INPP5D regulates inflammasome activation in human microglia.

Microglia and neuroinflammation play an important role in the development and progression of Alzheimer's disease (AD). Inositol polyphosphate-5-phosphatase D (INPP5D/SHIP1) is a myeloid-expressed gene genetically-associated with AD. Through unbiased analyses of RNA and protein profiles in INPP5D-disrupted iPSC-derived human microglia, we find that reduction in INPP5D activity is associated with molecular profiles consistent with disrupted autophagy and inflammasome activation. These findings are validated through targeted pharmacological experiments which demonstrate that reduced INPP5D activity induces the formation of the NLRP3 inflammasome, cleavage of CASP1, and secretion of IL-1ß and IL-18. Further, in-depth analyses of human brain tissue across hundreds of individuals using a multi-analytic approach provides evidence that a reduction in function of INPP5D in microglia results in inflammasome activation in AD. These findings provide insights into the molecular mechanisms underlying microglia-mediated processes in AD and highlight the inflammasome as a potential therapeutic target for modulating INPP5D-mediated vulnerability to AD.

Cell-type-specific regulation of APOE and CLU levels in human neurons by the Alzheimer's disease risk gene SORL1.

SORL1 is implicated in the pathogenesis of Alzheimer's disease (AD) through genetic studies. To interrogate the roles of SORL1 in human brain cells, SORL1-null induced pluripotent stem cells (iPSCs) were differentiated to neuron, astrocyte, microglial, and endothelial cell fates. Loss of SORL1 leads to alterations in both overlapping and distinct pathways across cell types, with the greatest effects in neurons and astrocytes. SORL1 loss induces a neuron-specific reduction in apolipoprotein E (APOE) and clusterin (CLU) and altered lipid profiles. Analyses of iPSCs derived from a large cohort reveal a neuron-specific association between SORL1, APOE, and CLU levels, a finding validated in postmortem brain. Enhancement of retromer-mediated trafficking rescues tau phenotypes observed in SORL1-null neurons but does not rescue APOE levels. Pathway analyses implicate transforming growth factor ß (TGF-ß)/SMAD signaling in SORL1 function, and modulating SMAD signaling in neurons alters APOE RNA levels in a SORL1-dependent manner. Taken together, these data provide a mechanistic link between strong genetic risk factors for AD.

Moving beyond amyloid and tau to capture the biological heterogeneity of Alzheimer's disease.

Alzheimer's disease (AD) manifests along a spectrum of cognitive deficits and levels of neuropathology. Genetic studies support a heterogeneous disease mechanism, with around 70 associated loci to date, implicating several biological processes that mediate risk for AD. Despite this heterogeneity, most experimental systems for testing new therapeutics are not designed to capture the genetically complex drivers of AD risk. In this review, we first provide an overview of those aspects of AD that are largely stereotyped and those that are heterogeneous, and we review the evidence supporting the concept that different subtypes of AD are important to consider in the design of agents for the prevention and treatment of the disease. We then dive into the multifaceted biological domains implicated to date in AD risk, highlighting studies of the diverse genetic drivers of disease. Finally, we explore recent efforts to identify biological subtypes of AD, with an emphasis on the experimental systems and data sets available to support progress in this area.

INPP5D/SHIP1 regulates inflammasome activation in human microglia.

Microglia and neuroinflammation are implicated in the development and progression of Alzheimer's disease (AD). To better understand microglia-mediated processes in AD, we studied the function of INPP5D/SHIP1, a gene linked to AD through GWAS. Immunostaining and single nucleus RNA sequencing confirmed that INPP5D expression in the adult human brain is largely restricted to microglia. Examination of prefrontal cortex across a large cohort revealed reduced full length INPP5D protein levels in AD patient brains compared to cognitively normal controls. The functional consequences of reduced INPP5D activity were evaluated in human induced pluripotent stem cell derived microglia (iMGLs), using both pharmacological inhibition of the phosphatase activity of INPP5D and genetic reduction in copy number. Unbiased transcriptional and proteomic profiling of iMGLs suggested an upregulation of innate immune signaling pathways, lower levels of scavenger receptors, and altered inflammasome signaling with INPP5D reduction. INPP5D inhibition induced the secretion of IL-1ß and IL-18, further implicating inflammasome activation. Inflammasome activation was confirmed through visualization of inflammasome formation through ASC immunostaining in INPP5D-inhibited iMGLs, increased cleaved caspase-1 and through rescue of elevated IL-1ß and IL-18 with caspase-1 and NLRP3 inhibitors. This work implicates INPP5D as a regulator of inflammasome signaling in human microglia.

Cell-type-specific regulation of APOE levels in human neurons by the Alzheimer's disease risk gene SORL1.

