Progranulin (PGRN) expression in ALS: An immunohistochemical study

Journal of the Neurological Sciences 276 (2009) 9–13

Contents lists available at ScienceDirect

Journal of the Neurological Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n s

Progranulin (PGRN) expression in ALS: An immunohistochemical study D. Irwin, C.F. Lippa ⁎, A. Rosso Department of Neurology, Drexel University College of Medicine, New College Building, 245 N 15th St Philadelphia PA 19102, USA

a r t i c l e

i n f o

Article history: Received 3 July 2007 Received in revised form 12 July 2008 Accepted 11 August 2008 Available online 11 October 2008 Keywords: Amyotrophic lateral sclerosis Progranulin Frontotemporal dementia Motor neuron disease Immunohistochemistry

a b s t r a c t Mutations in the gene progranulin (PGRN) were recently identified as the cause of some forms of frontotemporal dementia with ubiquitin-positive intraneuronal inclusion pathology (FTLD-U). The DNAbinding protein, TDP-43, was determined to be a component of these ubiquitinated inclusions in FTLD-U and amyotrophic lateral sclerosis (ALS) with dementia (ALS-D). These findings raise many interesting questions as to the shared pathology and possible common pathologic process between ALS and FTLD-U. This study examines the immunoexpression of PGRN in ALS patients using immunohistochemical analysis of post-mortem tissue. Available brain and spinal cord sections of eight ALS patients, including one case with severe dementia, and eighteen control-aged brains were stained with anti-PGRN antibodies. We found increased staining for PGRN in motor tracts with vacuolar degeneration and glial cells in ALS sample spinal cord and brainstem sections compared to controls. Variable upper motor neuron staining and reactive glia were seen in ALS motor cortex samples. Frontal lobe and hippocampal sections showed no consistent differences from control tissues with the exception of the ALS-dementia case, which showed PGRN immunoexpression in non-motor cortical areas. These results describe a pattern of increased PGRN expression in areas of active degeneration in ALS. The meaning of this association is unclear, but may indicate a potential role for PGRN in the variable expression of motor and cognitive deficits in the ALS–FTD spectrum. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Recently, a subset of frontotemporal dementia cases with ubiquitinated inclusions (FTLD-U) has been found to result from null mutations in the gene for the growth factor progranulin (PGRN) [1,2]. One study found that 10% of an FTD population has PGRN mutations resulting in loss of functional PGRN [3]. PGRN is a 68.5 kDa (appears as 90 kDa on western blotting due to heavy glycosylation) secreted growth factor which acts through extracellular kinases and plays a role in neurodevelopment and wound healing [4]. PGRN has been widely studied for its role in malignancy including breast, ovarian, renal, and glioma carcinomas [4]. Over expression of PGRN can increase malignant potential in breast cancer [5]. Intact PGRN exerts anti-inflammatory effects in wound healing by inhibiting tumor necrosis factor, while proteolytic peptides of PGRN, granulins, are pro-inflammatory via activation of interleukin-8 (IL-8) [4]. Regulation of this process occurs through secretory leukocyte protease inhibitor (SLPI), which protects PGRN from proteolysis, and elastase, which cleaves PGRN into pro-inflammatory granulins [4,6]. The role of PGRN in the adult nervous system is not as well understood. Its actions in wound healing and inflammation may be important in neurodegeneration [6,7]. PGRN also has neurotrophic properties, as it has been found to induce growth in a neuronal cell line [7]. In the normal brain, PGRN is expressed in pyramidal and ⁎ Corresponding author. Tel.: +1 215 762 4761; fax: +1 215 762 3161. E-mail address: [email protected] (C.F. Lippa). 0022-510X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2008.08.024

granule neurons in the hippocampus and purkinje cells of the cerebellum [7]. It has also been described in activated microglial cells [8,9] and cortical pyramidal cells [8]. Amyotrophic lateral sclerosis (ALS) is now thought to exist in a continuum with FTLD-U because of common clinical and pathologic features. The dramatic and devastating motor findings in ALS have overshadowed an increasingly described cognitive component [10]. Large-scale studies have found evidence of cognitive dysfunction in up to 50% of ALS cases [11–13]. Cognitive changes range from subclinical (only detected with detailed neuropsychological testing) to profound dementia similar to FTD [11,14]. In addition, the discovery of a common substrate for the ubiquitinated inclusion bodies seen in both ALS and FTLD-U, the intranuclear DNA-binding protein TDP-43 [15,16], further strengthens the association of these two diseases. Despite these common findings, PGRN expression has not been extensively studied in ALS. The purpose of this study was to examine the expression of PGRN in the nervous system of ALS patients through immunohistochemical methods to investigate a potential role of PGRN in ALS pathology. 2. Methods 2.1. Subjects We examined available regions from confirmed cases of ALS (n = 8) including one case with severe dementia (ALS-D). Case demographics

10

D. Irwin et al. / Journal of the Neurological Sciences 276 (2009) 9–13

include: all Caucasian, four males and four females, mean age at death was 57.0 years (range = 50–66), mean duration of illness was 3.8 years (range = 3–5). The ALS-D case did not receive extensive neuropsychia-

tric testing, but did display many features of frontal executive dysfunction and severe dementia. Available regions from controlaged brain cases (n = 18) were examined for comparison. Control cases

