Alpha-Synuclein Pre-Formed Fibrils Injected into Prefrontal Cortex Primarily Spread to Cortical and Subcortical Structures.

BACKGROUND: Parkinson's disease dementia (PDD) and dementia with Lewy bodies (DLB) are characterized by diffuse spread of alpha-synuclein (α-syn) throughout the brain. Patients with PDD and DLB have a neuropsychological pattern of deficits that include executive dysfunction, such as abnormalities in planning, timing, working memory, and behavioral flexibility. The prefrontal cortex (PFC) plays a major role in normal executive function and often develops α-syn aggregates in DLB and PDD. OBJECTIVE: To investigate the long-term behavioral and cognitive consequences of α-syn pathology in the cortex and characterize pathological spread of α-syn. METHODS: We injected human α-syn pre-formed fibrils into the PFC of wild-type male mice. We then assessed the behavioral and cognitive effects between 12- and 21-months post-injection and characterized the spread of pathological α-syn in cortical, subcortical, and brainstem regions. RESULTS: We report that PFC PFFs: 1) induced α-syn aggregation in multiple cortical and subcortical regions with sparse aggregation in midbrain and brainstem nuclei; 2) did not affect interval timing or spatial learning acquisition but did mildly alter behavioral flexibility as measured by intraday reversal learning; and 3) increased open field exploration. CONCLUSIONS: This model of cortical-dominant pathology aids in our understanding of how local α-syn aggregation might impact some symptoms in PDD and DLB.

Alpha-synuclein pre-formed fibrils injected into prefrontal cortex primarily spread to cortical and subcortical structures and lead to isolated behavioral symptoms.

Parkinson's disease dementia (PDD) and dementia with Lewy bodies (DLB) are characterized by diffuse spread of alpha-synuclein (α-syn) throughout the brain. Patients with PDD and DLB have a neuropsychological pattern of deficits that include executive dysfunction, such as abnormalities in planning, timing, working memory, and behavioral flexibility. The prefrontal cortex (PFC) plays a major role in normal executive function and often develops α-syn aggregates in DLB and PDD. To investigate the consequences of α-syn pathology in the cortex, we injected human α-syn pre-formed fibrils into the PFC of wildtype mice. We report that PFC PFFs: 1) induced α-syn aggregation in multiple cortical and subcortical regions with sparse aggregation in midbrain and brainstem nuclei; 2) did not affect interval timing or spatial learning acquisition but did mildly alter behavioral flexibility as measured by intraday reversal learning; 3) increased open field exploration; and 4) did not affect susceptibility to an inflammatory challenge. This model of cortical-dominant pathology aids in our understanding of how local α-syn aggregation might impact some symptoms in PDD and DLB.

Pathological α-synuclein recruits LRRK2 expressing pro-inflammatory monocytes to the brain.

BACKGROUND: Leucine rich repeat kinase 2 (LRRK2) and SNCA are genetically linked to late-onset Parkinson's disease (PD). Aggregated α-synuclein pathologically defines PD. Recent studies identified elevated LRRK2 expression in pro-inflammatory CD16+ monocytes in idiopathic PD, as well as increased phosphorylation of the LRRK2 kinase substrate Rab10 in monocytes in some LRRK2 mutation carriers. Brain-engrafting pro-inflammatory monocytes have been implicated in dopaminergic neurodegeneration in PD models. Here we examine how α-synuclein and LRRK2 interact in monocytes and subsequent neuroinflammatory responses. METHODS: Human and mouse monocytes were differentiated to distinct transcriptional states resembling macrophages, dendritic cells, or microglia, and exposed to well-characterized human or mouse α-synuclein fibrils. LRRK2 expression and LRRK2-dependent Rab10 phosphorylation were measured with monoclonal antibodies, and myeloid cell responses to α-synuclein fibrils in R1441C-Lrrk2 knock-in mice or G2019S-Lrrk2 BAC mice were evaluated by flow cytometry. Chemotaxis assays were performed with monocyte-derived macrophages stimulated with α-synuclein fibrils and microglia in Boyden chambers. RESULTS: α-synuclein fibrils robustly stimulate LRRK2 and Rab10 phosphorylation in human and mouse macrophages and dendritic-like cells. In these cells, α-synuclein fibrils stimulate LRRK2 through JAK-STAT activation and intrinsic LRRK2 kinase activity in a feed-forward pathway that upregulates phosphorylated Rab10. In contrast, LRRK2 expression and Rab10 phosphorylation are both suppressed in microglia-like cells that are otherwise highly responsive to α-synuclein fibrils. Corroborating these results, LRRK2 expression in the brain parenchyma occurs in pro-inflammatory monocytes infiltrating from the periphery, distinct from brain-resident microglia. Mice expressing pathogenic LRRK2 mutations G2019S or R1441C have increased numbers of infiltrating pro-inflammatory monocytes in acute response to α-synuclein fibrils. In primary cultured macrophages, LRRK2 kinase inhibition dampens α-synuclein fibril and microglia-stimulated chemotaxis. CONCLUSIONS: Pathologic α-synuclein activates LRRK2 expression and kinase activity in monocytes and induces their recruitment to the brain. These results predict that LRRK2 kinase inhibition may attenuate damaging pro-inflammatory monocyte responses in the brain.

