Mitochondrial function is modulated by its interaction with the endoplasmic reticulum (ER). Recent research indicates that these contacts are disrupted in familial models of amyotrophic lateral sclerosis (ALS). We report here that this impairment in the crosstalk between mitochondria and the ER impedes the use of glucose-derived pyruvate as mitochondrial fuel, causing a shift to fatty acids to sustain energy production. Over time, this deficiency alters mitochondrial electron flow and the active/dormant status of complex I in spinal cord tissues, but not in the brain. These findings suggest mitochondria-associated ER membranes (MAM domains) play a crucial role in regulating cellular glucose metabolism and that MAM dysfunction may underlie the bioenergetic deficits observed in ALS.

Large-scale genetic studies have identified numerous genetic risk factors that suggest a central role for innate immune cells in susceptibility to Alzheimer’s disease (AD). CD33, an immunomodulatory transmembrane sialic acid binding protein expressed on myeloid cells, was identified as one such genetic risk factor associated with Alzheimer’s disease. Several studies explored the molecular outcomes of genetic variation at the CD33 locus. It has been determined that the risk variant associated with AD increases the expression of the large isoform of CD33 (CD33M) in innate immune cells and alters its biological functions. CD33 is thought to signal via the interaction of its ITIM motif and the protein tyrosine phosphatase, SHP-1. Here, we utilize different molecular and computational approaches to investigate how AD-associated genetic variation in CD33 affects its interaction with SHP-1 in human microglia and microglia-like cells. Our findings demonstrate a genotype-dependent interaction between CD33 and SHP-1, which may functionally contribute to the AD risk associated with this CD33 variant. We also found that CD33-PTPN6 (SHP-1) gene–gene interactions impact AD-related traits, while CD33-PTPN11 (SHP-2) interactions do not.

Mitochondrial defects are a common hallmark of familial and sporadic forms of amyotrophic lateral sclerosis (ALS). However, the origin of these defects, including reduced pyruvate metabolism and reduced oxygen consumption, is poorly understood. These metabolic functions are regulated in specialized endoplasmic reticulum (ER) domains in close contact with mitochondria, called mitochondrial-associated ER membranes (MAM). Recently it has been shown that MAM domains are disrupted in ALS, but the connection between MAM dysregulation and mitochondrial defects in ALS cells remains unclear. Using human embryonic stem cell (ESC)-derived motor neurons (hMNs) and mouse models with ALS-pathogenic mutations in superoxide dismutase 1 (SOD1), we found that the glycolytic deficiency in ALS is a direct consequence of the progressive disruption of MAM structure and function that hinders the use of glucose-derived pyruvate as a mitochondrial fuel and triggers a shift in mitochondrial substrates from pyruvate to fatty acids. This glycolytic deficiency, over time, induces significant alterations in mitochondrial electron flow and in the active/dormant (A/D) status of complex I in spinal cord, but not in brain. These data agree with a role for MAM in the maintenance and regulation of cellular glucose metabolism and suggest that MAM disruption in ALS could be the underlying cause of the bioenergetic deficits observed in the disease.

