Extracellular Prion Protein Aggregates in Nine Gerstmann–Sträussler–Scheinker Syndrome Subjects with Mutation P102L: A Micromorphological Study and Comparison with Literature Data

Gerstmann–Sträussler–Scheinker syndrome (GSS) is a hereditary neurodegenerative disease characterized by extracellular aggregations of pathological prion protein (PrP) forming characteristic plaques. Our study aimed to evaluate the micromorphology and protein composition of these plaques in relation to age, disease duration, and co-expression of other pathogenic proteins related to other neurodegenerations. Hippocampal regions of nine clinically, neuropathologically, and genetically confirmed GSS subjects were investigated using immunohistochemistry and multichannel confocal fluorescent microscopy. Most pathognomic prion protein plaques were small (2–10 µm), condensed, globous, and did not contain any of the other investigated proteinaceous components, particularly dystrophic neurites. Equally rare (in two cases out of nine) were plaques over 50 µm having predominantly fibrillar structure and exhibit the presence of dystrophic neuritic structures; in one case, the plaques also included bulbous dystrophic neurites. Co-expression with hyperphosphorylated protein tau protein or amyloid beta-peptide (Aβ) in GSS PrP plaques is generally a rare observation, even in cases with comorbid neuropathology. The dominant picture of the GSS brain is small, condensed plaques, often multicentric, while presence of dystrophic neuritic changes accumulating hyperphosphorylated protein tau or Aβ in the PrP plaques are rare and, thus, their presence probably constitutes a trivial observation without any relationship to GSS development and progression.

Improved modeling of human AD with an automated culturing platform for iPSC neurons, astrocytes and microglia

Advancement in human induced pluripotent stem cell (iPSC) neuron and microglial differentiation protocols allow for disease modeling using physiologically relevant cells. However, iPSC differentiation and culturing protocols have posed challenges to maintaining consistency. Here, we generated an automated, consistent, and long-term culturing platform of human iPSC neurons, astrocytes, and microglia. Using this platform we generated a iPSC AD model using human derived cells, which showed signs of Aβ plaques, dystrophic neurites around plaques, synapse loss, dendrite retraction, axon fragmentation, phospho-Tau induction, and neuronal cell death in one model. We showed that the human iPSC microglia internalized and compacted Aβ to generate and surround the plaques, thereby conferring some neuroprotection. We investigated the mechanism of action of anti-Aβ antibodies protection and found that they protected neurons from these pathologies and were most effective before pTau induction. Taken together, these results suggest that this model can facilitate target discovery and drug development efforts.

Accumulation of saposin in dystrophic neurites is linked to impaired lysosomal functions in Alzheimer’s disease brains

Neuritic plaques in Alzheimer’s disease (AD) brains refer to β-amyloid (Aβ) plaques surrounded by dystrophic neurites (DNs), activated microglia and reactive astrocytes. Most recently, we showed that DNs form sequentially in three layers during plaque growth. Although lysosomal proteins such as LAMP1 are found in DNs, it is not clear how many and how early lysosomal proteins are involved in forming neuritic plaques. To answer this unmet question, we examined APP knock-in (APPNL-G-F), 5xFAD and APP/PS1ΔE9 mouse brains and found that the lysosomal activator proteins saposins (SAPs) and LAMP1 were accumulated to surround Aβ plaques at the earliest stage, namely the 1st layer of DNs. Noticeably, lysosomal hydrolases were not detectable in these early DNs, suggesting that DNs at this early stage likely enrich dysfunctional lysosomes. In old AD mouse brains and in the later stage of human AD brains, SAP-C+-DNs and LAMP1+-DNs were gradually reduced in concomitant with the growth of amyloid plaques. Remarkably, the observed LAMP1 immunoreactivity near plaques in aged AD mouse and human brains were actually associated with disease-associated microglia rather than neuronal sources, likely reflecting more severely impaired lysosomal functions in neurons. Western blot analyses showed increased levels of SAP-C in AD mouse brains, and Aβ oligomers induced elevated levels of SAP-C in cellular assays. The elevated protein levels of SAP-C in AD mouse brains during plaque growth potentially contributed lysosomal membrane leakage and loss of hydrolases. Together, our study indicates that lysosomal functions are impaired by being entrapped in DNs early during plaque growth, and this may viciously facilitate growth of amyloid plaques.

