Xiaolai Zhou

The ALS/FTLD associated protein C9orf72 associates with SMCR8 and WDR41 to regulate the autophagy-lysosome pathway.

Hexanucleotide repeat expansion in the C9orf72 gene is a leading cause of frontotemporal lobar degeneration (FTLD) with amyotrophic lateral sclerosis (ALS). Reduced expression of C9orf72 has been proposed as a possible disease mechanism. However, the cellular function of C9orf72 remains to be characterized. Here we report the identification of two binding partners of C9orf72: SMCR8 and WDR41. We show that WDR41 interacts with the C9orf72/SMCR8 heterodimer and WDR41 is tightly associated with the Golgi complex. We further demonstrate that C9orf72/SMCR8/WDR41 associates with the FIP200/Ulk1 complex, which is essential for autophagy initiation. C9orf72 deficient mice, generated using the CRISPR/Cas9 system, show severe inflammation in multiple organs, including lymph node, spleen and liver. Lymph node enlargement and severe splenomegaly are accompanied with macrophage infiltration. Increased levels of autophagy and lysosomal proteins and autophagy defects were detected in both the spleen and liver of C9orf72 deficient mice, supporting an in vivo role of C9orf72 in regulating the autophagy/lysosome pathway. In summary, our study elucidates potential physiological functions of C9orf72 and disease mechanisms of ALS/FTLD.

Prosaposin facilitates sortilin-independent lysosomal trafficking of progranulin

Prosaposin directly interacts with progranulin and facilitates progranulin lysosomal trafficking via the trafficking receptors M6PR and LRP1, independent of the previously identified progranulin trafficking pathway mediated by sortilin.

TGFβ1 inhibits IFNγ-mediated microglia activation and protects mDA neurons from IFNγ-driven neurotoxicity.

Microglia‐mediated neuroinflammation has been reported as a common feature of familial and sporadic forms of Parkinson′s disease (PD), and a growing body of evidence indicates that onset and progression of PD correlates with the extent of neuroinflammatory responses involving Interferon γ (IFNγ). Transforming growth factor β1 (TGFβ1) has been shown to be a major player in the regulation of microglia activation states and functions and, thus, might be a potential therapeutic agent by shaping microglial activation phenotypes during the course of neurodegenerative diseases such as PD. In this study, we demonstrate that TGFβ1 is able to block IFNγ‐induced microglia activation by attenuating STAT1 phosphorylation and IFNγRα expression. Moreover, we identified a set of genes involved in microglial IFNγ signaling transduction that were significantly down‐regulated upon TGFβ1 treatment, resulting in decreased sensitivity of microglia toward IFNγ stimuli. Interestingly, genes mediating negative regulation of IFNγ signaling, such as SOCS2 and SOCS6, were up‐regulated after TGFβ1 treatment. Finally, we demonstrate that TGFβ1 is capable of protecting midbrain dopaminergic (mDA) neurons from IFNγ‐driven neurotoxicity in mixed neuron‐glia cultures derived from embryonic day 14 (E14) midbrain tissue. Together, these data underline the importance of TGFβ1 as a key immunoregulatory factor for microglia by silencing IFNγ‐mediated microglia activation and, thereby, rescuing mDA neurons from IFNγ‐induced neurotoxicity. Interferon γ (IFNγ) is a potent pro‐inflammatory factor that triggers the activation of microglia and the subsequent release of neurotoxic factors. Transforming growth factor β1 (TGFβ1) is able to inhibit the IFNγ‐mediated activation of microglia, which is characterized by the release of nitric oxide (NO) and tumor necrosis factor α (TNFα). By decreasing the expression of IFNγ‐induced genes as well as the signaling receptor IFNγR1, TGFβ1 reduces the responsiveness of microglia towards IFNγ. In mixed neuron‐glia cultures, TGFβ1 protects midbrain dopaminergic (mDA) neurons from IFNγ‐induced neurotoxicity.

Expression of Tgfβ1 and Inflammatory Markers in the 6-hydroxydopamine Mouse Model of Parkinson’s Disease