Background: Genome‐wide association studies (GWAS) have identified genetic loci that robustly associate with Alzheimer’s Disease (AD), many of which are preferentially or exclusively expressed in innate immune cells. Among the identified AD risk genes is CD33: a transmembrane, sialic acid‐binding protein expressed on the surface of myeloid cells including microglia, the innate immune cells of the CNS. The function of microglia is highly responsive to and regulated by metabolic changes, which allows them to rapidly change phenotype and maintain brain health. However, the examination of human microglial metabolism as a driving factor in AD pathogenesis is an underappreciated concept.Method: To understand the molecular function of the CD33 protein in AD, our laboratory used a cutting‐edge mass spectrometry approach to identify binding partners of the CD33 sialic acid binding domain. We have also optimized a method using PBMCs from individuals of diverse age, sex, and ethnicity from which we can isolate monocytes to be differentiated to microglia‐like cells (MDMi).Result: Our mass spectrometry‐based approach revealed a novel interaction between CD33 and the glucose transporter GLUT1 that was further validated by proximity ligation assay (PLA) and co‐immunoprecipitation (co‐IP) in a human monocytic cell line. These findings prompted us to investigate CD33 genotype‐specific effects on microglial metabolism using human monocytes differentiated into microglia‐like cells (MDMi). We generated MDMi with different CD33 genetic backgrounds and measured their respiratory bioenergetics using a Seahorse extracellular flux analyzer. Interestingly, we found that the balance of ATP production via oxidative phosphorylation and glycolysis significantly varies with CD33 genotype at baseline. In a similar way, we found differences in glucose uptake and lactate secretion based on CD33 genotype, with AD‐risk (CD33^CC) MDMi taking up more glucose yet secreting less lactate compared to AD‐protective (CD33^AA) MDMi.Conclusion: Together, these results provide insight on microglia‐specific metabolic changes due to variations in the AD‐associated risk gene CD33. This novel link between genetic, immune, and metabolic risk factors in AD has the potential to reveal new therapeutic and immunomodulatory strategies with which to reduce susceptibility to this neurodegenerative disease.

Mechanisms linking CD33 variants to Alzheimer Disease (AD) are poorly defined. Here, we combine structural, cellular, and genetic analyses to delineate how the CD33 ^M splice isoform, upregulated in carriers of CD33 risk alleles, modulates microglial function. We show that CD33 ^M ectodomain dimerizes, enabling binding of large multi-sialylated molecules. We demonstrate that another AD risk protein - clusterin (CLU) ± Aβ oligomers (but not ApoE) binds with nanomolar avidity to CD33 ^M , but not CD33 ^m . We show that in human monocytes CD33 ^M :CLU binding induces CD33 ^M ITIM phosphorylation, recruits SHP-1, suppresses Aβ phagocytosis, and impairs clearance of amyloid plaques. We identify a soluble CD33 ^M ectodomain fragment (sCD33 ^M ) - absent from CD33 ^m -expressing cells - which could contribute to the role of CD33 ^M in AD. Genetic analyses confirm that CD33:CLU interaction modulates amyloid burden, cognition, and disease risk. These findings define a mechanistic CLU:Aβ:CD33 ^M axis, highlighting CD33 ^M dimerization and ligand-binding sites as potential therapeutic targets.

Background: The innate immune system has emerged as a central component of Alzheimer's disease (AD), with more than 75% of AD genetic loci being key innate immune genes. One such AD risk gene is CD33: a transmembrane, sialic‐acid binding protein that is specifically expressed by microglia in the brain. Previous studies suggest that CD33 regulates the inflammatory response by modifying microglial metabolism and oxidative phosphorylation. However, it remains unknown how CD33 genetic variation regulates microglial metabolism and how this contributes to AD pathogenesis. Method: To address this question, we generated human microglia‐like cells from primary monocytes with different CD33 genetic backgrounds and measured their respiratory bioenergetics and metabolic outcomes. Result: We found that the balance of ATP production via oxidative phosphorylation and glycolysis varies significantly with CD33 genotype. Compared to the AD‐risk genotype, those with the AD‐protective genotype had higher glycolytic activity but lower glucose uptake, specifically due to higher hexokinase activity, while no difference in lactate production or secretion was observed. To gain mechanistic insight, we used a mass spectrometry‐based approach to identify binding partners of the CD33 sialic acid binding domain, revealing a novel interaction between CD33 and the glucose transporter GLUT1. This result was further validated by PLA and co‐IP in a human monocytic cell line. Finally, using brain bulk RNA sequencing data from the ROSMAP cohort of AD patients and healthy elderly controls, we found CD33:GLUT1 gene interaction to be significantly associated with disease, underscoring the importance of this novel link between genetic, immune, and metabolic risk factors in AD. Conclusion: Together, these results provide insight on microglia‐specific metabolic changes due to variations in the AD‐associated risk gene CD33. This novel link between genetic, immune, and metabolic risk factors in AD has the potential to reveal new therapeutic and immunomodulatory strategies with which to reduce susceptibility to this neurodegenerative disease.