related synaptic dysfunction and neurotransmitter dynamics as well as studies of the normal and pathogenic functions of these genes with PD-linked mutations.

New Developments in Genetic rat models of Parkinson's Disease.

Preclinical research on Parkinson's disease has relied heavily on mouse and rat animal models. Initially, PD animal models were generated primarily by chemical neurotoxins that induce acute loss of dopaminergic neurons in the substantia nigra. On the discovery of genetic mutations causally linked to PD, mice were used more than rats to generate laboratory animals bearing PD-linked mutations because mutagenesis was more difficult in rats. Recent advances in technology for mammalian genome engineering and optimization of viral expression vectors have increased the use of genetic rat models of PD. Emerging research tools include "knockout" rats with disruption of genes in which mutations have been causally linked to PD, including LRRK2, α-synuclein, Parkin, PINK1, and DJ-1. Rats have also been increasingly used for transgenic and viral-mediated overexpression of genes relevant to PD, particularly α-synuclein. It may not be realistic to obtain a single animal model that completely reproduces every feature of a human disease as complex as PD. Nevertheless, compared with mice with the same mutations, many genetic rat animal models of PD better reproduce key aspects of PD including progressive loss of dopaminergic neurons in the substantia nigra, locomotor behavior deficits, and age-dependent formation of abnormal α-synuclein protein aggregates. Here we briefly review new developments in genetic rat models of PD that may have greater potential for identifying underlying mechanisms, for discovering novel therapeutic targets, and for developing greatly needed treatments to slow or halt disease progression. © 2018 International Parkinson and Movement Disorder Society.

Analysis of α-Synuclein Pathology in PINK1 Knockout Rat Brains.

Mutations in PTEN induced kinase 1 (PINK1) cause autosomal recessive Parkinson's disease (PD). The main pathological hallmarks of PD are loss of dopaminergic neurons in the substantia nigra pars compacta and the formation of protein aggregates containing α-synuclein. Previous studies of PINK1 knockout (PINK1-/-) rats have reported mitochondrial dysfunction, locomotor behavioral deficits, loss of neurons in the substantia nigra and α-synuclein aggregates in various brain regions. We sought to characterize PINK1-/- rats in more detail specifically with respect to α-synuclein pathology because abnormal α-synuclein has been implicated genetically, biophysically and neuropathologically as a mechanism of PD pathogenesis. Moreover, the spontaneous formation of α-synuclein aggregates without α-synuclein overexpression, injection or toxin administration is a rare and important characteristic for an animal model of PD or other synucleinopathies, such as dementia with Lewy bodies and multiple system atrophy. We observed α-synuclein-immunoreactive aggregates in various brain regions of PINK1-/- rats including cortex, thalamus, striatum and ventral midbrain, but nowhere in wild-type (WT) rats. Co-immunofluorescence showed that the α-synuclein-immunoreactive aggregates are both thioflavin S and ubiquitin positive. Many cells in the brains of PINK1-/- rats but not WT rats contained protease-resistant α-synuclein. Total synuclein protein levels were unchanged; however, biochemical fractionation showed a significant shift of α-synuclein from the cytosolic fraction to the synaptic vesicle-enriched fraction of PINK1-/- brain homogenates compared to WT. This data indicates that PINK1 deficiency results in abnormal α-synuclein localization, protease resistance and aggregation in vivo. The PINK1-/- rat could be a useful animal model to study the role of abnormal α-synuclein in PD-related neurodegeneration.

Parkin and PINK1 functions in oxidative stress and neurodegeneration.

Loss-of-function mutations in the genes encoding Parkin and PINK1 are causally linked to autosomal recessive Parkinson's disease (PD). Parkin, an E3 ubiquitin ligase, and PINK1, a mitochondrial-targeted kinase, function together in a common pathway to remove dysfunctional mitochondria by autophagy. Presumably, deficiency for Parkin or PINK1 impairs mitochondrial autophagy and thereby increases oxidative stress due to the accumulation of dysfunctional mitochondria that release reactive oxygen species. Parkin and PINK1 likely have additional functions that may be relevant to the mechanisms by which mutations in these genes cause neurodegeneration, such as regulating inflammation, apoptosis, or dendritic morphogenesis. Here we briefly review what is known about functions of Parkin and PINK1 related to oxidative stress and neurodegeneration.