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AimTau truncation (tr-tau) by active caspase-6 (aCasp-6) generates tau fragments that may be toxic. Yet the relationship between aCasp-6, different forms of tr-tau and hyperphosphorylated tau (p-tau) accumulation in human brains with Alzheimer's disease (AD) and other tauopathies remains unclear.MethodsWe generated two neoepitope monoclonal antibodies against tr-tau sites (D402 and D13) targeted by aCasp-6. Then, we used five-plex immunofluorescence to quantify the neuronal and astroglial burden of aCasp-6, tr-tau, p-tau and their co-occurrence in healthy controls, AD and primary tauopathies.ResultsCasp-6 activation was strongest in AD and Pick's disease (PiD) but almost absent in 4-repeat (4R) tauopathies. In neurons, the tr-tau burden was much more abundant in AD and PiD than in 4R tauopathies and disproportionally higher when normalising by p-tau pathology. Tr-tau astrogliopathy was detected in low numbers in 4R tauopathies. Unexpectedly, about half of tr-tau positive neurons in AD and PiD lacked p-tau aggregates, a finding we confirmed using several p-tau antibodies.ConclusionsEarly modulation of aCasp-6 to reduce tr-tau pathology is a promising therapeutic strategy for AD and PiD but is unlikely to benefit 4R tauopathies. The large percentage of tr-tau-positive neurons lacking p-tau suggests that many vulnerable neurons to tau pathology go undetected when using conventional p-tau antibodies. Therapeutic strategies against tr-tau pathology could be necessary to modulate the extent of tau abnormalities in AD. The disproportionally higher burden of tr-tau in AD and PiD supports the development of biofluid biomarkers against tr-tau to detect AD and PiD and differentiate them from 4R tauopathies at a patient level.

Alzheimer's disease (AD) is neuropathologically characterized by the intracellular accumulation of hyperphosphorylated tau and the extracellular deposition of amyloid-β plaques, which affect certain brain regions in a progressive manner. The locus coeruleus (LC), a small nucleus in the pons of the brainstem, is widely recognized as one of the earliest sites of neurofibrillary tangle formation in AD. Patients with AD exhibit significant neuronal loss in the LC, resulting in a marked reduction of its size and function. The LC, which vastly innervates several regions of the brain, is the primary source of the neurotransmitter norepinephrine (NE) in the central nervous system. Considering that NE is a major modulator of behavior, contributing to neuroprotection and suppression of neuroinflammation, degeneration of the LC in AD and the ultimate dysregulation of the LC-NE system has detrimental effects in the brain. In this review, we detail the neuroanatomy and function of the LC, its essential role in neuroprotection, and how this is dysregulated in AD. We discuss AD-related neuropathologic changes in the LC and mechanisms by which LC neurons are selectively vulnerable to insult. Further, we elucidate the neurotoxic effects of LC de-innervation both locally and at projection sites, and how this augments disease pathology, progression and severity. We summarize how preservation of the LC-NE system could be used in the treatment of AD and other neurodegenerative diseases affected by LC degeneration.

Primary tauopathies are characterized neuropathologically by inclusions containing abnormal forms of the microtubule-associated protein tau (MAPT) and clinically by diverse neuropsychiatric, cognitive, and motor impairments. Autosomal dominant mutations in the MAPT gene cause heterogeneous forms of frontotemporal lobar degeneration with tauopathy (FTLD-Tau). Common and rare variants in the MAPT gene increase the risk for sporadic FTLD-Tau, including progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). We generated a collection of fibroblasts from 140 MAPT mutation/risk variant carriers, PSP, CBD, and cognitively normal controls; 31 induced pluripotent stem cell (iPSC) lines from MAPT mutation carriers, non-carrier family members, and autopsy-confirmed PSP patients; 33 genome engineered iPSCs that were corrected or mutagenized; and forebrain neural progenitor cells (NPCs). Here, we present a resource of fibroblasts, iPSCs, and NPCs with comprehensive clinical histories that can be accessed by the scientific community for disease modeling and development of novel therapeutics for tauopathies.

IntroductionSleep-wake disturbances are a common and early feature in Alzheimer's disease (AD). The impact of early tau pathology in wake-promoting neurons (WPNs) remains unclear.MethodsWe performed stereology in postmortem brains from AD individuals and healthy controls to identify quantitative differences in morphological metrics in WPNs. Progressive supranuclear palsy (PSP) and corticobasal degeneration were included as disease-specific controls.ResultsThe three nuclei studied accumulate considerable amounts of tau inclusions and showed a decrease in neurotransmitter-synthetizing neurons in AD, PSP, and corticobasal degeneration. However, substantial neuronal loss was exclusively found in AD.DiscussionWPNs are extremely vulnerable to AD but not to 4 repeat tauopathies. Considering that WPNs are involved early in AD, such degeneration should be included in the models explaining sleep-wake disturbances in AD and considered when designing a clinical intervention. Sparing of WPNs in PSP, a condition featuring hyperinsomnia, suggest that interventions to suppress the arousal system may benefit patients with PSP.

