Progranulin Deficiency Reduces CDK4/6/pRb Activation and Survival of Human Neuroblastoma SH-SY5Y Cells

Abstract

Null mutations in GRN are associated with frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP). However, the influence of progranulin (PGRN) deficiency in neurodegeneration is largely unknown. In neuroblastoma cells, silencing of GRN gene causes significantly reduced cell survival after serum withdrawal. The following observations suggest that alterations of the CDK4/6/retinoblastoma protein (pRb) pathway, secondary to changes in PI3K/Akt and ERK1/2 activation induced by PGRN deficiency, are involved in the control of serum deprivation-induced apoptosis: (i) inhibiting CDK4/6 levels or their associated kinase activity by sodium butyrate or PD332991 sensitized control SH-SY5Y cells to serum deprivation-induced apoptosis without affecting survival of PGRN-deficient cells; (ii) CDK4/6/pRb seems to be downstream of the PI3K/Akt and ERK1/2 signaling pathways since their specific inhibitors, LY294002 and PD98059, were able to decrease CDK6-associated kinase activity and induce death of control SH-SY5Y cells; (iii) PGRN-deficient cells show reduced stimulation of PI3K/Akt, ERK1/2, and CDK4/6 activities compared with control cells in the absence of serum; and (iv) supplementation of recombinant human PGRN was able to rescue survival of PGRN-deficient cells. These observations highlight the important role of PGRN-mediated stimulation of the PI3K/Akt-ERK1/2/CDK4/6/pRb pathway in determining the cell fate survival/death under serum deprivation.

Processing of progranulin into granulins involves multiple lysosomal proteases and is affected in frontotemporal lobar degeneration

“…These results suggest that cysteine proteases such as CTSB and/or CTSL process full-length PGRN to mainly produce multi-granulin fragments that contain granulin F and to a lesser extent individual granulin F (similar to our in vitro assay), while AEP may act on one or more of these fragments to produce individual granulin F. It is therefore possible that the modest reduction in granulin F levels seen upon dual inhibition of CTSB and CTSL is due to reduced availability of multi-granulin fragments for AEP to process. To our knowledge, this is the rst study to identify cleavage sites in the inter-granulin linkers required to liberate granulin F. Earlier studies in human primary cell cultures have also referred to a potential multi-granulin fragment likely containing granulin F [48,49]. Future studies on these multi-granulin fragments will be essential to tease out the complex regulation of PGRN processing.…”

“…Holler et al, 2017, demonstrated a reduction in certain granulins in brain, however, the severity of degeneration and astrogliosis within these regions was not assessed [19]. Since these degenerating regions are characterized by neuronal loss and in ltration of in ammatory astrocytes and microglia, the latter of which expresses high levels of AEP [49], it is possible that gliotic cells are responsible for the increased granulin F. Moreover, since PGRN is also a secreted protein we cannot yet differentiate the cell autonomous and cell non-autonomous contributions of PGRN processing in disease. Whether the increased granulin F contributes to or results from FTLD pathophysiology also remains to be seen.…”

Processing of progranulin into granulins involves multiple lysosomal proteases and is affected in frontotemporal lobar degeneration

“…Previously, Holler et al, 2017, demonstrated a reduction in certain granulins in brain, however, the severity of degeneration and astrogliosis within these regions was not assessed [19]. Since these degenerating regions are characterized by neuronal loss and in ltration of in ammatory astrocytes and microglia, the latter of which expresses high levels of AEP [49], it is possible that gliotic cells are responsible for the increased granulin F. Moreover, since PGRN is also a secreted protein we cannot yet differentiate the cell autonomous and cell non-autonomous contributions of PGRN processing in disease. Whether the increased granulin F contributes to or results from FTLD pathophysiology also remains to be seen.…”

Progranulin Deficiency Induces Mitochondrial Dysfunction in Frontotemporal Lobar Degeneration with TDP-43 Inclusions

“…Alvin P. Joselin and Jane Y. Wu from the Center for Genetic Medicine (Northwestern University, Chicago, IL, USA). These lines were generated using the pSUPERIOR RNAi construct containing a sequence of 19 nucleotides targeting human GRN , as was previously described [ 14 , 17 ]. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% ( v / v ) heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, MA, USA) and 1% penicillin/streptomycin (P/S; Thermo Fisher Scientific, MA, USA).…”

“…Recent evidence has shown that PGRN plays an important role in regulating mitochondrial homeostasis and activity [ 12 , 13 ]. Furthermore, previous results from our lab showed that PGRN insufficiency regulated the intrinsic/mitochondrial apoptosis pathway in GRN knockdown (KD) SH-SY5Y neuroblastoma cells [ 14 , 15 ] and peripheral cells from FTLD-TDP patients carrying a LOF GRN mutation (c.709-1G > A) [ 16 ]. Altogether, this evidence suggests that mitochondrial dysfunction could also contribute to neurodegeneration in FTLD-TDP linked to PGRN deficiency.…”