Lysosomal proteolysis of amyloid beta is impeded by fibrils grown in both acidic and neutral pH environments

Aggregation of amyloid-beta (A) into extracellular plaques is a well-known hallmark of Alzheimers disease (AD). Similarly, autophagic vacuoles, autophagosomes, and other residual bodies within dystrophic neurites, though more difficult to detect, are characteristic features of AD. To explore the potential intersection between these observations, we conducted experiments to assess whether A fibril formation disrupts lysosomal proteolysis. Fibrils constituted from either A 1-40 or A 1-42 were grown under both neutral and acidic pH. The extent of proteolysis by individual cathepsins (L, D, B, and H) was monitored by both thioflavin T fluorescence and liquid-chromatography combined with mass spectrometry. The results show that all A fibrils are resistant to cathepsin digestion, with significant amounts of undigested material remaining for samples of fibrils grown in both neutral and acidic pH. Further analysis revealed that the neutral-grown fibrils are proteolytically resistant throughout the sequence, while the acid-grown fibrils prevented digestion primarily in the C-terminal portion of the sequence. Fibrils grown from A 1-42 are generally more resistant to degradation compared to A 1-40. Overall, the results indicate that A fibrils formed in the neutral pH environments found in intracellular or extracellular spaces may pose the greatest difficulty for complete digestion by the lysosome, particularly when the fibrils are comprised of A 1-42.

Plaque associated microglia hyper-secrete extracellular vesicles and accelerate tau propagation in a humanized APP mouse model

Background Recent studies suggest that microglia contribute to tau pathology progression in Alzheimer’s disease. Amyloid plaque accumulation transforms microglia, the primary innate immune cells in the brain, into neurodegenerative microglia (MGnD), which exhibit enhanced phagocytosis of plaques, apoptotic neurons and dystrophic neurites containing aggregated and phosphorylated tau (p-tau). It remains unclear how microglia promote disease progression while actively phagocytosing pathological proteins, therefore ameliorating pathology. Methods Adeno-associated virus expressing P301L tau mutant (AAV-P301L-tau) was stereotaxically injected into the medial entorhinal cortex (MEC) in C57BL/6 (WT) and humanized APP mutant knock-in homozygote (AppNL-G-F) mice at 5 months of age. Mice were fed either chow containing a colony stimulating factor-1 receptor inhibitor (PLX5622) or control chow from 4 to 6 months of age to test the effect of microglia depletion. Animals were tested at 6 months of age for immunofluorescence, biochemistry, and FACS of microglia. In order to monitor microglial extracellular vesicle secretion in vivo, a novel lentiviral EV reporter system was engineered to express mEmerald-CD9 (mE-CD9) specifically in microglia, which was injected into the same region of MEC. Results Expressing P301L tau mutant in the MEC induced tau propagation to the granule cell layer of the hippocampal dentate gyrus, which was significantly exacerbated in AppNL-G-F mice compared to WT control mice. Administration of PLX5622 depleted nearly all microglia in mouse brains and dramatically reduced propagation of p-tau in WT and to a greater extent in AppNL-G-F mice, although it increased plaque burden and plaque-associated p-tau+ dystrophic neurites. Plaque-associated MGnD microglia strongly expressed an EV marker, tumor susceptibility gene 101, indicative of heightened synthesis of EVs. Intracortical injection of mE-CD9 lentivirus successfully induced microglia-specific expression of mE-CD9+ EV particles, which were significantly enhanced in Mac2+ MGnD microglia compared to Mac2− homeostatic microglia. Finally, consecutive intracortical injection of mE-CD9 lentivirus and AAV-P301L-tau into AppNL-G-F mice revealed encapsulation of p-tau in microglia-specific mE-CD9+ EVs as determined by super-resolution microscopy and immuno-electron microscopy. Discussion Our findings suggest that MGnD microglia hyper-secrete p-tau+ EVs while compacting Aβ plaques and clearing NP tau, which we propose as a novel mechanistic link between amyloid plaque deposition and exacerbation of tau propagation in AppNL-G-F mice.