Protein-protein interactions between E3 ubiquitin ligases and protein termini help shape the proteome. These interactions are sensitive to proteolysis, which alters the ensemble of cellular N and C termini. Here we describe a mechanism wherein caspase activity reveals latent C termini that are then recognized by the E3 ubiquitin ligase CHIP. Using expanded knowledge of CHIP's binding specificity, we predicted hundreds of putative interactions arising from caspase activity. Subsequent validation experiments confirmed that CHIP binds the latent C termini at tauD421 and caspase-6D179. CHIP binding to tauD421, but not tauFL, promoted its ubiquitination, while binding to caspase-6D179 mediated ubiquitin-independent inhibition. Given that caspase activity generates tauD421 in Alzheimer's disease (AD), these results suggested a concise model for CHIP regulation of tau homeostasis. Indeed, we find that loss of CHIP expression in AD coincides with the accumulation of tauD421 and caspase-6D179. These results illustrate an unanticipated link between caspases and protein homeostasis.

The brainstem nuclei of the reticular formation (RF) are critical for regulating homeostasis, behavior, and cognition. RF degenerates in tauopathies including Alzheimer disease (AD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). Although the burden of phopho-tau inclusion is high across these diseases, suggesting a similar vulnerability pattern, a distinct RF-associated clinical phenotype in these diseases indicates the opposite. To compare patterns of RF selective vulnerability to tauopathies, we analyzed 5 RF nuclei in tissue from 14 AD, 14 CBD, 10 PSP, and 3 control cases. Multidimensional quantitative analysis unraveled discernable differences on how these nuclei are vulnerable to AD, CBD, and PSP. For instance, PSP and CBD accrued more tau inclusions than AD in locus coeruleus, suggesting a lower vulnerability to AD. However, locus coeruleus neuronal loss in AD was so extreme that few neurons remained to develop aggregates. Likewise, tau burden in gigantocellular nucleus was low in AD and high in PSP, but few GABAergic neurons were present in AD. This challenges the hypothesis that gigantocellular nucleus neuronal loss underlies REM behavioral disorders because REM behavioral disorders rarely manifests in AD. This study provides foundation for characterizing the clinical consequences of RF degeneration in tauopathies and guiding customized treatment.

Clarifying the mechanisms connecting neurofibrillary tangle (NFT) neurotoxicity to neuronal dysfunction in humans is likely to be pivotal for developing effective treatments for Alzheimer's disease (AD). To model the temporal progression of AD in humans, we used a collection of brains with controls and individuals from each Braak stage to quantitatively investigate the correlation between intraneuronal caspase activation or macroautophagy markers, NFT burden, and neuronal loss, in the dorsal raphe nucleus and locus coeruleus, the earliest vulnerable areas to NFT accumulation. We fit linear regressions with each count as outcomes, with Braak score and age as the predictors. In progressive Braak stages, intraneuronal active caspase-6 positivity increases both alone and overlapping with NFTs. Likewise, the proportion of NFT-bearing neurons showing autophagosomes increases. Overall, caspases may be involved in upstream cascades in AD and are associated with higher NFTs. Macroautophagy changes correlate with increasing NFT burden from early AD stages.

AimsHyperphosphorylated tau neuronal cytoplasmic inclusions (ht-NCI) are the best protein correlate of clinical decline in Alzheimer's disease (AD). Qualitative evidence identifies ht-NCI accumulating in the isodendritic core before the entorhinal cortex. Here, we used unbiased stereology to quantify ht-NCI burden in the locus coeruleus (LC) and dorsal raphe nucleus (DRN), aiming to characterize the impact of AD pathology in these nuclei with a focus on early stages.MethodsWe utilized unbiased stereology in a sample of 48 well-characterized subjects enriched for controls and early AD stages. ht-NCI counts were estimated in 60-μm-thick sections immunostained for p-tau throughout LC and DRN. Data were integrated with unbiased estimates of LC and DRN neuronal population for a subset of cases.ResultsIn Braak stage 0, 7.9% and 2.6% of neurons in LC and DRN, respectively, harbour ht-NCIs. Although the number of ht-NCI+ neurons significantly increased by about 1.9× between Braak stages 0 to I in LC (P = 0.02), we failed to detect any significant difference between Braak stage I and II. Also, the number of ht-NCI+ neurons remained stable in DRN between all stages 0 and II. Finally, the differential susceptibility to tau inclusions among nuclear subdivisions was more notable in LC than in DRN.ConclusionsLC and DRN neurons exhibited ht-NCI during AD precortical stages. The ht-NCI increases along AD progression on both nuclei, but quantitative changes in LC precede DRN changes.

BackgroundImmunofluorescence (IF) plays a major role in quantifying protein expression in situ and understanding cell function. It is widely applied in assessing disease mechanisms and in drug discovery research. Automation of IF analysis can transform studies using experimental cell models. However, IF analysis of postmortem human tissue relies mostly on manual interaction, often subjected to low-throughput and prone to error, leading to low inter and intra-observer reproducibility. Human postmortem brain samples challenges neuroscientists because of the high level of autofluorescence caused by accumulation of lipofuscin pigment during aging, hindering systematic analyses. We propose a method for automating cell counting and classification in IF microscopy of human postmortem brains. Our algorithm speeds up the quantification task while improving reproducibility.New methodDictionary learning and sparse coding allow for constructing improved cell representations using IF images. These models are input for detection and segmentation methods. Classification occurs by means of color distances between cells and a learned set.ResultsOur method successfully detected and classified cells in 49 human brain images. We evaluated our results regarding true positive, false positive, false negative, precision, recall, false positive rate and F1 score metrics. We also measured user-experience and time saved compared to manual countings.Comparison with existing methodsWe compared our results to four open-access IF-based cell-counting tools available in the literature. Our method showed improved accuracy for all data samples.ConclusionThe proposed method satisfactorily detects and classifies cells from human postmortem brain IF images, with potential to be generalized for applications in other counting tasks.

