Immunoelectron microscopic and biochemical studies of caspase-cleaved tau in a mouse model of tauopathy

Neurofibrillary tangles (NFTs) have been implicated in mediating neuronal death and disease progression in human tauopathies; however, mounting in vivo data suggest that NFTs may not be the primary initiators of neurotoxicity. Caspase activity has been implicated in processes associated with the development of tauopathy, but the position that caspase activation holds in neurodegenerative cascades remains uncertain. Using multiphoton real-time imaging microscopy, de Calignon et al recently demonstrated that caspase activation precedes and leads to tangle formation within 24 hours in the rTg4510 mouse model of tauopathy. Here, we used immunoelectron microscopy to determine whether caspase-cleaved tau was present in NFTs of rTg4510 mice. Using a caspase-cleaved tau-specific antibody (TauC3), we found very little immunogold labeling in NFTs in the brains of rTg4510 mice. By immunohistochemistry, the number of TauC3-positive neurons was far less than the numbers of neurons stained with the MC1 antibody, which recognizes abnormal conformations of tau. Biochemically, caspase-cleaved tau was barely detectable in fractions of rTg4510 mouse brain extracts. Our data suggest that caspase activation might be one of multiple routes through which NFT formation occurs, rather than an obligatory initiation step in pathologic tau production in rTg4510 mice.     

Synaptic alterations in the rTg4510 mouse model of tauopathy

Synapse loss, rather than the hallmark amyloid‐β (Aβ) plaques or tau‐filled neurofibrillary tangles (NFT), is considered the most predictive pathological feature associated with cognitive status in the Alzheimer's disease (AD) brain. The role of Aβ in synapse loss is well established, but despite data linking tau to synaptic function, the role of tau in synapse loss remains largely undetermined. Here we test the hypothesis that human mutant P301L tau overexpression in a mouse model (rTg4510) will lead to age‐dependent synaptic loss and dysfunction. Using array tomography and two methods of quantification (automated, threshold‐based counting and a manual stereology‐based technique) we demonstrate that overall synapse density is maintained in the neuropil, implicating synapse loss commensurate with the cortical atrophy known to occur in this model. Multiphoton in vivo imaging reveals close to 30% loss of apical dendritic spines of individual pyramidal neurons, suggesting these cells may be particularly vulnerable to tau‐induced degeneration. Postmortem, we confirm the presence of tau in dendritic spines of rTg4510‐YFP mouse brain by array tomography. These data implicate tau‐induced loss of a subset of synapses that may be accompanied by compensatory increases in other synaptic subtypes, thereby preserving overall synapse density. Biochemical fractionation of synaptosomes from rTg4510 brain demonstrates a significant decrease in expression of several synaptic proteins, suggesting a functional deficit of remaining synapses in the rTg4510 brain. Together, these data show morphological and biochemical synaptic consequences in response to tau overexpression in the rTg4510 mouse model. J. Comp. Neurol., 521:1334–1353, 2013. © 2012 Wiley Periodicals, Inc.     

Fluorodopa is a Promising Fluorine‐19 MRI Probe for Evaluating Striatal Dopaminergic Function in a Rat Model of Parkinson's Disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra projecting to the striatum. It has been estimated that approximately 80% of the striatal dopamine and 50% of nigral dopaminergic neurons are lost before the onset of typical motor symptoms, indicating that early diagnosis of PD using noninvasive imaging is feasible. Fluorine‐19 (¹⁹F) magnetic resonance imaging (MRI) represents a highly sensitive, easily available, low‐background, and cost‐effective approach to evaluate dopaminergic function using non‐radioactive fluorine‐containing dopaminergic agents. The aim of this study was to find a potent ¹⁹F MRI probe to evaluate dopaminergic presynaptic function in the striatum. To select candidates for ¹⁹F MRI probes, we investigated the following eight non‐radioactive fluorine‐containing dopaminergic agents: fluorodopa (F‐DOPA), F‐tyrosine, haloperidol, GBR13069 duhydrochloride, GBR12909 duhydrochloride, 3‐bis‐(4‐fluorophenyl) methoxytropane hydrochloride, flupenthixol, and fenfluramine. In ¹⁹F nuclear magnetic resonance measurements, F‐tyrosine and F‐DOPA displayed a relatively higher signal‐to‐noise ratio value in brain homogenates than in others. F‐DOPA, but not F‐tyrosine, induced the rotational behavior in a 6‐hydroxydopamine (6‐OHDA)‐induced hemiparkinsonian rat model. In addition, a significantly high amount of F‐DOPA accumulated in the ipsilateral striatum of hemiparkinsonian rats after the injection. We performed ¹⁹F MRI in PC12 cells and isolated rat brain using a 7T MR scanner. Our findings suggest that F‐DOPA is a promising ¹⁹F MRI probe for evaluating dopaminergic presynaptic function in the striatum of hemiparkinsonian rats. © 2016 Wiley Periodicals, Inc.     