Parkinson’s disease (PD) is a neurodegenerative disorder that is characterized by loss of midbrain dopaminergic (mDA) neurons in the substantia nigra (SN). Microglia-mediated neuroinflammation has been described as a common hallmark of PD and is believed to further trigger the progression of neurodegenerative events. Injections of 6-hydroxydopamine (6-OHDA) are widely used to induce degeneration of mDA neurons in rodents as an attempt to mimic PD and to study neurodegeneration, neuroinflammation as well as potential therapeutic approaches. In the present study, we addressed microglia and astroglia reactivity in the SN and the caudatoputamen (CPu) after 6-OHDA injections into the medial forebrain bundle (MFB), and further analyzed the temporal and spatial expression patterns of pro-inflammatory and anti-inflammatory markers in this mouse model of PD. We provide evidence that activated microglia as well as neurons in the lesioned SN and CPu express Transforming growth factor β1 (Tgfβ1), which overlaps with the downregulation of pro-inflammatory markers Tnfα, and iNos, and upregulation of anti-inflammatory markers Ym1 and Arg1. Taken together, the data presented in this study suggest an important role for Tgfβ1 as a lesion-associated factor that might be involved in regulating microglia activation states in the 6-OHDA mouse model of PD in order to prevent degeneration of uninjured neurons by microglia-mediated release of neurotoxic factors such as Tnfα and nitric oxide (NO).

Impaired prosaposin lysosomal trafficking in frontotemporal lobar degeneration due to progranulin mutations

Haploinsufficiency of progranulin (PGRN) due to mutations in the granulin (GRN) gene causes frontotemporal lobar degeneration (FTLD), and complete loss of PGRN leads to a lysosomal storage disorder, neuronal ceroid lipofuscinosis (NCL). Accumulating evidence suggests that PGRN is essential for proper lysosomal function, but the precise mechanisms involved are not known. Here, we show that PGRN facilitates neuronal uptake and lysosomal delivery of prosaposin (PSAP), the precursor of saposin peptides that are essential for lysosomal glycosphingolipid degradation. We found reduced levels of PSAP in neurons both in mice deficient in PGRN and in human samples from FTLD patients due to GRN mutations. Furthermore, mice with reduced PSAP expression demonstrated FTLD-like pathology and behavioural changes. Thus, our data demonstrate a role of PGRN in PSAP lysosomal trafficking and suggest that impaired lysosomal trafficking of PSAP is an underlying disease mechanism for NCL and FTLD due to GRN mutations.

Elevated TMEM106B levels exaggerate lipofuscin accumulation and lysosomal dysfunction in aged mice with progranulin deficiency

Mutations resulting in haploinsufficiency of progranulin (PGRN) cause frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP), a devastating neurodegenerative disease. Accumulating evidence suggest a crucial role of progranulin in maintaining proper lysosomal function during aging. TMEM106B has been identified as a risk factor for frontotemporal lobar degeneration with progranulin mutations and elevated mRNA and protein levels of TMEM106B are associated with increased risk for frontotemporal lobar degeneration. Increased levels of TMEM106B alter lysosomal morphology and interfere with lysosomal degradation. However, how progranulin and TMEM106B interact to regulate lysosomal function and frontotemporal lobar degeneration (FTLD) disease progression is still unclear. Here we report that progranulin deficiency leads to increased TMEM106B protein levels in the mouse cortex with aging. To mimic elevated levels of TMEM106B in frontotemporal lobar degeneration (FTLD) cases, we generated transgenic mice expressing TMEM106B under the neuronal specific promoter, CamKII. Surprisingly, we found that the total protein levels of TMEM106B are not altered despite the expression of the TMEM106B transgene at mRNA and protein levels, suggesting a tight regulation of TMEM106B protein levels in the mouse brain. However, progranulin deficiency results in accumulation of TMEM106B protein from the transgene expression during aging, which is accompanied by exaggerated lysosomal abnormalities and increased lipofuscin accumulation. In summary, our mouse model nicely recapitulates the interaction between progranulin and TMEM106B in human patients and supports a critical role of lysosomal dysfunction in the frontotemporal lobar degeneration (FTLD) disease progression.

Regulated Intramembrane Proteolysis of the Frontotemporal Lobar Degeneration Risk Factor, TMEM106B, by Signal Peptide Peptidase-like 2a (SPPL2a)

Background: TMEM106B polymorphisms are associated with some forms of dementia. Results: A pathway for the sequential processing of TMEM106B on the lysosome membrane has been identified. Conclusion: TMEM106B undergoes processing via removal of its lumenal domain, followed by intramembrane cleavage by the protease SPPL2a. Significance: This may represent a mechanism for regulation of TMEM106B levels. The sequential processing of single pass transmembrane proteins via ectodomain shedding followed by intramembrane proteolysis is involved in a wide variety of signaling processes, as well as maintenance of membrane protein homeostasis. Here we report that the recently identified frontotemporal lobar degeneration risk factor TMEM106B undergoes regulated intramembrane proteolysis. We demonstrate that TMEM106B is readily processed to an N-terminal fragment containing the transmembrane and intracellular domains, and this processing is dependent on the activities of lysosomal proteases. The N-terminal fragment is further processed into a small, rapidly degraded intracellular domain. The GxGD aspartyl proteases SPPL2a and, to a lesser extent, SPPL2b are responsible for this intramembrane cleavage event. Additionally, the TMEM106B paralog TMEM106A is also lysosomally localized; however, it is not a specific substrate of SPPL2a or SPPL2b. Our data add to the growing list of proteins that undergo intramembrane proteolysis and may shed light on the regulation of the frontotemporal lobar degeneration risk factor TMEM106B.