IntroductionAlzheimer's disease (AD) progression follows a specific spreading pattern, emphasizing the need to characterize those brain areas that degenerate first. The brainstem's locus coeruleus (LC) is the first area to develop neurofibrillary changes (neurofibrillary tangles [NFTs]).MethodsThe methods include unbiased stereological analyses in human brainstems to estimate LC volume and neuronal population in controls and individuals across all AD stages.ResultsAs the Braak stage increases by 1 unit, the LC volume decreases by 8.4%. Neuronal loss started only midway through AD progression. Age-related changes spare the LC.DiscussionThe long gap between NFT accumulation and neuronal loss suggests that a second trigger may be necessary to induce neuronal death in AD. Imaging studies should determine whether LC volumetry can replicate the stage-wise atrophy observed here and how these changes are specific to AD. LC volumetry may develop into a screening biomarker for selecting high-yield candidates to undergo expensive and less accessible positron emission tomography scans and to monitor AD progression from presymptomatic stages.

Pharmacological interventions in Alzheimer's disease (AD) are likely to be more efficacious if administered early in the course of the disease, foregoing the spread of irreversible changes in the brain. Research findings underline an early vulnerability of the isodendritic core (IC) network to AD neurofibrillary lesions. The IC constitutes a phylogenetically conserved subcortical system including the locus coeruleus in pons, dorsal raphe nucleus, and substantia nigra in the midbrain, and nucleus basalis of Meynert in basal forebrain. Through their ascending projections to the cortex, the IC neurons regulate homeostasis and behavior by synthesizing aminergic and cholinergic neurotransmitters. Here we reviewed the evidence demonstrating that neurons of the IC system show neurofibrillary tangles in the earliest stages of AD, prior to cortical pathology, and how this involvement may explain pre-amnestic symptoms, including depression, agitation, and sleep disturbances in AD patients. In fact, clinical and animal studies show a significant reduction of AD cognitive and behavioral symptoms following replenishment of neurotransmitters associated with the IC network. Therefore, the IC network represents a unique candidate for viable therapeutic intervention and should become a high priority for research in AD.

Despite a massive research effort to elucidate Alzheimers disease (AD) in recent decades, effective treatment remains elusive. This failure may relate to an oversimplification of the pathogenic processes underlying AD and also lack of understanding of AD progression during its long latent stages. Although evidence shows that the two specific neuropathological hallmarks in AD (neuronal loss and protein accumulation), which are opposite in nature, do not progress in parallel, the great majority of studies have focused on only one of these aspects. Furthermore, research focusing on single structures is likely to render an incomplete picture of AD pathogenesis because as AD involves complete brain networks, potential compensatory mechanisms within the network may ameliorate impairment of the system to a certain extent. Here, we describe an approach for enabling integrative analysis of the dual-nature lesions, simultaneously, in all components of one of the brain networks most vulnerable to AD. This approach is based on significant development of methods previously described mainly by our group that were optimized and complemented for this study. It combines unbiased stereology with immunohistochemistry and immunofluorescence, making use of advanced graphics computing for three-dimensional (3D) volume reconstructions. Although this study was performed in human brainstem and focused in AD, it may be applied to the study of any neurological disease characterized by dual-nature lesions, in humans and animal models. This approach does not require a high level of investment in new equipment and a significant number of specimens can be processed and analyzed within a funding cycle.

Post-translational modifications play a key role in tau protein aggregation and related neurodegeneration. Because hyperphosphorylation alone does not necessarily cause tau aggregation, other post-translational modifications have been recently explored. Tau acetylation promotes aggregation and inhibits tau's ability to stabilize microtubules. Recent studies have shown co-localization of acetylated and phosphorylated tau in AD and some 4R tauopathies. We developed a novel monoclonal antibody against acetylated tau at lysine residue 274, which recognizes both 3R and 4R tau, and used immunohistochemistry and immunofluorescence to probe 22 cases, including AD and another eight familial or sporadic tauopathies. Acetylated tau was identified in all tauopathies except argyrophilic grain disease (AGD). AGD is an age-associated, common but atypical 4R tauopathy, not always associated with clinical progression. Pathologically, AGD is characterized by neuropil grains, pre-neurofibrillary tangles, and oligodendroglial coiled bodies, all recognized by phospho-tau antibodies. The lack of acetylated tau in these inclusions suggests that AGD represents a distinctive tauopathy. Our data converge with previous findings to raise the hypothesis that AGD could play a protective role against the spread of AD-related tau pathology. Tau acetylation as a key modification for the propagation tau toxicity deserves further investigation.