In vivo imaging of inflammation in the peripheral nervous system by (19)F MRI

Visualization of neuroinflammation is still a major task in neuroscience and neurology since inflammatory processes play a central pathophysiological role in many disorders of the nervous system but are not yet covered by conventional imaging techniques.Recently, ¹⁹F magnetic resonance imaging (MRI) was introduced as a new cellular imaging technology. In the present study, we established ¹⁹F high field MRI for cell tracking in the peripheral nervous system (PNS) of rats using dedicated MR coils. To mimic focal neuroinflammation, lysolecithin was locally injected into the left sciatic nerve inducing demyelination followed by severe infiltration of monocytes/macrophages from the circulation. Systemic administration of perfluorocarbons (PFC) led to a fluorine signal along the proximal stretch of the affected sciatic nerves in in vivo¹⁹F MRI which was not seen on the right healthy side. The preferential in vivo uptake of PFC by circulating mononuclear cells was confirmed by density gradient centrifugation of the blood. Removal of nerves with consecutive ex vivo¹⁹F MRI and additional ¹⁹F spectroscopy for quantification corroborated the localization of the ¹⁹F marker within the injured nerves (1.07 × 10¹⁸ ± 1.00 × 10¹⁸ mean detectable fluorine spins) while contralateral naive nerves did not exhibit any detectable fluorine signal. Histological assessment confirmed the presence of numerous ED1-positive macrophages within the nerve lesions. Control experiments showed that intraneural application of saline led to an inflammatory reaction restricted to the perineurium which could also be detected by ¹⁹F MRI. In conclusion, we show that ¹⁹F MRI is a promising new technology to visualize hematogenous macrophage responses in the nervous system.     

Accelerated kindling epileptogenesis in Tg4510 tau transgenic mice,but not in tau knockout mice

The biologic processes underlying epileptogenesis following a brain insult are not fully understood, but several lines of evidence suggest that hyperphosphorylation of tau may be an important factor in these processes. To provide further insight into the causal relationship between tau and epileptogenesis, this study applied amygdala kindling to rTg4510 mice that, concurrent with other pathologies, overexpress phosphorylated tau, tau knockout mice, or their respective wild‐type controls. Mice were electrically stimulated twice daily, 5 days per week for 3 weeks. Electroencephalography was recorded to measure the primary afterdischarge duration, and the behavioral progression of kindling‐induced seizures was assessed. rTg4510 mice (n = 10) had increased primary afterdischarge durations (p < 0.001), and significantly more rapid progression of kindling (p < 0.001), compared with wild‐type mice (n = 10). Tau knockout mice (n = 7), however, did not differ from their wild‐type counterparts (n = 8) on any of the seizure outcomes. These results suggest that Tg4510 mice are more vulnerable to epileptogenesis, but that the presence of tau itself is not necessary for kindling epileptogenesis to occur.     

Regional multimodal relationships between tau,hypometabolism, atrophy,and fractional anisotropy in atypical Alzheimer's disease

Alzheimer's disease (AD) can present with atypical clinical forms where the prominent domain of deficit is not memory, that is, atypical AD. Atypical AD patients show cortical atrophy on MRI, hypometabolism on [¹⁸F]fluorodeoxyglucose (FDG) PET, tau uptake on [¹⁸F]AV‐1451 PET, and white matter tract degeneration on diffusion tensor imaging (DTI). How these disease processes relate to each other locally and distantly remains unclear. We aimed to examine multimodal neuroimaging relationships in individuals with atypical AD, using univariate and multivariate techniques at region‐ and voxel‐level. Forty atypical AD patients underwent MRI, FDG‐PET, tau‐PET, beta‐amyloid PET, and DTI. Patients were all beta‐amyloid positive. Partial Pearson's correlations were performed between tau and FDG standardized uptake value ratios, gray matter MRI‐volumes and white matter tract fractional anisotropy. Sparse canonical correlation analysis was applied to identify multivariate relationships between the same quantities. Voxel‐level associations across modalities were also assessed. Tau showed strong local negative correlations with FDG metabolism in the occipital and frontal lobes. Tau in frontal and parietal regions was negatively associated with temporoparietal gray matter MRI‐volume. Fractional anisotropy in a set of posterior white matter tracts, including the splenium of the corpus callosum, cingulum, and posterior thalamic radiation, was negatively correlated with parietal and occipital tau, atrophy and, predominantly, with hypometabolism. These results support the view that tau is the driving force behind neurodegeneration in atypical AD, and that a breakdown in structural connectivity is related to cortical neurodegeneration, particularly hypometabolism.     

