Targeting 14-3-3θ-mediated TDP-43 pathology in amyotrophic lateral sclerosis and frontotemporal dementia mice.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are characterized by cytoplasmic deposition of the nuclear TAR-binding protein 43 (TDP-43). Although cytoplasmic re-localization of TDP-43 is a key event in the pathogenesis of ALS/FTD, the underlying mechanisms remain unknown. Here, we identified a non-canonical interaction between 14-3-3θ and TDP-43, which regulates nuclear-cytoplasmic shuttling. Neuronal 14-3-3θ levels were increased in sporadic ALS and FTD with TDP-43 pathology. Pathogenic TDP-43 showed increased interaction with 14-3-3θ, resulting in cytoplasmic accumulation, insolubility, phosphorylation, and fragmentation of TDP-43, resembling pathological changes in disease. Harnessing this increased affinity of 14-3-3θ for pathogenic TDP-43, we devised a gene therapy vector targeting TDP-43 pathology, which mitigated functional deficits and neurodegeneration in different ALS/FTD mouse models expressing mutant or non-mutant TDP-43, including when already symptomatic at the time of treatment. Our study identified 14-3-3θ as a mediator of cytoplasmic TDP-43 localization with implications for ALS/FTD pathogenesis and therapy.

Treatment with the copper compound CuATSM has no significant effect on motor neuronal pathology in patients with ALS.

AIMS: Although the orally available brain-penetrant copper compound CuATSM has demonstrated promising effects in SOD1-linked mouse models, the impact of CuATSM on disease pathology in patients with amyotrophic lateral sclerosis (ALS) remains unknown. METHODS: The present study set out to address this deficit by performing the first pilot comparative analysis of ALS pathology in patients that had been administered CuATSM and riluzole [N = 6 cases composed of ALS-TDP (n = 5) and ALS-SOD1 (n = 1)] versus riluzole only [N = 6 cases composed of ALS-TDP (n = 4) and ALS-SOD1 (n = 2)]. RESULTS: Our results revealed no significant difference in neuron density or TDP-43 burden in the motor cortex and spinal cord of patients that had received CuATSM compared with patients that had not. In patients that had received CuATSM, p62-immunoreactive astrocytes were observed in the motor cortex and reduced Iba1 density was found in the spinal cord. However, no significant difference in measures of astrocytic activity and SOD1 immunoreactivity was found with CuATSM treatment. DISCUSSION: These findings, in this first postmortem investigation of patients with ALS in CuATSM trials, demonstrate that in contrast to that seen in preclinical models of disease, CuATSM does not significantly alleviate neuronal pathology or astrogliosis in patients with ALS.

Distribution Patterns of Astrocyte Populations in the Human Cortex.

Astrocytes are a major class of glial cell in the central nervous system that have a diverse range of types and functions thought to be based on their anatomical location, morphology and cellular properties. Recent studies highlight that astrocyte dysfunction contributes to the pathogenesis of neurological conditions. However, few studies have described the pattern, distribution and density of astrocytes in the adult human cortex. This study mapped the distribution and density of astrocytes immunolabelled with a range of cytoskeletal and membrane markers in the human frontal cortex. Distinct and overlapping astrocyte populations were determined. The frontal cortex from ten normal control cases (75 ± 9 years) was immunostained with glial fibrillary acidic protein (GFAP), aldehyde dehydrogenase-1 L1 (ALDH1L1), connexin-43 (Cx43), aquaporin-4 (AQP4), and glutamate transporter 1 (GLT-1). All markers labelled populations of astrocytes in the grey and white matter, separate cortical layers, subpial and perivascular regions. All markers were informative for labelling different cellular properties and cellular compartments of astrocytes. ALDH1L1 labelled the largest population of astrocytes, and Cx43-immunopositive astrocytes were found in all cortical layers. AQP4 and GLT-1 labelled distal astrocytic process and end-feet in the same population of astrocytes (98% of GLT-1-immunopositive astrocytes contained AQP4). In contrast, GFAP, the most widely used marker, predominantly labelled astrocytes in superficial cortical layers. This study highlights the diversity of astrocytes in the human cortex, providing a reference map of the distribution of distinct and overlapping astrocyte populations which can be used for comparative purposes in various disease, inflammatory and injury states involving astrocytes.

Biomarker discovery and development for frontotemporal dementia and amyotrophic lateral sclerosis.