Background: Melanin‐concentrating hormone neurons (MCH) are a key component of the subcortical brain circuits controlling sleep and wake states, and integrating them with other vital functions like motivated and feeding behavior into a 24‐hour circadian rhythm. Together with orexinergic neurons (OX), they represent the major neuronal population of the lateral hypothalamic area (LHA). Our group previously reported a profound degeneration of OX neurons in Alzheimer's disease (AD), and a reduction of OX production in progressive supranuclear palsy (PSP). In this report we provide for the first time a comprehensive and quantitative morphological study of MCH neurons in patients with AD, PSP and normal controls. Method: Post‐mortem human brain tissue of subjects with AD, PSP and controls was examined using unbiased stereology. Double immunohistochemistry with MCH‐ and tau‐antibodies on formalin‐fixed, celloidin embedded tissue was performed. Result: There was no significant difference in the total number of MCH neurons between AD, PSP and controls, but a significant loss of non‐MCH neurons in AD patients (p=0.019). The proportion of MCH neurons was significantly higher in AD (p=0.0047). No such a difference was found in PSP. In PSP, but not AD, the proportion of tau+ MCH neurons was lower than the proportion of tau+ non‐MCH neurons (p=0.002). When comparing AD to PSP, the proportion of tau+MCH neurons was higher in AD (p<0.001). Conclusion: High burden of tau‐inclusions, but comparably milder loss of MCH neurons in AD, together with previously reported orexinergic neuronal loss may lead to a hyperexcitability of the MCH system in AD. This may contribute to the well‐known impairment of vigilance, reduction of NREM sleep and REM sleep disruption in AD. In contrast, we found virtually no involvement of MCH neurons in PSP, which reflects a specific vulnerability of these neurons to AD pathology. Clinically, PSP features a hyperarousal state, indicating that the activity of the arousal system prevails over a deficient sleep promoting system. We conclude that the involvement of other sleep promoting nuclei contributes to the insomnia in PSP. Further experimental research is needed to understand the molecular pathways involved in the specific vulnerability of MCH neurons in AD. [ABSTRACT FROM AUTHOR]

Background: Tau truncation by active caspase‐6 facilitates tau pathogenesis by producing toxic fragments prone to self‐aggregation. We previously demonstrated that neuronal active caspase‐6 co‐occurs with phosphorylated tau (p‐tau) during the earliest stages of Alzheimer's disease (AD) and that the percentage of neurons positive for both active caspase‐6 and p‐tau increases as the disease progresses. Although caspase‐6‐mediated cleavage of tau appears to be a key contributor to tau pathology, questions concerning the relationship between caspase‐6 activation, tau cleavage, and the formation of p‐tau aggregates remain. Methods: We developed novel neoepitope antibodies against tau truncated by caspase‐6, performed 5‐plex immunofluorescence on tissue microarrays—sampled from the middle frontal gyrus (MFG) and the inferior temporal gyrus (ITG)—and quantified neuronal and glial positivity for active caspase‐6, tau truncated at D402 and D13 (tr‐tau; C‐ and N‐terminal, respectively), and tau phosphorylated at Ser202 (p‐tau; CP13) in AD, argyrophilic grain disease (AGD), corticobasal degeneration (CBD), Pick's disease (PiD), progressive supranuclear palsy (PSP), and healthy aging controls. Results: Percentages of neurons positive for p‐tau were as to be expected in all 17 cases. AD showed the highest burden of neuronal positivity for active caspase‐6, followed by PiD (Figure 1, Table 1). Mean percentages of neurons positive for D402 and D13 were markedly higher (1.80 to 236x) in AD and PiD relative to the 4‐repeat tauopathies tested (Figure 1, Table 1). Surprisingly, in AD, only ∼44.36% of these neurons showed p‐tau positivity (Figure 2). Regarding glial positivity, active caspase‐6 and tr‐tau were present in PSP but scarce in CBD (Figure 3, Table 2). Conclusions: (1) Caspase‐6 activation and cleavage of tau is more prominent in AD and, to a lesser extent, PiD. Caspase‐6 modulation could be a promising therapeutic for AD, and possibly PiD, but not 4‐repeat tauopathies. (2) In AD, there is a substantial number of neurons with tr‐tau inclusions that lack p‐tau, which suggests that biofluid biomarkers of tr‐tau (specifically at N‐terminal sites) would allow for better detection of AD and differentiation of AD from 4‐repeat tauopathies. [ABSTRACT FROM AUTHOR]

Background: Tau post‐translational modifications (PTMs) are associated with progressive tau accumulation and neuronal loss in tauopathies, including forms of frontotemporal lobar degeneration (FTLD) and Alzheimer's disease (AD). Proteolytic cleavage of tau by active caspases, including caspase‐6, represents an underexplored tau PTM implicated in tau pathology by generating tau species with an enhanced propensity for self‐aggregation and accumulation into neuronal cytoplasmic inclusions. Method: To elucidate the presence and temporal course of caspase activation, tau cleavage, and neuronal death, we generated two neoepitope monoclonal antibodies (mAbs) against caspase‐6 tau proteolytic sites (D13 and D402) and cortical neurons (iNs) from induced pluripotent stem cells (iPSCs) with the frontotemporal dementia (FTD)‐causing V337M MAPT mutation (tauV337M). Result: FTLD tauV337M and AD postmortem brains showed positivity for both cleaved tau mAbs as well as active caspase‐6 (Fig. 1). Three month tauV337M iNs showed a three‐fold increase in caspase‐cleaved D402 and D13 tau protein levels and increased p‐tau and oligomeric tau species compared to controls (Fig. 2). Three month tauV337M iNs also showed a significantly higher rate of neuronal death than the tauWT counterpart. Treatment with staurosporine (STS), a stress inducer, led to a 5‐fold increase of cytotoxicity levels in STS‐treated tauV337M iNs compared to vehicle‐treated tauV337M iNs. Although STS‐induced cytotoxicity was anticipated, the increase was significantly higher in STS‐treated tauV337M iNs than in STS‐treated control iNs. Similarly, we observed a 4‐fold increase of caspase‐6 activity in STS‐treated tauV337M iNs compared to vehicle‐treated tauV337M iNs that was reversed following co‐treatment with the caspase‐6 inhibitor z‐VEID‐fmk (Fig. 2). Conclusion: We propose a model in which time‐dependent accumulation of caspase‐cleaved tau in V337M MAPT neurons promotes neurotoxicity that is reversed by caspase‐6 inhibition. Caspase‐cleaved tau may be a biomarker of tauopathy, and caspases could be viable targets for therapeutic intervention against tau pathogenesis in FTLD and other tauopathies. [ABSTRACT FROM AUTHOR]