Structural and functional changes in tau mutant mice neurons are not linked to the presence of NFTs

In the rTg4510 mouse model, expression of the mutant human tau variant P301L leads to development of neurofibrillary tangles (NFTs), neuronal death, and memory impairment, reminiscent of the pathology observed in human tauopathies. In the present study, we examined the effects of mutant tau expression on the electrophysiology and morphology of individual neurons using whole-cell patch-clamp recordings and biocytin filling of pyramidal cells in cortical slices prepared from rTg4510 (TG) and wild-type (WT) littermate mice. Among the TG cells, 42% contained a clear Thioflavin-S positive inclusion in the soma and were categorized as NFT positive (NFT+), while 58% had no discernable inclusion and were categorized as NFT negative (NFT−). The resting membrane potential (V_r) was significantly depolarized (+ 8 mV) in TG cells, and as a consequence, evoked repetitive action potential (AP) firing rates were also significantly increased. Further, single APs were significantly shorter in duration in TG cells and the depolarizing voltage deflection or “sag” evoked by hyperpolarization was significantly greater in amplitude. In addition to these functional electrophysiological changes, TG cells exhibited significant morphological alterations, including loss or significant atrophy of the apical tuft, reduced dendritic complexity and length, and reduced spine density. Importantly, NFT− and NFT+ TG cells were indistinguishable with regard to both morphological and electrophysiological properties. Our observations show that expression of mutated tau results in significant structural and functional changes in neurons, but that these changes occur independent of mature NFT formation.     

Soluble tau species, not neurofibrillary aggregates, disrupt neural system integration in a tau transgenic model

Neurofibrillary tangles are a feature of Alzheimer disease and other tauopathies, and although they are generally believed to be markers of neuronal pathology, there is little evidence evaluating whether tangles directly impact neuronal function. To investigate the response of cells in hippocampal circuits to complex behavioral stimuli, we used an environmental enrichment paradigm to induce expression of an immediate-early gene, Arc, in the rTg4510 mouse model of tauopathy. These mice reversibly overexpress P301L tau and exhibit substantial neurofibrillary tangle deposition, neuronal loss, and memory deficits. Using fluorescent in situ hybridization to detect Arc messenger RNA, we found that rTg4510 mice have impaired hippocampal Arc expression both without stimulation and in response to environmental enrichment; this likely reflects the combination of functional impairments of existing neurons and loss of neurons. However, tangle-bearing cells were at least as likely as non-tangle-bearing neurons to exhibit Arc expression in response to enrichment. Transgene suppression with doxycycline for 6 weeks resulted in increased percentages of Arc-positive cells in rTg4510 brains compared with untreated transgenics, restoring enrichment-induced Arc messenger RNA levels to that of wild-type controls despite the continued presence of neurofibrillary pathology. We interpret these data to indicate that soluble tau contributes to impairment of hippocampal function, although tangles do not preclude neurons from responding in a functional circuit.     

Human tau increases amyloid β plaque size but not amyloid β‐mediated synapse loss in a novel mouse model of Alzheimer's disease

Alzheimer's disease is characterized by the presence of aggregates of amyloid beta (Aβ) in senile plaques and tau in neurofibrillary tangles, as well as marked neuron and synapse loss. Of these pathological changes, synapse loss correlates most strongly with cognitive decline. Synapse loss occurs prominently around plaques due to accumulations of oligomeric Aβ. Recent evidence suggests that tau may also play a role in synapse loss but the interactions of Aβ and tau in synapse loss remain to be determined. In this study, we generated a novel transgenic mouse line, the APP/PS1/rTg21221 line, by crossing APP/PS1 mice, which develop Aβ‐plaques and synapse loss, with rTg21221 mice, which overexpress wild‐type human tau. When compared to the APP/PS1 mice without human tau, the cross‐sectional area of ThioS+ dense core plaques was increased by ~50%. Along with increased plaque size, we observed an increase in plaque‐associated dystrophic neurites containing misfolded tau, but there was no exacerbation of neurite curvature or local neuron loss around plaques. Array tomography analysis similarly revealed no worsening of synapse loss around plaques, and no change in the accumulation of Aβ at synapses. Together, these results indicate that adding human wild‐type tau exacerbates plaque pathology and neurite deformation but does not exacerbate plaque‐associated synapse loss.     