Regulation of cathepsin D activity by the FTLD protein progranulin

and suggest that PGRN and CTSD might function together to regulate lysosomal activities. In support of this hypothesis, granulin motifs are found in cathepsin-like cysteine proteases in plants [11, 16]. These lines of evidence led us to postulate that an interaction between PGRN and CTSD may exist. To test the physical interaction between PGRN and CTSD, FLAG-tagged CTSD was co-transfected with untagged PGRN in HEK293T cells. Cell lysates were immunoprecipitated with anti-FLAG antibodies. PGRN signal is detected in anti-FLAG CTSD immunoprecipitates but not in the controls (Fig. 1a), suggesting a physical interaction between PGRN and CTSD. Since CTSD is known to interact with prosaposin (PSAP) [6, 10], which we previously showed to bind to PGRN as well [17], it is possible that the PGRN and CTSD interaction might be bridged by endogenous PSAP in HEK293T cells. To rule out this possibility, we compared the interaction between PGRN and CTSD in control N2a cells or N2a cells with PSAP expression depleted using the CRISPR/Cas9 system [17]. PSAP ablation has no effect on PGRN-CTSD binding in the coimmunoprecipitation assay (Fig. 1b), indicating that the interaction between PGRN and CTSD is not mediated by PSAP. Co-immunoprecipitation studies between individual granulins and CTSD suggest that multiple granulin motifs interact with CTSD (Supplemental Fig. 2a and 2b). With the physical interaction between PGRN and CTSD confirmed, next, we wanted to investigate its functional relevance. Since CTSD deficiency results in much more severe lysosomal phenotypes than PGRN deficiency [4], we hypothesized that PGRN might regulate CTSD activities. Therefore, we measured CTSD activities in 2-monthold wild type (WT) and PGRN-deficient mice before the appearance of any obvious lysosomal abnormalities or glial activation. Indeed, liver and spleen lysates from Progranulin (PGRN) protein, encoded by the granulin (GRN) gene, has been recently implicated in several neurodegenerative diseases [2, 5]. While haploinsufficiency of PGRN leads to frontotemporal lobar degeneration (FTLD) [2, 5], the most prevalent form of early onset dementia after Alzheimer’s disease (AD), complete loss of PGRN is known to cause neuronal ceroid lipofuscinosis (NCL) [1, 13], a group of lysosomal storage diseases. PGRN is a secreted glycoprotein of 7.5 granulin repeats [2, 5]. However, within the cell, PGRN is localized to lysosomes through two independent trafficking pathways [8, 17]. Furthermore, GRN is transcriptionally co-regulated with a number of essential lysosomal genes by the transcriptional factor TFEB [3]. While all these studies suggest an essential role of PGRN in regulating lysosomal function, how PGRN does so is still unclear. Cathepsin D (CTSD) is a lysosomal aspartic-type protease involved in many neurodegenerative diseases [14]. Mutations in the cathepsin D gene (CTSD) result in NCL in humans [9]. Interestingly, mice deficient in CTSD also develops TDP-43 aggregates (Supplementary Fig. 1) [7], a hallmark of FTLD with GRN mutations. FTLD patients with GRN mutations exhibit typical pathological features of NCL [7]. These data support that lysosomal dysfunction might serve as a common mechanism for FTLD and NCL

The interaction between progranulin and prosaposin is mediated by granulins and the linker region between saposin B and C

The frontotemporal lobar degeneration (FTLD) protein progranulin (PGRN) is essential for proper lysosomal function. PGRN localizes in the lysosomal compartment within the cell. Prosaposin (PSAP), the precursor of lysosomal saposin activators (saposin A, B, C, D), physically interacts with PGRN. Previously, we have shown that PGRN and PSAP facilitate each others lysosomal trafficking. Here, we report that the interaction between PSAP and PGRN requires the linker region of saposin B and C (BC linker). PSAP protein with the BC linker mutated, fails to interact with PGRN and deliver PGRN to lysosomes in the biosynthetic and endocytic pathways. On the other hand, PGRN interacts with PSAP through multiple granulin motifs. Granulin D and E bind to PSAP with similar affinity as full‐length PGRN.