Background: Caspases are being explored as targets for pharmacological treatment of tauopathies due to their role in tau cleavage, which leads to tau aggregation and neuronal loss. Tau truncated at Asp421, a residue cleaved by effector caspases, is found in four common tauopathies: Alzheimer's disease (AD), corticobasal degeneration (CBD), Pick's disease (PiD), and progressive supranuclear palsy (PSP) (Newman et al., 2005). We previously demonstrated that active caspase‐6 colocalizes with neurons containing phosphorylated tau during early AD stages and that the number of neurons positive for both active caspase‐6 and p‐tau increases throughout AD's progression. However, how caspase‐6 activity differs between tauopathies involving different tau isoforms is unknown. This study compares caspase‐6 activation and caspase‐6 cleavage of tau between AD, argyrophilic grain disease (AGD), CBD, PiD, and PSP using novel neoepitope antibodies against caspase‐6‐cleaved tau. Method: Using multiplex immunofluorescence on tissue microarrays, we measured levels of neuronal active caspase‐6, tau cleaved at C‐terminal residue D402 (D402), tau cleaved at N‐terminal residue D13 (D13), and tau phosphorylated at Ser202 (p‐tau; CP13) in the middle frontal gyrus (MFG) of well‐characterized AD, AGD, CBD, PiD, and PSP cases and controls. We used semi‐automated methods to calculate the percentage of neurons positive for each marker. Result: We found similar percentages of neuronal p‐tau in every tauopathy except AGD, as expected. Only AD and PiD showed substantial percentages of neuronal caspase‐6‐cleaved tau (Figure 1, Table 1). The mean percentages of neuronal D402 and D13 were up to 121.88‐fold higher in AD and up to 16.75‐fold higher in PiD compared to the other tauopathies (Figure 1, Table 1). In PiD, on average, 79.82‐87.61% of neurons positive for D402 or D13 were also positive for p‐tau. Interestingly, in AD, only an average of 50.70‐58.98% of neurons positive for D402 or D13 showed p‐tau positivity (Figure 2). Conclusion: 1) Caspase‐6‐cleavage of tau appears to be a more defining feature of tauopathies involving 3‐repeat isoforms of tau than 4‐repeat tauopathies. 2) Caspase‐6‐cleaved tau inclusions do not necessarily colocalize with p‐tau inclusions. 3) Future research on the therapeutic potential of caspase modulators should focus on AD and PiD. [ABSTRACT FROM AUTHOR]

Background: Sleep disorders represent one of the leading causes of life impairment and a major therapeutic challenge in patients with Alzheimer's disease (AD) and tauopathies. However, the disease specific neurobiological basis of these sleep disturbances remains mostly elusive. Melanin‐concentrating hormone (MCH) neurons are a conserved neuronal population in the lateral and posterior hypothalamus with critical sleep/wake and metabolic control functions in animals, but largely underexplored in humans. Here, we used unbiased stereology to examine the involvement of MCH neurons in Alzheimer's disease (AD) as a 3R‐tauopathy, and progressive supranuclear palsy (PSP) as a 4R‐tauopathy featuring hyperinsomnia. Method: We used unbiased stereology to quantify total and MCH‐positive neuronal numbers and tau burden in the region of interest (ROI) of 5 AD and 6 PSP brains (for detailed demographics see Table 1). Data were analyzed using Wilcoxon signed rank and Mann‐Whitney U tests. Result: Both AD and PSP subjects show significant neuronal tau‐burden in the ROI. There was no statistically significant difference in the absolute number of MCH‐neurons and non‐MCH‐neurons (Figure 1), or proportion of MCH neurons in the total neuronal population between AD (26.5%) and PSP (24.9%). Whereas there was no difference in the proportion of tau+ non‐MCH neurons between AD and PSP, the proportion of tau+ MCH‐neurons was significantly higher in AD relative to PSP (p=0.0043) (Figure 2). Contrary to AD, where MCH and non‐MCH neurons had similar tau‐burden (18.8% and 17.1%, respectively), in PSP there was a significantly lower involvement of MCH‐neurons (4.9%) in comparison to the total neuronal population (19.9%, p=0.0313) (Figure 2). In the PSP group there was a positive correlation between Braak stages and the proportion of tau+ MCH neurons (p=0.023), but not total tau+ neurons. Conclusion: Our preliminary data show a trend for lower neuronal numbers in AD and in PSP, lower neuronal numbers correlated with higher Braak stages. Together, it suggest an early vulnerability of MHC to AD and a moderate vulnerability to PSP. We are analyzing further cases to increase the power of analysis. Different involvement of MCH‐neurons in AD and PSP may reflect the specific patterns of sleep disorders in those tauopathies. [ABSTRACT FROM AUTHOR]

