Targeting tauopathy with engineered tau-degrading intrabodies

BACKGROUND: The accumulation of pathological tau is the main component of neurofibrillary tangles and other tau aggregates in several neurodegenerative diseases, referred to as tauopathies. Recently, immunotherapeutic approaches targeting tau have been demonstrated to be beneficial in decreasing tauopathy in animal models. We previously found that passive immunotherapy with anti-tau antibody to human tau or expression of an anti-tau secreted single-chain variable fragment (scFv) in the central nervous system of a mouse model of tauopathy decreased but did not remove all tau-associated pathology. Although these and other studies demonstrate that conventional immunotherapeutic approaches targeting tau can influence tau pathogenesis, the majority of pathological tau remains in the cytosol of cells, not typically accessible to an extracellular antibody. Therefore, we reasoned targeting intracellular tau might be more efficacious in preventing or decreasing tauopathy. METHODS: By utilizing our anti-tau scFv, we generated anti-tau intrabodies for the expression in the cytosol of neurons. To enhance the degradation capacity of conventional intrabodies, we engineered chimeric anti-tau intrabodies fused to ubiquitin harboring distinct mutations that shuttle intracellular tau for either the proteasome or lysosomal mediated degradation. To evaluate the efficacy in delaying or eliminating tauopathy, we expressed our tau degrading intrabodies or controls in human tau transgenic mice by adeno-associated virus prior to overt tau pathology and after tau deposition. RESULTS: Our results demonstrate, the expression of chimeric anti-tau intrabodies significantly reduce tau protein levels in primary neuronal cultures expression human tau relative to a non-modified anti-tau intrabody. We found the expression of engineered tau-degrading intrabodies destined for proteasomal-mediated degradation are more effective in delaying or eliminating tauopathy than a conventional intrabody in aged human tau transgenic mice. CONCLUSION: This study, harnesses the strength of intrabodies that are amendable for targeting specific domains or modifications with the cell-intrinsic mechanisms that regulate protein degradation providing a new immunotherapeutic approach with potentially improved efficacy

TREM2 deficiency attenuates neuroinflammation and protects against neurodegeneration in a mouse model of tauopathy

Significance Alzheimer’s disease (AD) is the most common cause of dementia and is a major public health problem for which there is currently no disease-modifying treatment. There is an urgent need for greater understanding of the molecular mechanisms underlying neurodegeneration in patients to create better therapeutic options. Recently, genetic studies uncovered novel AD risk variants in the microglial receptor, triggering receptor expressed on myeloid cells 2 (TREM2). Previous studies suggested that loss of TREM2 function worsens amyloid-β (Aβ) plaque-related toxicity. In contrast, we observe TREM2 deficiency mitigates neuroinflammation and protects against brain atrophy in the context of tau pathology. These findings indicate dual roles for TREM2 and microglia in the context of amyloid versus tau pathology, which are important to consider for potential treatments targeting TREM2.</jats:p

Glial contributions to neurodegeneration in tauopathies

Abstract Tauopathies are a broad set of neurodegenerative dementias characterized by aggregation of the tau protein into filamentous inclusions that can be found in neurons and glial cells. Activated microglia, astrocytes and elevated levels of proinflammatory molecules are also pathological hallmarks that are found in brain regions affected by tau pathology. There has been abundant research in recent years to understand the role of gliosis and neuroinflammation in neurodegenerative diseases, particularly in Alzheimer’s disease (AD) which is the most common form of dementia. AD is a tauopathy characterized by both extracellular amyloid-β plaques in addition to intracellular neurofibrillary tangles and neuropil threads containing aggregated tau protein. Accumulating evidence suggests that neuroinflammation offers a possible mechanistic link between these pathologies. Additionally, there appears to be a role for neuroinflammation in aggravating tau pathology and neurodegeneration in tauopathies featuring tau deposits as the predominant pathological signature. In this review, we survey the literature regarding inflammatory mechanisms that may impact neurodegeneration in AD and related tauopathies. We consider a physical role for microglia in the spread of tau pathology as well as the non-cell autonomous effects of secreted proinflammatory cytokines, specifically interleukin 1 beta, interleukin 6, tumor necrosis factor alpha and complement proteins. These molecules appear to have direct effects on tau pathophysiology and overall neuronal health. They also indirectly impact neuronal homeostasis by altering glial function. We conclude by proposing a complex role for gliosis and neuroinflammation in accelerating the progression of AD and other tauopathies

New insights into the role of TREM2 in Alzheimer’s disease

Abstract Alzheimer’s disease (AD) is the leading cause of dementia. The two histopathological markers of AD are amyloid plaques composed of the amyloid-β (Aβ) peptide, and neurofibrillary tangles of aggregated, abnormally hyperphosphorylated tau protein. The majority of AD cases are late-onset, after the age of 65, where a clear cause is still unknown. However, there are likely different multifactorial contributors including age, enviornment, biology and genetics which can increase risk for the disease. Genetic predisposition is considerable, with heritability estimates of 60–80%. Genetic factors such as rare variants of TREM2 (triggering receptor expressed on myeloid cells-2) strongly increase the risk of developing AD, confirming the role of microglia in AD pathogenesis. In the last 5 years, several studies have dissected the mechanisms by which TREM2, as well as its rare variants affect amyloid and tau pathologies and their consequences in both animal models and in human studies. In this review, we summarize increases in our understanding of the involvement of TREM2 and microglia in AD development that may open new therapeutic strategies targeting the immune system to influence AD pathogenesis

Microglial roles in Alzheimer's disease: An agent‐based model to elucidate microglial spatiotemporal response to beta‐amyloid