COVID-19-associated necrotizing encephalopathy presenting without active respiratory symptoms: a case report with histopathology.

Acute necrotizing encephalopathy (ANE) is a rare complication of coronavirus disease 2019 (COVID-19) secondary to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The condition is typically diagnosed based on characteristic neuroimaging findings in the context of active viral respiratory symptoms. We present a rare case of COVID-19-associated ANE presenting with expressive aphasia and encephalopathy in the absence of active respiratory symptoms. Initial evaluation revealed bilateral thalamic lesions and a mild neutrophilic-predominant pleocytosis on cerebrospinal fluid analysis, the latter of which has not been described in previously published cases. Presence of these atypical features prompted extensive diagnostic evaluation. Metagenomic next-generation sequencing on cerebrospinal fluid did not detect the presence of pathogenic nucleic acids. Thalamic biopsy revealed perivascular neutrophilic inflammation suggestive of small vessel vasculitis with surrounding hemorrhage and necrosis. Ultimately, the diagnosis was made following detection of SARS-CoV-2 serologies and after exclusion of alternative etiologies. The patient was successfully treated with a short course of high-dose methylprednisolone with favorable outcome.

Cortical alpha-synuclein preformed fibrils do not affect interval timing in mice.

One hallmark feature of Parkinson's disease (PD) is Lewy body pathology associated with misfolded alpha-synuclein. Previous studies have shown that striatal injection of alpha-synuclein preformed fibrils (PFF) can induce misfolding and aggregation of native alpha-synuclein in a prion-like manner, leading to cell death and motor dysfunction in mouse models. Here, we tested whether alpha-synuclein PFFs injected into the medial prefrontal cortex results in deficits in interval timing, a cognitive task which is disrupted in human PD patients and in rodent models of PD. We injected PFF or monomers of human alpha-synuclein into the medial prefrontal cortex of mice pre-injected with adeno-associated virus (AAV) coding for overexpression of human alpha-synuclein or control protein. Despite notable medial prefrontal cortical synucleinopathy, we did not observe consistent deficits in fixed-interval timing. These results suggest that cortical alpha-synuclein does not reliably disrupt fixed-interval timing.

Genetic background influences LRRK2-mediated Rab phosphorylation in the rat brain.

Pathogenic missense mutations in the leucine-rich repeat kinase 2 gene, encoding LRRK2, results in the upregulation of Rab10 and Rab12 phosphorylation in different cells and tissues. Here, we evaluate levels of the LRRK2 kinase substrates pT73-Rab10 and pS106-Rab12 proteins in rat brain tissues from different genetic backgrounds. Whereas lines of Sprague Dawley rats have equivalent levels of pT73-Rab10 and pS106-Rab12 similar to Lrrk2 knockout rats, Long-Evans rats have levels of pT73-Rab10 and pS106-Rab12 comparable to G2019S-LRRK2 BAC transgenic rats. Strong LRRK2 kinase inhibitors are ineffective at reducing pT73-Rab10 and pS106-Rab12 levels in the Sprague Dawley rats, but potently reduce pT73-Rab10 and pS106-Rab12 levels in Long-Evans rats. Oral administration of the PFE-360 LRRK2 kinase inhibitor fails to provide neuroprotection from dopaminergic neurodegeneration caused by rAAV2/1-mediated overexpression of A53T-αsynuclein in Sprague Dawley rats. These results highlight substantial differences in LRRK2-mediated Rab10 and Rab12 phosphorylation in commonly utilized rat genetic backgrounds and suggest LRRK2 may not play a central role in Rab phosphorylation or mutant αsynuclein toxicity in Sprague Dawley rats.

Sensitivity and specificity of phospho-Ser129 α-synuclein monoclonal antibodies.