Abstract: Purpose: Intracerebral delivery of anti-epileptic compounds represents a novel strategy for the treatment of refractory epilepsy. Adenosine is a possible candidate for local delivery based on its proven anti-epileptic effects. Neural stem cells constitute an ideal cell source for intracerebral transplantation and long-term drug delivery. In order to develop a cell-based system for the long-term delivery of adenosine, we isolated neural progenitor cells from adenosine kinase deficient mice (Adk −/−) and compared their differentiation potential and adenosine release properties with corresponding wild-type cells. Methods: Fetal neural progenitor cells were isolated from the brains of Adk −/− and C57BL/6 mice fetuses and expanded in vitro. Before and after neural differentiation, supernatants were collected and assayed for adenosine release using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Results: Adk −/− cells secreted significantly more adenosine compared to wild-type cells at any time point of differentiation. Undifferentiated Adk −/− cells secreted 137±5ng adenosine per 105 cells during 24h in culture, compared to 11±1ng released from corresponding wild-type cells. Adenosine release was maintained after differentiation as differentiated Adk −/− cells continued to release significantly more adenosine per 24h (47±1ng per 105 cells) compared to wild-type cells (3±0.2ng per 105 cells). Conclusions: Fetal neural progenitor cells isolated from Adk −/− mice – but not those from C57BL/6 mice – release amounts of adenosine considered to be of therapeutic relevance. [ABSTRACT FROM AUTHOR]

