Frontotemporal dementia (FTD) encompasses a group of neurodegenerative disorders characterized by cognitive and behavioral impairments. Heterozygous mutations in progranulin (PGRN) cause familial FTD and result in decreased PGRN expression, while homozygous mutations result in complete loss of PGRN expression and lead to the neurodegenerative lysosomal storage disorder neuronal ceroid lipofuscinosis (NCL). However, how dose-dependent PGRN mutations contribute to these two different diseases is not well understood. Using iPSC-derived human cortical neurons from FTD patients harboring PGRN mutations, we demonstrate that PGRN mutant neurons exhibit decreased nuclear TDP-43 and increased insoluble TDP-43, as well as enlarged electron-dense vesicles, lipofuscin accumulation, fingerprint-like profiles and granular osmiophilic deposits, suggesting that both FTD and NCL-like pathology are present in PGRN patient neurons as compared to isogenic controls. PGRN mutant neurons also show impaired lysosomal proteolysis and decreased activity of the lysosomal enzyme cathepsin D. Furthermore, we find that PGRN interacts with cathepsin D, and that PGRN increases the activity of cathepsin D but not cathepsins B or L. Finally, we show that granulin E, a cleavage product of PGRN, is sufficient to increase cathepsin D activity. This functional relationship between PGRN and cathepsin D provides a possible explanation for overlapping NCL-like pathology observed in patients with mutations in PGRN or CTSD, the gene encoding cathepsin D. Together, our work identifies PGRN as an activator of lysosomal cathepsin D activity, and suggests that decreased cathepsin D activity due to loss of PGRN contributes to both FTD and NCL pathology in a dose-dependent manner.

The pathologic hallmark of Parkinson's disease is the accumulation of a-synuclein-containing Lewy bodies/neurites almost exclusively in neurons, and rarely in glial cells. However, emerging evidence suggests that glia such as astrocytes play an important role in the development of a-synuclein pathology. Using induced pluripotent stem-derived dopaminergic neurons and astrocytes from healthy subjects and patients carrying mutations in lysosomal ATP13A2, a monogenic form of synucleinopathy, we found that astrocytes rapidly internalized a-synuclein, and exhibited higher lysosomal degradation rates compared with neurons. Moreover, coculturing astrocytes and neurons led to decreased accumulation of a-synuclein in neurons and consequently diminished interneuronal transfer of a-synuclein. These protective functions of astrocytes were attenuated by ATP13A2 deficiency, suggesting that the loss of ATP13A2 function in astrocytes at least partially contributes to neuronal a-synuclein pathology. Together, our results highlight the importance of lysosomal function in astrocytes in the pathogenesis of synucleinopathies.

Frontotemporal dementia (FTD) is a common neurogenerative disorder characterized by progressive degeneration in the frontal and temporal lobes. Heterozygous mutations in the gene encoding progranulin (PGRN) are a common genetic cause of FTD. Recently, PGRN has emerged as an important regulator of lysosomal function. Here, we examine the impact of PGRN mutations on the processing of full-length prosaposin to individual saposins, which are critical regulators of lysosomal sphingolipid metabolism. Using FTD-PGRN patient-derived cortical neurons differentiated from induced pluripotent stem cells, as well as post-mortem tissue from patients with FTLD-PGRN, we show that PGRN haploinsufficiency results in impaired processing of prosaposin to saposin C, a critical activator of the lysosomal enzyme glucocerebrosidase (GCase). Additionally, we found that PGRN mutant neurons had reduced lysosomal GCase activity, lipid accumulation and increased insoluble α-synuclein relative to isogenic controls. Importantly, reduced GCase activity in PGRN mutant neurons is rescued by treatment with saposin C. Together, these findings suggest that reduced GCase activity due to impaired processing of prosaposin may contribute to pathogenesis of FTD resulting from PGRN mutations.

Improvements have been made in the diagnosis of Alzheimer’s disease (AD), manifesting mostly in the development of in vivo imaging methods that allow for the detection of pathological changes in AD by magnetic resonance imaging (MRI) and positron emission tomography (PET) scans. Many of these imaging methods, however, use agents that probe amyloid fibrils and plaques–species that do not correlate well with disease progression and are not present at the earliest stages of the disease. Amyloid β oligomers (AβOs), rather, are now widely accepted as the Aβ species most germane to AD onset and progression. Here we report evidence further supporting the role of AβOs as pathological instigators of AD and introduce promising anti-AβO diagnostic probes capable of distinguishing the 5xFAD mouse model from wild type mice by PET and MRI. In a developmental study, Aβ oligomers in 5xFAD mice were found to appear at 3 months of age, just prior to the onset of memory dysfunction, and spread as memory worsened. The increase of AβOs is prominent in the subiculum and correlates with concomitant development of reactive astrocytosis. The impact of these AβOs on memory is in harmony with findings that intraventricular injection of synthetic AβOs into wild type mice induced hippocampal dependent memory dysfunction within 24 h. Compelling support for the conclusion that endogenous AβOs cause memory loss was found in experiments showing that intranasal inoculation of AβO-selective antibodies into 5xFAD mice completely restored memory function, measured 30–40 days post-inoculation. These antibodies, which were modified to give MRI and PET imaging probes, were able to distinguish 5xFAD mice from wild type littermates. These results provide strong support for the role of AβOs in instigating memory loss and salient AD neuropathology, and they demonstrate that AβO selective antibodies have potential both for therapeutics and for diagnostics.

The life cycle of cultured proliferating cells is characterized by fluctuations in cell population density induced by periodic subculturing. This leads to corresponding changes in micro- and macroenvironment of the cells, accompanied by altered cellular metabolism, growth rate and locomotion. Studying cell density-dependent morphological, physiological and biochemical fluctuations is relevant for understanding basic cellular mechanisms and for uncovering the intrinsic variation of commonly used tissue culture experimental models. Using multiple cell lines, we found that expression levels of the autophagic markers p62 and LC3II, and lysosomal enzyme cathepsin D were altered in highly confluent cells as a consequence of nutrient depletion and cell crowding, which led to inactivation of the mTOR signaling pathway. Furthermore, both Lamp1 and active focal adhesion kinase (FAK) were reduced in high-density cells, while chemical inhibition or deletion of FAK led to alterations in lysosomal and autophagic proteins, as well as in the mTOR signaling. This was accompanied by alterations in the Hippo signaling pathway, while cell cycle checkpoint regulator p-cdc2 remained unaffected in at least one studied cell line. On the other hand, allometric scaling of cellular compartments in growing cell populations resulted in biochemically detectable changes in the plasma membrane proteins Na+K+-ATPase and cadherin, and nuclear proteins HDAC1 and Lamin B1. Finally, we demonstrate how treatment-induced changes in cell density and corresponding modulation of susceptible proteins may lead to ambiguous experimental outcomes, or erroneous interpretation of cell culture data. Together, our data emphasize the need to recognize cell density as an important experimental variable in order to improve scientific rigor of cell culture-based studies.