Increased cerebral vascular reactivity in the tau expressing rTg4510 mouse: evidence against the role of tau pathology to impair vascular health in Alzheimer's disease

Vascular abnormalities are a key feature of Alzheimer''s disease (AD). Imaging of cerebral vascular reactivity (CVR) is a powerful tool to investigate vascular health in clinical populations although the cause of reduced CVR in AD patients is not fully understood. We investigated the specific role of tau pathology in CVR derangement in AD using the rTg4510 mouse model. We observed an increase in CVR in cortical regions with tau pathology. These data suggest that tau pathology alone does not produce the clinically observed decreases in CVR and implicates amyloid pathology as the dominant etiology of impaired CVR in AD patients.     

Diagnostic value of brain MRI and 18F‐FDG PET in the differentiation of parkinsonian type multiple system atrophy from Parkinson’s disease

Background and purpose: Differentiation between parkinsonian type multiple system atrophy (MSA‐P) and Parkinson’s disease (PD) is important but often difficult. We investigated the diagnostic value of brain magnetic resonance imaging (MRI) and ¹⁸F‐fluorodeoxyglucose positron emission tomography (¹⁸F‐FDG PET) in differentiating MSA‐P from PD. Methods: Twenty‐four patients with MSA‐P (16 probable and 8 possible) and eight patients with PD were included in this study. Results: For analysis using the putaminal findings, the sensitivities were 58.3% by visual analysis of brain MRI, 95.8% by visual analysis of ¹⁸F‐FDG PET, and 79.2% by statistical parametric mapping (SPM) analysis of ¹⁸F‐FDG PET in differentiating MSA‐P from PD; the specificity was 100% for each analysis. Using the putaminal findings, visual analysis of ¹⁸F‐FDG PET had a higher sensitivity compared with brain MRI (P = 0.004) and SPM analysis of ¹⁸F‐FDG PET revealed a tendency towards higher sensitivity compared with brain MRI (P = 0.063). For analysis using both putaminal and infratentorial findings, the sensitivities were 79.2% by visual analysis of brain MRI, 95.8% by visual analysis of ¹⁸F‐FDG PET, 95.8% by SPM analysis of ¹⁸F‐FDG PET in differentiating MSA‐P from PD; the specificity was 100% for each analysis. Conclusion: Both brain MRI and ¹⁸F‐FDG PET showed diagnostic usefulness in differentiating MSA‐P from PD, with ¹⁸F‐FDG PET being more sensitive than brain MRI.     

Hyperdynamic microtubules, cognitive deficits, and pathology are improved in tau transgenic mice with low doses of the microtubule-stabilizing agent BMS-241027

Tau is a microtubule (MT)-stabilizing protein that is altered in Alzheimer's disease (AD) and other tauopathies. It is hypothesized that the hyperphosphorylated, conformationally altered, and multimeric forms of tau lead to a disruption of MT stability; however, direct evidence is lacking in vivo. In this study, an in vivo stable isotope-mass spectrometric technique was used to measure the turnover, or dynamicity, of MTs in brains of living animals. We demonstrated an age-dependent increase in MT dynamics in two different tau transgenic mouse models, 3xTg and rTg4510. MT hyperdynamicity was dependent on tau expression, since a reduction of transgene expression with doxycycline reversed the MT changes. Treatment of rTg4510 mice with the epothilone, BMS-241027, also restored MT dynamics to baseline levels. In addition, MT stabilization with BMS-241027 had beneficial effects on Morris water maze deficits, tau pathology, and neurodegeneration. Interestingly, pathological and functional benefits of BMS-241027 were observed at doses that only partially reversed MT hyperdynamicity. Together, these data suggest that tau-mediated loss of MT stability may contribute to disease progression and that very low doses of BMS-241027 may be useful in the treatment of AD and other tauopathies.     