Metformin attenuates plaque-associated tau pathology and reduces amyloid-β burden in APP/PS1 mice

Background The neuropathological hallmarks of Alzheimer’s disease (AD) are amyloid-β (Aβ) plaques and neurofibrillary tangles (NFTs). The amyloid cascade theory is the leading hypothesis of AD pathology. Aβ deposition precedes the aggregation of tau pathology and Aβ pathology precipitates tau pathology. Evidence also indicates the reciprocal interactions between amyloid and tau pathology. However, the detailed relationship between amyloid and tau pathology in AD remains elusive. Metformin might have a positive effect on cognitive impairments. However, whether metformin can reduce AD-related pathologies is still unconclusive. Methods Brain extracts containing tau aggregates were unilaterally injected into the hippocampus and the overlying cerebral cortex of 9-month-old APPswe/PS1DE9 (APP/PS1) mice and age-matched wild-type (WT) mice. Metformin was administrated in the drinking water for 2 months. Aβ pathology, tau pathology, plaque-associated microgliosis, and autophagy marker were analyzed by immunohistochemical staining and immunofluorescence analysis 2 months after injection of proteopathic tau seeds. The effects of metformin on both pathologies were explored. Results We observed tau aggregates in dystrophic neurites surrounding Aβ plaques (NP tau) in the bilateral hippocampi and cortices of tau-injected APP/PS1 mice but not WT mice. Aβ plaques promoted the aggregation of NP tau pathology. Injection of proteopathic tau seeds exacerbated Aβ deposits and decreased the number of microglia around Aβ plaques in the hippocampus and cortex of APP/PS1 mice. Metformin ameliorated the microglial autophagy impairment, increased the number of microglia around Aβ plaques, promoted the phagocytosis of NP tau, and reduced Aβ load and NP tau pathology in APP/PS1 mice. Conclusion These findings indicate the existence of the crosstalk between amyloid and NP tau pathology. Metformin promoted the phagocytosis of pathological Aβ and tau proteins by enhancing microglial autophagy capability. It reduced Aβ deposits and limited the spreading of NP tau pathology in APP/PS1 mice, which exerts a beneficial effect on both pathologies.

Different Morphology of Neuritic Plaques in the Archicortex of Alzheimer’s Disease with Comorbid Synucleinopathy: A Pilot Study

Background: Bulbous neuritic changes in neuritic plaques have already been described, and their possible effect on the clinical course of the disease has been discussed. OBJECTIVE: In our study, we focused on the location and density of these structures in patients with only Alzheimer’s disease (AD) and patients with AD in comorbidity with synucleinopathies. Methods: Utilizing immunohistochemistry and confocal microscopy, we evaluated differences of neocortical and archicortical neuritic plaques and the frequency of bulbous changes in the archicortex of 14 subjects with Alzheimer’s disease (AD), 10 subjects with the Lewy body variant of Alzheimer's disease (AD/DLB), and 4 subjects with Alzheimer's disease with amygdala Lewy bodies (AD/ALB). Also, the progression and density of neuritic changes over the time course of the disease were evaluated. Results: We found structural differences in bulbous dystrophic neurites more often in AD/DLB and AD/ALB than in pure AD cases. The bulbous neuritic changes were more prominent in the initial and progressive phases and were reduced in cases with a long clinical course. Conclusion: Our results indicate that there is a prominent difference in the shape and composition of neocortical and archicortical neuritic plaques and, moreover, that bulbous neuritic changes can be observed at a higher rate in AD/DLB and AD/ALB subjects compared to pure AD subjects. This observation probably reflects that these subacute changes are more easily seen in the faster clinical course of AD patients with comorbidities.