Frontotemporal dementia refers to a group of neurodegenerative disorders characterized by behaviour and language alterations and focal brain atrophy. Amyotrophic lateral sclerosis is a rapidly progressing neurodegenerative disease characterized by loss of motor neurons resulting in muscle wasting and paralysis. Frontotemporal dementia and amyotrophic lateral sclerosis are considered to exist on a disease spectrum given substantial overlap of genetic and molecular signatures. The predominant genetic abnormality in both frontotemporal dementia and amyotrophic lateral sclerosis is an expanded hexanucleotide repeat sequence in the C9orf72 gene. In terms of brain pathology, abnormal aggregates of TAR-DNA-binding protein-43 are predominantly present in frontotemporal dementia and amyotrophic lateral sclerosis patients. Currently, sensitive and specific diagnostic and disease surveillance biomarkers are lacking for both diseases. This has impeded the capacity to monitor disease progression during life and the development of targeted drug therapies for the two diseases. The purpose of this review is to examine the status of current biofluid biomarker discovery and development in frontotemporal dementia and amyotrophic lateral sclerosis. The major pathogenic proteins implicated in different frontotemporal dementia and amyotrophic lateral sclerosis molecular subtypes and proteins associated with neurodegeneration and the immune system will be discussed. Furthermore, the use of mass spectrometry-based proteomics as an emerging tool to identify new biomarkers in frontotemporal dementia and amyotrophic lateral sclerosis will be summarized.

Lipidome changes in alcohol-related brain damage.

Alcohol-related brain injury is characterized by cognitive deficits and brain atrophy with the prefrontal cortex particularly susceptible. White matter in the human brain is lipid rich and a major target of damage from chronic alcohol abuse; yet, there is sparse information on how these lipids are affected. Here, we used untargeted lipidomics as a discovery tool to describe these changes in the prefrontal, middle temporal, and visual cortices of human subjects with alcohol use disorder and controls. Significant changes to the lipidome, predominantly in the prefrontal and visual cortices, and differences between the white and grey matter of each brain region were identified. These effects include broad decreases to phospholipids and ceramide, decreased polyunsaturated fatty acids, decreased sphingadiene backbones, and selective decreases in cholesteryl ester fatty acid chains. Our findings show that chronic alcohol abuse results in selective changes to the neurolipidome, which likely reflects both the directs effects on the brain and concurrent effects on the liver.

Glycoprotein Pathways Altered in Frontotemporal Dementia With Autoimmune Disease.

Behavioral variant frontotemporal dementia (bvFTD) is a younger onset form of neurodegeneration initiated in the frontal and/or temporal lobes with a slow clinical onset but rapid progression. bvFTD is highly complex biologically with different pathological signatures and genetic variants that can exhibit a spectrum of overlapping clinical manifestations. Although the role of innate immunity has been extensively investigated in bvFTD, the involvement of adaptive immunity in bvFTD pathogenesis is poorly understood. We analyzed blood serum proteomics to identify proteins that are associated with autoimmune disease in bvFTD. Eleven proteins (increased: ATP5B, CALML5, COLEC11, FCGBP, PLEK, PLXND1; decreased: APOB, ATP8B1, FAM20C, LOXL3, TIMD4) were significantly altered in bvFTD with autoimmune disease compared to those without autoimmune disease. The majority of these proteins were enriched for glycoprotein-associated proteins and pathways, suggesting that the glycome is targeted in bvFTD with autoimmune disease.

Association Between Globular Glial Tauopathies and Frontotemporal Dementia-Expanding the Spectrum of Gliocentric Disorders: A Review.