Abstract Alzheimer's disease (AD) is characterized by beta‐amyloid (Aβ) plaques in the brain and widespread neuronal damage. Because of the high drug attrition rates in AD, there is increased interest in characterizing neuroimmune responses to Aβ plaques. In response to AD pathology, microglia are innate phagocytotic immune cells that transition into a neuroprotective state and form barriers around plaques. We seek to understand the role of microglia in modifying Aβ dynamics and barrier formation. To quantify the influence of individual microglia behaviors (activation, chemotaxis, phagocytosis, and proliferation) on plaque size and barrier coverage, we developed an agent‐based model to characterize the spatiotemporal interactions between microglia and Aβ. Our model qualitatively reproduces mouse data trends where the fraction of microglia coverage decreases as plaques become larger. In our model, the time to microglial arrival at the plaque boundary is significantly negatively correlated (p < 0.0001) with plaque size, indicating the importance of the time to microglial activation for regulating plaque size. In addition, in silico behavioral knockout simulations show that phagocytosis knockouts have the strongest impact on plaque size, but modest impacts on microglial coverage and activation. In contrast, the chemotaxis knockouts had a strong impact on microglial coverage with a more modest impact on plaque volume and microglial activation. These simulations suggest that phagocytosis, chemotaxis, and replication of activated microglia have complex impacts on plaque volume and coverage, whereas microglial activation remains fairly robust to perturbations of these functions. Thus, our work provides insights into the potential and limitations of targeting microglial activation as a pharmacological strategy for the treatment of AD

Glucocerebrosidase activity and lipid levels are related to protein pathologies in Parkinson’s disease

Abstract Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) are progressive neurodegenerative diseases characterized by the accumulation of misfolded α-synuclein in the form of Lewy pathology. While most cases are sporadic, there are rare genetic mutations that cause disease and more common variants that increase incidence of disease. The most prominent genetic mutations for PD and DLB are in the GBA1 and LRRK2 genes. GBA1 mutations are associated with decreased glucocerebrosidase activity and lysosomal accumulation of its lipid substrates, glucosylceramide and glucosylsphingosine. Previous studies have shown a link between this enzyme and lipids even in sporadic PD. However, it is unclear how the protein pathologies of disease are related to enzyme activity and glycosphingolipid levels. To address this gap in knowledge, we examined quantitative protein pathology, glucocerebrosidase activity and lipid substrates in parallel from 4 regions of 91 brains with no neurological disease, idiopathic, GBA1-linked, or LRRK2-linked PD and DLB. We find that several biomarkers are altered with respect to mutation and progression to dementia. We found mild association of glucocerebrosidase activity with disease, but a strong association of glucosylsphingosine with α-synuclein pathology, irrespective of genetic mutation. This association suggests that Lewy pathology precipitates changes in lipid levels related to progression to dementia

LRRK2 kinase inhibition reverses G2019S mutation-dependent effects on tau pathology progression

Abstract Background Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson’s disease (PD). These mutations elevate the LRRK2 kinase activity, making LRRK2 kinase inhibitors an attractive therapeutic. LRRK2 kinase activity has been consistently linked to specific cell signaling pathways, mostly related to organelle trafficking and homeostasis, but its relationship to PD pathogenesis has been more difficult to define. LRRK2-PD patients consistently present with loss of dopaminergic neurons in the substantia nigra but show variable development of Lewy body or tau tangle pathology. Animal models carrying LRRK2 mutations do not develop robust PD-related phenotypes spontaneously, hampering the assessment of the efficacy of LRRK2 inhibitors against disease processes. We hypothesized that mutations in LRRK2 may not be directly related to a single disease pathway, but instead may elevate the susceptibility to multiple disease processes, depending on the disease trigger. To test this hypothesis, we have previously evaluated progression of α-synuclein and tau pathologies following injection of proteopathic seeds. We demonstrated that transgenic mice overexpressing mutant LRRK2 show alterations in the brain-wide progression of pathology, especially at older ages. Methods Here, we assess tau pathology progression in relation to long-term LRRK2 kinase inhibition. Wild-type or LRRK2G2019S knock-in mice were injected with tau fibrils and treated with control diet or diet containing LRRK2 kinase inhibitor MLi-2 targeting the IC50 or IC90 of LRRK2 for 3–6 months. Mice were evaluated for tau pathology by brain-wide quantitative pathology in 844 brain regions and subsequent linear diffusion modeling of progression. Results Consistent with our previous work, we found systemic alterations in the progression of tau pathology in LRRK2G2019S mice, which were most pronounced at 6 months. Importantly, LRRK2 kinase inhibition reversed these effects in LRRK2G2019S mice, but had minimal effect in wild-type mice, suggesting that LRRK2 kinase inhibition is likely to reverse specific disease processes in G2019S mutation carriers. Additional work may be necessary to determine the potential effect in non-carriers. Conclusions This work supports a protective role of LRRK2 kinase inhibition in G2019S carriers and provides a rational workflow for systematic evaluation of brain-wide phenotypes in therapeutic development

Additional file 1 of LRRK2 kinase inhibition reverses G2019S mutation-dependent effects on tau pathology progression

Additional file 1. Table S1: Characterization of PHF preparations from AD brains. Figure S1: Long-term MLi-2 impacts lung, but not the gross kidney morphology. Figure S2: Quantitative pathology workflow. Figure S3: Quantitative pathology analysis from all mice. Figure S4: Sex differences in tau pathology in wild-type mice. Figure S5: Representative staining from 3 MPI mice. Figure S6: Wild-type compared to LRRK2G2019S mice at 3 MPI. Figure S7: Tau pathology compared by treatment group at 3 MPI. Figure S8: Representative staining from 6 MPI mice. Figure S9: Examination of linear diffusion fits by hemisphere. Figure S10: Microglia quantification in caudal cortex of 6 MPI mice