SORL1 is strongly implicated in the pathogenesis of Alzheimer's disease (AD) through human genetic studies that point to an association of reduced SORL1 levels with higher risk for AD. To interrogate the role(s) of SORL1 in human brain cells, SORL1 null iPSCs were generated, followed by differentiation to neuron, astrocyte, microglia, and endothelial cell fates. Loss of SORL1 led to alterations in both overlapping and distinct pathways across cell types, with the greatest effects in neurons and astrocytes. Intriguingly, SORL1 loss led to a dramatic neuron-specific reduction in APOE levels. Further, analyses of iPSCs derived from a human aging cohort revealed a neuron-specific linear correlation between SORL1 and APOE RNA and protein levels, a finding validated in human post-mortem brain. Pathway analysis implicated intracellular transport pathways and TGF- ß/SMAD signaling in the function of SORL1 in neurons. In accord, enhancement of retromer-mediated trafficking and autophagy rescued elevated phospho-tau observed in SORL1 null neurons but did not rescue APOE levels, suggesting that these phenotypes are separable. Stimulation and inhibition of SMAD signaling modulated APOE RNA levels in a SORL1-dependent manner. These studies provide a mechanistic link between two of the strongest genetic risk factors for AD.

Gasdermin-E mediates mitochondrial damage in axons and neurodegeneration.

Mitochondrial dysfunction and axon loss are hallmarks of neurologic diseases. Gasdermin (GSDM) proteins are executioner pore-forming molecules that mediate cell death, yet their roles in the central nervous system (CNS) are not well understood. Here, we find that one GSDM family member, GSDME, is expressed by both mouse and human neurons. GSDME plays a role in mitochondrial damage and axon loss. Mitochondrial neurotoxins induced caspase-dependent GSDME cleavage and rapid localization to mitochondria in axons, where GSDME promoted mitochondrial depolarization, trafficking defects, and neurite retraction. Frontotemporal dementia (FTD)/amyotrophic lateral sclerosis (ALS)-associated proteins TDP-43 and PR-50 induced GSDME-mediated damage to mitochondria and neurite loss. GSDME knockdown protected against neurite loss in ALS patient iPSC-derived motor neurons. Knockout of GSDME in SOD1G93A ALS mice prolonged survival, ameliorated motor dysfunction, rescued motor neuron loss, and reduced neuroinflammation. We identify GSDME as an executioner of neuronal mitochondrial dysfunction that may contribute to neurodegeneration.

Identification of substrates of palmitoyl protein thioesterase 1 highlights roles of depalmitoylation in disulfide bond formation and synaptic function.

Loss-of-function mutations in the depalmitoylating enzyme palmitoyl protein thioesterase 1 (PPT1) cause neuronal ceroid lipofuscinosis (NCL), a devastating neurodegenerative disease. The substrates of PPT1 are largely undescribed, posing a limitation on molecular dissection of disease mechanisms and therapeutic development. Here, we provide a resource identifying >100 novel PPT1 substrates. We utilized Acyl Resin-Assisted Capture (Acyl RAC) and mass spectrometry to identify proteins with increased in vivo palmitoylation in PPT1 knockout (KO) mouse brains. We then validated putative substrates through direct depalmitoylation with recombinant PPT1. This stringent screen elucidated diverse PPT1 substrates at the synapse, including channels and transporters, G-protein-associated molecules, endo/exocytic components, synaptic adhesion molecules, and mitochondrial proteins. Cysteine depalmitoylation sites in transmembrane PPT1 substrates frequently participate in disulfide bonds in the mature protein. We confirmed that depalmitoylation plays a role in disulfide bond formation in a tertiary screen analyzing posttranslational modifications (PTMs). Collectively, these data highlight the role of PPT1 in mediating synapse functions, implicate molecular pathways in the etiology of NCL and other neurodegenerative diseases, and advance our basic understanding of the purpose of depalmitoylation.

Stem cell-derived neurons reflect features of protein networks, neuropathology, and cognitive outcome of their aged human donors.

We have generated a controlled and manipulable resource that captures genetic risk for Alzheimer's disease: iPSC lines from 53 individuals coupled with RNA and proteomic profiling of both iPSC-derived neurons and brain tissue of the same individuals. Data collected for each person include genome sequencing, longitudinal cognitive scores, and quantitative neuropathology. The utility of this resource is exemplified here by analyses of neurons derived from these lines, revealing significant associations between specific Aß and tau species and the levels of plaque and tangle deposition in the brain and, more importantly, with the trajectory of cognitive decline. Proteins and networks are identified that are associated with AD phenotypes in iPSC neurons, and relevant associations are validated in brain. The data presented establish this iPSC collection as a resource for investigating person-specific processes in the brain that can aid in identifying and validating molecular pathways underlying AD.