Importance: Globular glial tauopathies (GGTs), as defined by a consensus study in 2013, belong to the group of frontotemporal lobar degenerations and expand the spectrum of glial-predominant neurodegenerative diseases. Three neuropathological subtypes of GGT (types I-III) are characterized by phosphorylated tau-immunopositive inclusions that are predominantly in oligodendroglia and/or astroglia in the frontal, temporal, and/or precentral cortices. Type II is largely restricted to the corticospinal system. The low incidence of GGT (<10% of cases of frontotemporal lobar degeneration with tau pathology), together with its unusual combination of neuronal and nonneuronal pathology, has hindered identification and accurate diagnosis. This review collated clinical, demographic, neuropathological, and genetic data from 88 published GGT cases identified on PubMed to examine the association between GGT and frontotemporal dementia and associated disorders. Observations: Among 88 patients with GGT (46 female [52.3%]; mean [SD] age at disease onset, 65 [11] years), 44 patients (50.0%) had idiopathic disease, and 21 patients (23.9%) had a variation in the microtubule-associated protein tau (MAPT) gene. Those with idiopathic GGT compared with those with a variation in MAPT had a mean (SD) age at symptom onset of 70 (8) years vs 54 (9) years and a mean (SD) disease duration of 7 (3) years vs 6 (3) years, respectively. A similar sex distribution was observed among patients with GGT; however, female patients were typically 6 years older at symptom onset than male patients (mean [SD] age, 68 [11] years vs 62 [11] years, respectively). Disease duration was similar in both sexes (mean [SD], 6 [3] years for women and 6 [4] years for men). The most common predominant clinical features were primary progressive aphasia (22 patients [25.0%]), behavioral-variant frontotemporal dementia (20 patients [22.7%]), upper motor neuron signs (11 patients [12.5%]), memory impairment (7 patients [8.0%]), and Richardson syndrome (7 patients [8.0%]). Although some demographic differences between GGT subtypes were identified, the predictive value of the clinical presentation was low, calling into question the need for neuropathological subtyping. Further neuropathological studies are needed to clarify whether GGT type II should be interpreted as atypical progressive supranuclear palsy or a separate entity. Few cases (7 patients [8.0%]) had coexisting proteinopathies. Conclusions and Relevance: This review of the published data suggests an association between regional distribution of glial tau pathology and neuronal degeneration. Targeting glial tau accumulation or sustaining their neuron-supportive function might require different therapeutic or neuroprotective strategies and more accurate preclinical models to explore disease mechanisms and track progression. Emerging data support the important role of glia in the pathogenesis of neurodegenerative disorders, highlighting the need to raise awareness of GGT in clinical and research settings.

Coexisting Lewy body disease and clinical parkinsonism in amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is associated with a range of clinical phenotypes and shows progressive degeneration of upper and/or lower motor neurons, and phosphorylated 43 kDa TAR DNA-binding protein (pTDP-43) inclusions in motor and non-motor pathways. Parkinsonian features have been reported in up to 30% of ALS patients, and Lewy bodies, normally associated with Lewy body disease (LBD), have been reported in a small number of ALS cases, with unknown clinical relevance. This study investigates the prevalence of clinically relevant LBD in a prospectively studied ALS cohort to determine whether concomitant pathology contributes to the clinical heterogeneity. METHODS: All ALS cases held by the New South Wales Brain Bank (n = 97) were screened for coexisting LBD consistent with clinical disease (Braak ≥ stage IV). Relevant clinical and genetic associations were determined. RESULTS: Six cases had coexisting LBD Braak ≥ stage IV pathology. The age at symptom onset (69 ± 7 years) and disease duration (4 ± 3 years) in ALS cases with coexisting LBD did not differ from ALS cases. Three patients had lower limb onset and two patients had bulbar onset. Two patients developed the clinical features of Parkinson's disease, with one receiving a dual diagnosis. All cases had no known relevant family history or genetic abnormalities. CONCLUSION: The prevalence of clinically relevant LBD pathology in ALS is higher than in the general population, and has implications for clinical and neuropathological diagnoses and the identification of biomarkers.

Ground state depletion microscopy as a tool for studying microglia-synapse interactions.

Ground state depletion followed by individual molecule return microscopy (GSDIM) has been used in the past to study the nanoscale distribution of protein co-localization in living cells. We now demonstrate the successful application of GSDIM to archival human brain tissue sections including from Alzheimer's disease cases as well as experimental tissue samples from mouse and zebrafish larvae. Presynaptic terminals and microglia and their cell processes were visualized at a resolution beyond diffraction-limited light microscopy, allowing clearer insights into their interactions in situ. The procedure described here offers time and cost savings compared to electron microscopy and opens the spectrum of molecular imaging using antibodies and super-resolution microscopy to the analysis of routine formalin-fixed paraffin sections of archival human brain. The investigation of microglia-synapse interactions in dementia will be of special interest in this context.

Defining early changes in Alzheimer's disease from RNA sequencing of brain regions differentially affected by pathology.

Tau pathology in Alzheimer's disease (AD) spreads in a predictable pattern that corresponds with disease symptoms and severity. At post-mortem there are cortical regions that range from mildly to severely affected by tau pathology and neuronal loss. A comparison of the molecular signatures of these differentially affected areas within cases and between cases and controls may allow the temporal modelling of disease progression. Here we used RNA sequencing to explore differential gene expression in the mildly affected primary visual cortex and moderately affected precuneus of ten age-, gender- and RNA quality-matched post-mortem brains from AD patients and healthy controls. The two regions in AD cases had similar transcriptomic signatures but there were broader abnormalities in the precuneus consistent with the greater tau load. Both regions were characterised by upregulation of immune-related genes such as those encoding triggering receptor expressed on myeloid cells 2 and membrane spanning 4-domains A6A and milder changes in insulin/IGF1 signalling. The precuneus in AD was also characterised by changes in vesicle secretion and downregulation of the interneuronal subtype marker, somatostatin. The 'early' AD transcriptome is characterised by perturbations in synaptic vesicle secretion on a background of neuroimmune dysfunction. In particular, the synaptic deficits that characterise AD may begin with the somatostatin division of inhibitory neurotransmission.