Background: Tau post-translational modifications (PTMs) result in the gradual build-up of abnormal tau and neuronal degeneration in tauopathies, encompassing variants of frontotemporal lobar degeneration (FTLD) and Alzheimer's disease (AD). Tau proteolytically cleaved by active caspases, including caspase-6, may be neurotoxic and prone to self-aggregation. Also, our recent findings show that caspase-6 truncated tau represents a frequent and understudied aspect of tau pathology in AD in addition to phospho-tau pathology. In AD and Pick's disease, a large percentage of caspase-6 associated cleaved-tau positive neurons lack phospho-tau, suggesting that many vulnerable neurons to tau pathology go undetected when using conventional phospho-tau antibodies and possibly will not respond to phospho-tau based therapies. Therefore, therapeutic strategies against caspase cleaved-tau pathology could be necessary to modulate the extent of tau abnormalities in AD and other tauopathies., Methods: To understand the timing and progression of caspase activation, tau cleavage, and neuronal death, we created two mAbs targeting caspase-6 tau cleavage sites and probed postmortem brain tissue from an individual with FTLD due to the V337M MAPT mutation. We then assessed tau cleavage and apoptotic stress response in cortical neurons derived from induced pluripotent stem cells (iPSCs) carrying the FTD-related V337M MAPT mutation. Finally, we evaluated the neuroprotective effects of caspase inhibitors in these iPSC-derived neurons., Results: FTLD V337M MAPT postmortem brain showed positivity for both cleaved tau mAbs and active caspase-6. Relative to isogenic wild-type MAPT controls, V337M MAPT neurons cultured for 3 months post-differentiation showed a time-dependent increase in pathogenic tau in the form of caspase-cleaved tau, phospho-tau, and higher levels of tau oligomers. Accumulation of toxic tau species in V337M MAPT neurons was correlated with increased vulnerability to pro-apoptotic stress. Notably, this mutation-associated cell death was pharmacologically rescued by the inhibition of effector caspases., Conclusions: Our results suggest an upstream, time-dependent accumulation of caspase-6 cleaved tau in V337M MAPT neurons promoting neurotoxicity. These processes can be reversed by caspase inhibition. These results underscore the potential of developing caspase-6 inhibitors as therapeutic agents for FTLD and other tauopathies. Additionally, they highlight the promise of using caspase-cleaved tau as biomarkers for these conditions., Competing Interests: Declaration of competing interest Michelle R. Arkin is cofounder of Elgia Therapeutics, which is developing caspase-6 inhibitors for inflammatory diseases. The other authors have declared no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The SORL1 gene (SORLA) is strongly associated with risk of developing Alzheimer's disease (AD). SORLA is a regulator of endosomal trafficking in neurons and interacts with retromer, a complex that is a "master conductor" of endosomal trafficking. Small molecules can increase retromer expression in vitro, enhancing its function. We treated hiPSC-derived cortical neurons that are either fully deficient, haploinsufficient, or that harbor one copy of SORL1 variants linked to AD with TPT-260, a retromer-enhancing molecule. We show significant increases in retromer subunit VPS26B expression. We tested whether endosomal, amyloid, and TAU pathologies were corrected. We observed that the degree of rescue by TPT-260 treatment depended on the number of copies of functional SORL1 and which SORL1 variant was expressed. Using a disease-relevant preclinical model, our work illuminates how the SORL1-retromer pathway can be therapeutically harnessed., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Clarifying the mechanisms connecting neurofibrillary tangle (NFT) neurotoxicity to neuronal dysfunction in humans is likely to be pivotal for developing effective treatments for Alzheimer's disease (AD). To model the temporal progression of AD in humans, we used a collection of brains with controls and individuals from each Braak stage to quantitatively investigate the correlation between intraneuronal caspase activation or macroautophagy markers, NFT burden, and neuronal loss, in the dorsal raphe nucleus and locus coeruleus, the earliest vulnerable areas to NFT accumulation. We fit linear regressions with each count as outcomes, with Braak score and age as the predictors. In progressive Braak stages, intraneuronal active caspase-6 positivity increases both alone and overlapping with NFTs. Likewise, the proportion of NFT-bearing neurons showing autophagosomes increases. Overall, caspases may be involved in upstream cascades in AD and are associated with higher NFTs. Macroautophagy changes correlate with increasing NFT burden from early AD stages., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Connexin 43 (Cx43) is the main astrocytic connexin and forms the basis of the glial syncytium. The morphology of connexin-expressing cells can be best studied in transgenic mouse lines expressing cytoplasmic fluorescent reporters, since immunolabeling the plaques can obscure the shapes of the individual cells. The Cx43kiECFP mouse generated by Degen et al. (FASEBJ 26:4576, 2012) expresses cytosolic ECFP and has previously been used to establish that Cx43 may not be expressed by all astrocytes within a population, and this can vary in a region-dependent way. To establish this mouse line as a tool for future astrocyte and connexin research, we sought to consolidate reporter authenticity, studying cell types and within-region population heterogeneity. Applying anti-GFP, all cell types related to astroglia were positive-namely, protoplasmic astrocytes in the hippocampus, cortex, thalamus, spinal cord, olfactory bulb, cerebellum with Bergmann glia and astrocytes also in the molecular layer, and retinal Müller cells and astrocytes. Labeled cell types further comprise white matter astrocytes, olfactory ensheathing cells, radial glia-like stem cells, retinal pigment epithelium cells, ependymal cells, and meningeal cells. We furthermore describe a retinal Cx43-expressing amacrine cell morphologically reminiscent of ON-OFF wide-field amacrine cells, representing the first example of a mammalian CNS neuron-expressing Cx43 protein. In double staining with cell type-specific markers (GFAP, S100ß, glutamine synthetase), Cx43 reporter expression in the hippocampus and cortex was restricted to GFAP + astrocytes. Altogether, this mouse line is a highly reliable tool for studies of Cx43-expressing CNS cells and astroglial cell morphology. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Background: Immunofluorescence (IF) plays a major role in quantifying protein expression in situ and understanding cell function. It is widely applied in assessing disease mechanisms and in drug discovery research. Automation of IF analysis can transform studies using experimental cell models. However, IF analysis of postmortem human tissue relies mostly on manual interaction, often subjected to low-throughput and prone to error, leading to low inter and intra-observer reproducibility. Human postmortem brain samples challenges neuroscientists because of the high level of autofluorescence caused by accumulation of lipofuscin pigment during aging, hindering systematic analyses. We propose a method for automating cell counting and classification in IF microscopy of human postmortem brains. Our algorithm speeds up the quantification task while improving reproducibility., New Method: Dictionary learning and sparse coding allow for constructing improved cell representations using IF images. These models are input for detection and segmentation methods. Classification occurs by means of color distances between cells and a learned set., Results: Our method successfully detected and classified cells in 49 human brain images. We evaluated our results regarding true positive, false positive, false negative, precision, recall, false positive rate and F1 score metrics. We also measured user-experience and time saved compared to manual countings., Comparison With Existing Methods: We compared our results to four open-access IF-based cell-counting tools available in the literature. Our method showed improved accuracy for all data samples., Conclusion: The proposed method satisfactorily detects and classifies cells from human postmortem brain IF images, with potential to be generalized for applications in other counting tasks., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Despite a massive research effort to elucidate Alzheimer's disease (AD) in recent decades, effective treatment remains elusive. This failure may relate to an oversimplification of the pathogenic processes underlying AD and also lack of understanding of AD progression during its long latent stages. Although evidence shows that the two specific neuropathological hallmarks in AD (neuronal loss and protein accumulation), which are opposite in nature, do not progress in parallel, the great majority of studies have focused on only one of these aspects. Furthermore, research focusing on single structures is likely to render an incomplete picture of AD pathogenesis because as AD involves complete brain networks, potential compensatory mechanisms within the network may ameliorate impairment of the system to a certain extent. Here, we describe an approach for enabling integrative analysis of the dual-nature lesions, simultaneously, in all components of one of the brain networks most vulnerable to AD. This approach is based on significant development of methods previously described mainly by our group that were optimized and complemented for this study. It combines unbiased stereology with immunohistochemistry and immunofluorescence, making use of advanced graphics computing for three-dimensional (3D) volume reconstructions. Although this study was performed in human brainstem and focused in AD, it may be applied to the study of any neurological disease characterized by dual-nature lesions, in humans and animal models. This approach does not require a high level of investment in new equipment and a significant number of specimens can be processed and analyzed within a funding cycle., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Extracellular adenosine, a key regulator of neuronal excitability, is metabolized by astrocyte-based enzyme adenosine kinase (ADK). We hypothesized that ADK might be an upstream regulator of adenosine-based homeostatic brain functions by simultaneously affecting several downstream pathways. We therefore studied the relationship between ADK expression, levels of extracellular adenosine, synaptic transmission, intrinsic excitability, and brain-derived neurotrophic factor (BDNF)-dependent synaptic actions in transgenic mice underexpressing or overexpressing ADK. We demonstrate that ADK: 1) Critically influences the basal tone of adenosine, evaluated by microelectrode adenosine biosensors, and its release following stimulation; 2) determines the degree of tonic adenosine-dependent synaptic inhibition, which correlates with differential plasticity at hippocampal synapses with low release probability; 3) modulates the age-dependent effects of BDNF on hippocampal synaptic transmission, an action dependent upon co-activation of adenosine A2A receptors; and 4) influences GABAA receptor-mediated currents in CA3 pyramidal neurons. We conclude that ADK provides important upstream regulation of adenosine-based homeostatic function of the brain and that this mechanism is necessary and permissive to synaptic actions of adenosine acting on multiple pathways. These mechanistic studies support previous therapeutic studies and implicate ADK as a promising therapeutic target for upstream control of multiple neuronal signaling pathways crucial for a variety of neurological disorders.