α-Synuclein (α-syn) is an abundant presynaptic protein that is the primary constituent of inclusions that define Lewy body diseases (LBDs). In these inclusions, α-syn is phosphorylated at the serine-129 residue. Antibodies directed to this phosphorylation site are used to measure inclusion abundance and stage disease progression in preclinical models as well as in postmortem tissues in LBDs. While it is critical to reliably identify inclusions, phospho-specific antibodies often cross-react with nonspecific antigens. Four commercially available monoclonal antibodies, two from rabbits (clones EP1536Y and MJF-R13) and two from mice (81a and pSyn#64), have been the most widely used in detecting pS129-α-syn inclusions. Here, we systematically evaluated these antibodies in brain sections and protein lysates from rats and mice. All antibodies detected pS129-α-syn inclusions in the brain that were induced by preformed α-syn fibrils in wild-type rats and mice. Antibody titrations revealed that clones EP1536Y and 81a comparably labeled inclusions in both the perikarya and neuronal processes in contrast to clones MJF-R13 and pSyn#64 that incompletely labeled inclusions at various antibody concentrations. Except for EP1536Y, the clones produced nonspecific diffuse neuropil labeling in α-syn knockout mice as well as mice and rats injected with monomeric α-syn, with some nonspecific staining titrating with pS129-α-syn inclusions. By immunoblot, all the clones cross-reacted with proteins other than α-syn, warranting caution in interpretations of specificity. Clone EP1536Y uniquely and robustly detected endogenous pS129-α-syn in highly soluble protein fractions from the mouse brain. In summary, EP1536Y had the highest sensitivity and specificity for detecting pS129-α-syn.

Breathing new life into an old target: pulmonary disease drugs for Parkinson's disease therapy.

Increases in α-synuclein protein expression are suspected to increase the risk of the development of Parkinson's disease (PD). A recent study has demonstrated that ß2-adrenergic receptor (ß2AR) agonists decrease histone acetylation in the α-synuclein gene and suppress transcription. Coupled with the anti-inflammatory effects that are associated with ß2AR activation, this two-pronged attack holds promise for PD treatment and the development of new therapeutic approaches for this disease.

α-Synuclein fibril-induced inclusion spread in rats and mice correlates with dopaminergic Neurodegeneration.

Proteinaceous inclusions in neurons, composed primarily of α-synuclein, define the pathology in several neurodegenerative disorders. Neurons can internalize α-synuclein fibrils that can seed new inclusions from endogenously expressed α-synuclein. The factors contributing to the spread of pathology and subsequent neurodegeneration are not fully understood, and different compositions and concentrations of fibrils have been used in different hosts. Here, we systematically vary the concentration and length of well-characterized α-synuclein fibrils and determine their relative ability to induce inclusions and neurodegeneration in different hosts (primary neurons, C57BL/6J and C3H/HeJ mice, and Sprague Dawley rats). Using dynamic-light scattering profiles and other measurements to determine fibril length and concentration, we find that femptomolar concentrations of fibrils are sufficient to induce robust inclusions in primary neurons. However, a narrow and non-linear dynamic range characterizes fibril-mediated inclusion induction in axons and the soma. In mice, the C3H/HeJ strain is more sensitive to fibril exposures than C57BL/6J counterparts, with more inclusions and dopaminergic neurodegeneration. In rats, injection of fibrils into the substantia nigra pars compacta (SNpc) results in similar inclusion spread and dopaminergic neurodegeneration as injection of the fibrils into the dorsal striatum, with prominent inclusion spread to the amygdala and several other brain areas. Inclusion spread, particularly from the SNpc to the striatum, positively correlates with dopaminergic neurodegeneration. These results define biophysical characteristics of α-synuclein fibrils that induce inclusions and neurodegeneration both in vitro and in vivo, and suggest that inclusion spread in the brain may be promoted by a loss of neurons.

G2019S-LRRK2 Expression Augments α-Synuclein Sequestration into Inclusions in Neurons.