A Practical Approach to Differentiate the Frontotemporal Tauopathy Subtypes.

This study proposes a practical approach, using the minimum number of brain regions and stains, to consolidate previously published neuropathological criteria into one operationalized schema to differentiate subtypes of frontotemporal lobar degeneration with tau-immunopositive inclusions (FTLD-tau). This approach uses the superior frontal and precentral cortices and hippocampus stained for phosphorylated-tau, p62 and modified Bielschowsky silver, and the midbrain stained only for modified Bielschowsky silver. Accuracy of interrater reliability was determined by 10 raters in 24 FTLD-tau cases (Pick disease = 4, corticobasal degeneration = 9, progressive supranuclear palsy = 5, globular glial tauopathy = 6) including 4 with a mutation in MAPT collected with consent by Sydney Brain Bank. All brain regions and stains assessed proved informative for accurate pathological subtyping, and many neuropathological features were identified as common across the FTLD-tau subtypes. By identifying subtype-specific neuropathological features in the sections selected, 10 independent observers assigned the cases to a FTLD-tau subtype with almost perfect agreement between raters, emphasizing the requirement for the assessment of subtype-specific features for the accurate subtyping of FTLD-tau. This study consolidates current consensus diagnostic criteria for classifying FTLD-tau subtypes with an efficient, simple and accurate approach that can be implemented in future clinicopathological studies.

Altered serum protein levels in frontotemporal dementia and amyotrophic lateral sclerosis indicate calcium and immunity dysregulation.

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are neurodegenerative diseases that are considered to be on the same disease spectrum because of overlapping genetic, pathological and clinical traits. Changes in serum proteins in FTD and ALS are poorly understood, and currently no definitive biomarkers exist for diagnosing or monitoring disease progression for either disease. Here we applied quantitative discovery proteomics to analyze protein changes in FTD (N = 72) and ALS (N = 28) patient serum compared to controls (N = 22). Twenty three proteins were significantly altered in FTD compared to controls (increased-APOL1, C3, CTSH, EIF5A, MYH2, S100A8, SUSD5, WDR1; decreased-C1S, C7, CILP2, COMP, CRTAC1, EFEMP1, FBLN1, GSN, HSPG2, IGHV1, ITIH2, PROS1, SHBG, UMOD, VASN) and 14 proteins were significantly altered in ALS compared to controls (increased-APOL1, CKM, CTSH, IGHG1, IGKC, MYH2; decreased-C7, COMP, CRTAC1, EFEMP1, FBLN1, GSN, HSPG2, SHBG). There was substantial overlap in the proteins that were altered in FTD and ALS. These results were validated using western blotting. Gene ontology tools were used to assess functional pathways potentially dysregulated in the two diseases, and calcium ion binding and innate immunity pathways were altered in both diseases. When put together, these results suggest significant overlap in pathophysiological peripheral changes in FTD and ALS. This study represents the first proteomics side-by-side comparison of serum changes in FTD and ALS, providing new insights into under-recognized perturbed pathways and an avenue for biomarker development for FTD and ALS.

CNS cell type-specific gene profiling of P301S tau transgenic mice identifies genes dysregulated by progressive tau accumulation.

The microtubule-associated protein tau undergoes aberrant modification resulting in insoluble brain deposits in various neurodegenerative diseases, including frontotemporal dementia (FTD), progressive supranuclear palsy, and corticobasal degeneration. Tau aggregates can form in different cell types of the central nervous system (CNS) but are most prevalent in neurons. We have previously recapitulated aspects of human FTD in mouse models by overexpressing mutant human tau in CNS neurons, including a P301S tau variant in TAU58/2 mice, characterized by early-onset and progressive behavioral deficits and FTD-like neuropathology. The molecular mechanisms underlying the functional deficits of TAU58/2 mice remain mostly elusive. Here, we employed functional genomics (i.e. RNAseq) to determine differentially expressed genes in young and aged TAU58/2 mice to identify alterations in cellular processes that may contribute to neuropathy. We identified genes in cortical brain samples differentially regulated between young and old TAU58/2 mice relative to nontransgenic littermates and by comparative analysis with a dataset of CNS cell type-specific genes expressed in nontransgenic mice. Most differentially-regulated genes had known or putative roles in neurons and included presynaptic and excitatory genes. Specifically, we observed changes in presynaptic factors, glutamatergic signaling, and protein scaffolding. Moreover, in the aged mice, expression levels of several genes whose expression was annotated to occur in other brain cell types were altered. Immunoblotting and immunostaining of brain samples from the TAU58/2 mice confirmed altered expression and localization of identified and network-linked proteins. Our results have revealed genes dysregulated by progressive tau accumulation in an FTD mouse model.