Data‐driven approaches for tau‐PET imaging biomarkers in Alzheimer's disease

Previous positron emission tomography (PET) studies have quantified filamentous tau pathology using regions‐of‐interest (ROIs) based on observations of the topographical distribution of neurofibrillary tangles in post‐mortem tissue. However, such approaches may not take full advantage of information contained in neuroimaging data. The present study employs an unsupervised data‐driven method to identify spatial patterns of tau‐PET distribution, and to compare these patterns to previously published “pathology‐driven” ROIs. Tau‐PET patterns were identified from a discovery sample comprised of 123 normal controls and patients with mild cognitive impairment or Alzheimer's disease (AD) dementia from the Swedish BioFINDER cohort, who underwent [¹⁸F]AV1451 PET scanning. Associations with cognition were tested in a separate sample of 90 individuals from ADNI. BioFINDER [¹⁸F]AV1451 images were entered into a robust voxelwise stable clustering algorithm, which resulted in five clusters. Mean [¹⁸F]AV1451 uptake in the data‐driven clusters, and in 35 previously published pathology‐driven ROIs, was extracted from ADNI [¹⁸F]AV1451 scans. We performed linear models comparing [¹⁸F]AV1451 signal across all 40 ROIs to tests of global cognition and episodic memory, adjusting for age, sex, and education. Two data‐driven ROIs consistently demonstrated the strongest or near‐strongest effect sizes across all cognitive tests. Inputting all regions plus demographics into a feature selection routine resulted in selection of two ROIs (one data‐driven, one pathology‐driven) and education, which together explained 28% of the variance of a global cognitive composite score. Our findings suggest that [¹⁸F]AV1451‐PET data naturally clusters into spatial patterns that are biologically meaningful and that may offer advantages as clinical tools.     

Behavioral and stereological characterization of Hdc KO mice: Relation to Tourette syndrome

A premature termination codon in the human histidine decarboxylase (Hdc) gene has been identified in a family suffering from Guilles de la Tourette syndrome (GTS). In the current study we investigated if mice lacking the histamine producing enzyme HDC share the morphological and cytological phenotype with GTS patients by using magnetic resonance (MRI) and diffusion tensor imaging (DTI), unbiased stereology and immunohistochemistry. Behavior of Hdc knock‐out (Hdc KO) mice was assessed in an open field test. The results of stereological, volumetric and DTI analysis measurements showed no significant differences between control and Hdc KO mice. The numbers and distribution of GABAergic parvalbumin or nitric oxide‐expressing and cholinergic interneurons were normal in Hdc KO mice. Cortical morphology and layering in adult Hdc KO mice were also preserved. In open field test Hdc KO mice showed impaired exploratory activity and habituation when introduced to novel environment. Our results indicate that Hdc deficiency in mice does not disturb the development of striatal and cortical interneurons and does not lead to the morphological and cytological phenotypes characterized by humans with GTS. Nevertheless, histamine deficiency leads to behavioral alterations probably due to neurotransmitter dysbalance on the level of the striatum.     

Invited review: Frontotemporal dementia caused by microtubule‐associated protein tau gene (MAPT) mutations: a chameleon for neuropathology and neuroimaging

Hereditary frontotemporal dementia associated with mutations in the microtubule‐associated protein tau gene (MAPT) is a protean disorder. Three neuropathologic subtypes can be recognized, based on the presence of inclusions made of tau isoforms with three and four repeats, predominantly three repeats and mostly four repeats. This is relevant for establishing a correlation between structural magnetic resonance imaging and positron emission tomography using tracers specific for aggregated tau. Longitudinal studies will be essential to determine the evolution of anatomical alterations from the asymptomatic stage to the various phases of disease following the onset of symptoms.     

A common genetic mechanism underlying susceptibility to posttraumatic stress disorder

We hypothesize that susceptibility to post-traumatic stress disorder (PTSD) may be determined in part by aberrant microtubule-associated protein tau expression in neurons of critical brain structures. The following lines of evidence support this hypothesis. First, epidemiologic data suggest the involvement of genetic factors in the susceptibility to PTSD. Second, the common features of both abnormal tau expression and PTSD include amygdalar and hippocampal atrophy, upregulation of norepinephrine biosynthetic capacity in the surviving locus coeruleus neurons and dysfunction of N-methyl-D-aspartate-receptors. Finally, our experiments using rTg4510 mice, a model that over-expresses human mutant tau and develops age-dependent tauopathy, demonstrate that these animals display circling behavior thought to be related to states of anxiety. To detect the potential molecular mechanisms underlying PTSD episodes, laser-assisted/capture microdissection can be used with microarray analysis as an alternative approach to identify changes in gene expression in excitatory and/or inhibitory neurons in critical brain structures (i.e., hippocampus and amygdala) in response to the onset of PTSD.     