UNLABELLED: Pathologic inclusions define α-synucleinopathies that include Parkinson's disease (PD). The most common genetic cause of PD is the G2019S LRRK2 mutation that upregulates LRRK2 kinase activity. However, the interaction between α-synuclein, LRRK2, and the formation of α-synuclein inclusions remains unclear. Here, we show that G2019S-LRRK2 expression, in both cultured neurons and dopaminergic neurons in the rat substantia nigra pars compact, increases the recruitment of endogenous α-synuclein into inclusions in response to α-synuclein fibril exposure. This results from the expression of mutant G2019S-LRRK2, as overexpression of WT-LRRK2 not only does not increase formation of inclusions but reduces their abundance. In addition, treatment of primary mouse neurons with LRRK2 kinase inhibitors, PF-06447475 and MLi-2, blocks G2019S-LRRK2 effects, suggesting that the G2019S-LRRK2 potentiation of inclusion formation depends on its kinase activity. Overexpression of G2019S-LRRK2 slightly increases, whereas WT-LRRK2 decreases, total levels of α-synuclein. Knockdown of total α-synuclein with potent antisense oligonucleotides substantially reduces inclusion formation in G2019S-LRRK2-expressing neurons, suggesting that LRRK2 influences α-synuclein inclusion formation by altering α-synuclein levels. These findings support the hypothesis that G2019S-LRRK2 may increase the progression of pathological α-synuclein inclusions after the initial formation of α-synuclein pathology by increasing a pool of α-synuclein that is more susceptible to forming inclusions. SIGNIFICANCE STATEMENT: α-Synuclein inclusions are found in the brains of patients with many different neurodegenerative diseases. Point mutation, duplication, or triplication of the α-synuclein gene can all cause Parkinson's disease (PD). The G2019S mutation in LRRK2 is the most common known genetic cause of PD. The interaction between G2019S-LRRK2 and α-synuclein may uncover new mechanisms and targets for neuroprotection. Here, we show that expression of G2019S-LRRK2 increases α-synuclein mobility and enhances aggregation of α-synuclein in primary cultured neurons and in dopaminergic neurons of the substantia nigra pars compacta, a susceptible brain region in PD. Potent LRRK2 kinase inhibitors, which are being developed for clinical use, block the increased α-synuclein aggregation in G2019S-LRRK2-expressing neurons. These results demonstrate that α-synuclein inclusion formation in neurons can be blocked and that novel therapeutic compounds targeting this process by inhibiting LRRK2 kinase activity may slow progression of PD-associated pathology.

Leucine-rich Repeat Kinase 2 (LRRK2) Pharmacological Inhibition Abates α-Synuclein Gene-induced Neurodegeneration.

Therapeutic approaches to slow or block the progression of Parkinson disease (PD) do not exist. Genetic and biochemical studies implicate α-synuclein and leucine-rich repeat kinase 2 (LRRK2) in late-onset PD. LRRK2 kinase activity has been linked to neurodegenerative pathways. However, the therapeutic potential of LRRK2 kinase inhibitors is not clear because significant toxicities have been associated with one class of LRRK2 kinase inhibitors. Furthermore, LRRK2 kinase inhibitors have not been tested previously for efficacy in models of α-synuclein-induced neurodegeneration. To better understand the therapeutic potential of LRRK2 kinase inhibition in PD, we evaluated the tolerability and efficacy of a LRRK2 kinase inhibitor, PF-06447475, in preventing α-synuclein-induced neurodegeneration in rats. Both wild-type rats as well as transgenic G2019S-LRRK2 rats were injected intracranially with adeno-associated viral vectors expressing human α-synuclein in the substantia nigra. Rats were treated with PF-06447475 or a control compound for 4 weeks post-viral transduction. We found that rats expressing G2019S-LRRK2 have exacerbated dopaminergic neurodegeneration and inflammation in response to the overexpression of α-synuclein. Both neurodegeneration and neuroinflammation associated with G2019S-LRRK2 expression were mitigated by LRRK2 kinase inhibition. Furthermore, PF-06447475 provided neuroprotection in wild-type rats. We could not detect adverse pathological indications in the lung, kidney, or liver of rats treated with PF-06447475. These results demonstrate that pharmacological inhibition of LRRK2 is well tolerated for a 4-week period of time in rats and can counteract dopaminergic neurodegeneration caused by acute α-synuclein overexpression.

The G2019S LRRK2 mutation increases myeloid cell chemotactic responses and enhances LRRK2 binding to actin-regulatory proteins.

The Leucine rich repeat kinase 2 (LRRK2) gene is genetically and biochemically linked to several diseases that involve innate immunity. LRRK2 protein is highly expressed in phagocytic cells of the innate immune system, most notably in myeloid cells capable of mounting potent pro-inflammatory responses. Knockdown of LRRK2 protein in these cells reduces pro-inflammatory responses. However, the effect of LRRK2 pathogenic mutations that cause Parkinson's disease on myeloid cell function is not clear but could provide insight into LRRK2-linked disease. Here, we find that rats expressing G2019S LRRK2 have exaggerated pro-inflammatory responses and subsequent neurodegeneration after lipopolysaccharide injections in the substantia nigra, with a marked increase in the recruitment of CD68 myeloid cells to the site of injection. While G2019S LRRK2 expression did not affect immunological homeostasis, myeloid cells expressing G2019S LRRK2 show enhanced chemotaxis both in vitro in two-chamber assays and in vivo in response to thioglycollate injections in the peritoneum. The G2019S mutation enhanced the association between LRRK2 and actin-regulatory proteins that control chemotaxis. The interaction between G2019S LRRK2 and actin-regulatory proteins can be blocked by LRRK2 kinase inhibitors, although we did not find evidence that LRRK2 phosphorylated these interacting proteins. These results suggest that the primary mechanism of G2019S LRRK2 with respect to myeloid cell function in disease may be related to exaggerated chemotactic responses.