Neuroinflammation in frontotemporal dementia.

Frontotemporal dementia (FTD) refers to a group of progressive neurodegenerative disorders with different pathological signatures, genetic variability and complex disease mechanisms, for which no effective treatments exist. Despite advances in understanding the underlying pathology of FTD, sensitive and specific fluid biomarkers for this disease are lacking. As in other types of dementia, mounting evidence suggests that neuroinflammation is involved in the progression of FTD, including cortical inflammation, microglial activation, astrogliosis and differential expression of inflammation-related proteins in the periphery. Furthermore, an overlap between FTD and autoimmune disease has been identified. The most substantial evidence, however, comes from genetic studies, and several FTD-related genes are also implicated in neuroinflammation. This Review discusses specific evidence of neuroinflammatory mechanisms in FTD and describes how advances in our understanding of these mechanisms, in FTD as well as in other neurodegenerative diseases, might facilitate the development and implementation of diagnostic tools and disease-modifying treatments for FTD.

Recent Developments in TSPO PET Imaging as A Biomarker of Neuroinflammation in Neurodegenerative Disorders.

Neuroinflammation is an inflammatory response in the brain and spinal cord, which can involve the activation of microglia and astrocytes. It is a common feature of many central nervous system disorders, including a range of neurodegenerative disorders. An overlap between activated microglia, pro-inflammatory cytokines and translocator protein (TSPO) ligand binding was shown in early animal studies of neurodegeneration. These findings have been translated in clinical studies, where increases in TSPO positron emission tomography (PET) signal occur in disease-relevant areas across a broad spectrum of neurodegenerative diseases. While this supports the use of TSPO PET as a biomarker to monitor response in clinical trials of novel neurodegenerative therapeutics, the clinical utility of current TSPO PET radioligands has been hampered by the lack of high affinity binding to a prevalent form of polymorphic TSPO (A147T) compared to wild type TSPO. This review details recent developments in exploration of ligand-sensitivity to A147T TSPO that have yielded ligands with improved clinical utility. In addition to developing a non-discriminating TSPO ligand, the final frontier of TSPO biomarker research requires developing an understanding of the cellular and functional interpretation of the TSPO PET signal. Recent insights resulting from single cell analysis of microglial phenotypes are reviewed.

Cellular and regional vulnerability in frontotemporal tauopathies.

The frontotemporal tauopathies all deposit abnormal tau protein aggregates, but often of only certain isoforms and in distinguishing pathologies of five main types (neuronal Pick bodies, neurofibrillary tangles, astrocytic plaques, tufted astrocytes, globular glial inclusions and argyrophilic grains). In those with isoform specific tau aggregates glial pathologies are substantial, even though there is limited evidence that these cells normally produce tau protein. This review will assess the differentiating features and clinicopathological correlations of the frontotemporal tauopathies, the genetic predisposition for these different pathologies, their neuroanatomical selectivity, current observations on how they spread through the brain, and any potential contributing cellular and molecular changes. The findings show that diverse clinical phenotypes relate most to the brain region degenerating rather than the type of pathology involved, that different regions on the MAPT gene and novel risk genes are associated with specific tau pathologies, that the 4-repeat glial tauopathies do not follow individual patterns of spreading as identified for neuronal pathologies, and that genetic and pathological data indicate that neuroinflammatory mechanisms are involved. Each pathological frontotemporal tauopathy subtype with their distinct pathological features differ substantially in the cell type affected, morphology, biochemical and anatomical distribution of inclusions, a fundamental concept central to future success in understanding the disease mechanisms required for developing therapeutic interventions. Tau directed therapies targeting genetic mechanisms, tau aggregation and pathological spread are being trialled, although biomarkers that differentiate these diseases are required. Suggested areas of future research to address the regional and cellular vulnerabilities in frontotemporal tauopathies are discussed.