Absence of microglia or presence of peripherally-derived macrophages does not affect tau pathology in young or old hTau mice

Microglia are implicated in the pathophysiology of several neurodegenerative disorders, including Alzheimer's disease. While the role of microglia and peripheral macrophages in regulating amyloid beta pathology has been well characterized, the impact of these distinct cell subsets on tau pathology remains poorly understood. We and others have recently demonstrated that monocytes can engraft the brain and give rise to long-lived parenchymal macrophages, even under nonpathological conditions. We undertook the current study to investigate the regulation of tau pathology by microglia and peripheral macrophages using hTau transgenic mice, which do not exhibit microglial activation/pathology or macrophage engraftment. To assess the direct impact of microglia on tau pathology we developed a protocol for long-term microglial depletion in Cx3cr1^CreERR26^DTA mice and crossed them with hTau mice. We then depleted microglia up to 3 months in both young and old mice, but no net change in forebrain soluble oligomeric tau or total or phosphorylated levels of aggregated tau was recorded. To investigate the consequence of peripherally-derived parenchymal macrophages on tau aggregation we partially repopulated the hTau microglial pool with peripheral macrophages, but this also did not affect levels of tau oligomers or insoluble aggregates. Our study questions the direct involvement of microglia or peripheral macrophages in the development of tau pathology in the hTau model.     

Imaging brain deoxyglucose uptake and metabolism by glucoCEST MRI

2-Deoxy-D-glucose (2DG) is a known surrogate molecule that is useful for inferring glucose uptake and metabolism. Although ¹³C-labeled 2DG can be detected by nuclear magnetic resonance (NMR), its low sensitivity for detection prohibits imaging to be performed. Using chemical exchange saturation transfer (CEST) as a signal-amplification mechanism, 2DG and the phosphorylated 2DG-6-phosphate (2DG6P) can be indirectly detected in ¹H magnetic resonance imaging (MRI). We showed that the CEST signal changed with 2DG concentration, and was reduced by suppressing cerebral metabolism with increased general anesthetic. The signal changes were not affected by cerebral or plasma pH, and were not correlated with altered cerebral blood flow as demonstrated by hypercapnia; neither were they related to the extracellular glucose amounts as compared with injection of D- and L-glucose. In vivo ³¹P NMR revealed similar changes in 2DG6P concentration, suggesting that the CEST signal reflected the rate of glucose assimilation. This method provides a new way to use widely available MRI techniques to image deoxyglucose/glucose uptake and metabolism in vivo without the need for isotopic labeling of the molecules.     

Physiologically relevant factors influence tau phosphorylation by leucine‐rich repeat kinase 2

Hyperphosphorylation and aggregation of tau are observed in multiple neurodegenerative diseases termed tauopathies. Tau has also been implicated in the pathogenesis of Parkinson's disease (PD) and parkinsonisms. Some PD patients with mutations in the leucine‐rich repeat kinase 2 (LRRK2) gene exhibit tau pathology. Mutations in LRRK2 are a major risk factor for PD, but LRRK2 protein function remains unclear. The most common mutation, G2019S, is located in the kinase domain of LRRK2 and enhances kinase activity in vitro. This suggests that the kinase activity of LRRK2 may underlie its cellular toxicity. Recently, in vitro studies have suggested a direct interaction between tubulin‐bound tau and LRRK2 that results in tau phosphorylation at one identified site. Here we present data suggesting that microtubules (MTs) enhance LRRK2‐mediated tau phosphorylation at three different epitopes. We also explore the effect of divalent cations as catalytic cofactors for G2019S LRRK2‐mediated tau phosphorylation and show that manganese does not support kinase activity but inhibits the efficient ability of magnesium to catalyze LRRK2‐mediated phosphorylation of tau. These results suggest that cofactors such as MTs and cations in the cellular milieu have an important impact on LRRK2‐tau interactions and resultant tau phosphorylation. © 2015 Wiley Periodicals, Inc.