MicroRNAs are small non-coding RNAs that regulate post-transcriptional gene expression. In the short time since the discovery of microRNAs, the literature has burgeoned with studies focused on the biosynthesis of microRNAs, target prediction and binding, and mechanisms of translational repression by microRNAs. Given the prominent role of microRNAs in all areas of cell biology, it is not surprising that microRNAs are also linked to human diseases, including those of the nervous system. One of the least-studied areas of microRNA research is how their expression is regulated outside of development and cancer. Thus, we examined a role for regulation of microRNAs by neurotransmitter receptor activation in mouse brain. We focused on the group I metabotropic glutamate receptors by using intracerebroventricular injection of the selective agonist, (S)-3,5-dihydroxyphenylglycine (DHPG) in mouse brain. We then examined the expression of microRNAs in the cerebral cortex by Ambion and Invitrogen microarrays, and the expression of mature microRNA sequences by SABiosciences qPCR arrays, at 4, 8 and 24 h after DHPG injection. These studies revealed that the largest number of significantly regulated microRNAs was detected 8h after DHPG injection in the microarrays and qPCR arrays. We then used RNA blots to quantify microRNA expression, and in situ hybridization to examine cellular distribution of the microRNAs regulated by DHPG. Bioinformatic analysis of the microRNAs regulated 8 h after DHPG in all three arrays revealed KEGG pathways that are known to correlate with group I mGluR effects, as well as recently described and novel pathways. These studies are the first to show that DHGP regulates the expression of microRNAs in mouse cerebral cortex, and support the hypothesis that group I mGluRs may regulate microRNA expression in mouse brain., (Copyright © 2012 Elsevier Inc. All rights reserved.)

A ketogenic diet (KD) is a high-fat, low-carbohydrate metabolic regimen; its effectiveness in the treatment of refractory epilepsy suggests that the mechanisms underlying its anticonvulsive effects differ from those targeted by conventional antiepileptic drugs. Recently, KD and analogous metabolic strategies have shown therapeutic promise in other neurologic disorders, such as reducing brain injury, pain, and inflammation. Here, we have shown that KD can reduce seizures in mice by increasing activation of adenosine A1 receptors (A1Rs). When transgenic mice with spontaneous seizures caused by deficiency in adenosine metabolism or signaling were fed KD, seizures were nearly abolished if mice had intact A1Rs, were reduced if mice expressed reduced A1Rs, and were unaltered if mice lacked A1Rs. Seizures were restored by injecting either glucose (metabolic reversal) or an A1R antagonist (pharmacologic reversal). Western blot analysis demonstrated that the KD reduced adenosine kinase, the major adenosine-metabolizing enzyme. Importantly, hippocampal tissue resected from patients with medically intractable epilepsy demonstrated increased adenosine kinase. We therefore conclude that adenosine deficiency may be relevant to human epilepsy and that KD can reduce seizures by increasing A1R-mediated inhibition.

Purpose: Given the high incidence of refractory epilepsy, novel therapeutic approaches and concepts are urgently needed. To date, viral-mediated delivery and endogenous expression of antisense sequences as a strategy to prevent seizures have received little attention in epilepsy therapy development efforts. Here we validate adenosine kinase (ADK), the astrocyte-based key negative regulator of the brain's endogenous anticonvulsant adenosine, as a potential therapeutic target for antisense-mediated seizure suppression., Methods: We developed adenoassociated virus 8 (AAV8)-based gene therapy vectors to selectively modulate ADK expression in astrocytes. Cell type selectivity was achieved by expressing an Adk-cDNA in sense or antisense orientation under the control of an astrocyte-specific gfaABC1D promoter. Viral vectors where injected into the CA3 of wild-type mice or spontaneously epileptic Adk-tg transgenic mice that overexpress ADK in brain. After virus injection, ADK expression was assessed histologically and biochemically. In addition, intracranial electroencephalography (EEG) recordings were obtained., Key Findings: We demonstrate in wild-type mice that viral overexpression of ADK within astrocytes is sufficient to trigger spontaneous recurrent seizures in the absence of any other epileptogenic event, whereas ADK downregulation via AAV8-mediated RNA interference almost completely abolished spontaneous recurrent seizures in Adk-tg mice., Significance: Our data demonstrate that modulation of astrocytic ADK expression can trigger or prevent seizures, respectively. This is the first study to use an antisense approach to validate ADK as a rational therapeutic target for the treatment of epilepsy and suggests that gene therapies based on the knock down of ADK might be a feasible approach to control seizures in refractory epilepsy., (Wiley Periodicals, Inc. © 2011 International League Against Epilepsy.)

Studies examining the effects of hippocampal lesions on object recognition memory in rats have produced conflicting results. The present study investigated how methodological differences and lesion size may have contributed to these discrepancies. In Experiment 1 we compared rats with complete, partial (septal) and sham hippocampal lesions on a spontaneous object recognition task, using a protocol previously reported to result in deficits following large hippocampal lesions . Rats with complete and partial hippocampal lesions were unimpaired, suggesting the hippocampus is not required for object recognition memory. However, rats with partial lesions showed relatively poor performance raising the possibility that floor effects masked a deficit on this group. In Experiment 2, we used a second spontaneous object recognition protocol similar to that used by the two other studies that have reported deficits following hippocampal lesions . Rats with complete hippocampal lesions were significantly impaired, whereas rats with partial lesions were unimpaired. However, the complete lesion group showed less object exploration during the sample phase. Thus, the apparent recognition memory deficit in Experiment 2 may be attributable to differential encoding. Together, these findings suggest that the hippocampus is not required for intact spontaneous object recognition memory. These findings suggest that levels of object exploration during the sample phase may be a critical issue, and raise the possibility that previous reports of object recognition deficits may be due to differences in object exploration rather than deficits in object recognition per se.