Fig. 1. Photomicrographs of PGRN immunostaining in ALS and control spinal cord and brainstem tissue. A: ALS spinal cord showing intense immunostaining for PGRN in areas of vacuolar degeneration of long axonal tracts in the lateral corticospinal tracts (60×). B: Dystrophic ALS anterior horn cell with PGRN reactivity (60×). C: Control anterior horn cell displaying PGRN reactivity (60×). D: Descending pyramidal tract immunostaining for PGRN in ALS-dementia case pons (adjacent to crossing pontine fibers [60×]). E: Descending pyramidal tract immunostaining for PGRN in ALS medulla (60×). F: Control medulla pyramidal tract showing less PGRN immunoreactivity (60×). G: Control medulla inferior olive nucleus (10×) with PGRN reactive neurons.

D. Irwin et al. / Journal of the Neurological Sciences 276 (2009) 9–13

11

tissue. Slides were examined by two different evaluators and graded on a semi-quantitive scale from 0 to 3 on intensity of PGRN immunoreactivity. The scoring system used was 0 for no visible reactivity, 1 for trace staining, 2 for moderate reactivity, and 3 for extensive immunoreactivity. 2.4. Statistical analysis Mean staining scores were analyzed for statistical significance (p b 0.05) using a computerized 2-tailed t-test. 3. Results 3.1. Spinal cord

Fig. 2. Semi-quantitive analysis of PGRN immunostaining: Average staining intensity score of spinal cord and brainstem sections (p b 0.05).

were cognitively normal with no neurological illness. Samples were formalin fixed and embedded in paraffin. 2.2. Histological methods Paraffin embedded tissue samples were sectioned at a thickness of 6 µm and placed on glass slides. The tissue was deparaffinized in xylene, hydrated in graded ethanol solutions, and then stained with antibodies against recombinant human PGRN protein in a 1:250 dilution (R&D #AF2420, Minneapolis, MN) with an incubation time of 1 h, or recombinant human TDP-43 at 1:500 (ProteinTech Group, Chicago, IL) incubated overnight. Slides stained for TDP-43 were treated with antigen retrieval solution (DakoCytomation, Denmark) prior to application of the primary antibody. HRP-conjugated anti-goat IgG in a dilution of 1:400 (R&D, Minneapolis, MN) was used as the secondary antibody for PGRN and the DakoCytomation ABC kit was used for TDP-43. 3,3′ diaminobenzene (DAB) was used as the chromogen for both antibodies. The slides were counterstained using Hematoxylin. 2.3. Semi-quantitative measures A semi-quantitative scale was used as previously described [17] to estimate the level of PGRN reactivity within selected regions of sample

ALS patients displayed intense PGRN immunostaining in the anterior horn region and lateral corticospinal tracts of the spinal cord, (Fig. 1A) reaching statistical significance using semi-quantitative analysis (Fig. 2; Table 1). Areas of vacuolar degeneration in the lateral corticospinal tracts had the most intense reactions. In addition, reactive glial cells in this region also expressed PGRN. The anterior horn cells of ALS patients were completely degenerated in most sample sections; however, when present, were diminished in size and displayed variable PGRN immunoreactivity (Fig. 1B). Control spinal cord sections had diffuse glial staining along motor tracts with variable motor neuron staining (Fig. 1C). 3.2. Brainstem ALS corticospinal tracts in the brainstem showed similar immunoreactivity in degenerating axons with vacuolar changes as in spinal cord sections, (Fig. 1D, E) reaching statistical significance using semiquantitative analysis (Fig. 2). Interestingly, neurons of the inferior olivary nucleus in both control and ALS patient samples showed significant immunostaining to PGRN (Fig. 1G). Control brainstem sections displayed minimal diffuse PGRN reactivity (Fig. 1F). 3.3. Frontal cortex The ALS motor cortex showed PGRN immunoreactivity in the remaining pyramidal and Betz cells (Fig. 3B). In addition, reactive glial cells in exiting pyramidal tracts expressed PGRN. Control samples also showed reactivity in pyramidal cells (Fig. 3A) and diffuse glial staining.

Table 1 Data from semi-quantitative analysis of immunohistochemical staining ALS spinal cord motor tract

Control spinal cord motor tract

ALS brainstem motor tract

Control brainstem motor tract

ALS frontal cortex

Control frontal cortex

ALS hippocampus granule cells

Control hippocampus granule cells

ALS cerebellum Purkinje cells

Control cerebellum Purkinje cells

2.5a 1.5 2 0.5 2.5 2.5 2.5 2.5

0 0.5 1.5 0.5 1 1.5 0 0 0 0 2

2-medulla 2.5-medullaa 2.5-medulla 2-medulla 0.5-pons 2.5-pons 3-ponsa 2.5-pons 1.5-pons 1.5-pons 0-pons 3-midbraina 2-midbrain

2.0a 0.5 1.5 1.5

1 1.5 1 1.5

1.5a 1.5 1 1

0.5 0 0.5 2.5

3a 1

1.5 2.5

2.06

0.64

1.96

1.5-medulla 1-medulla 1-medulla 0.5-pons 0-pons 0-pons 0.5-pons 0.5-pons 0-pons 1-pons 0.5-pons 1-midbrain 0-midbrain 1.5-midbrain 1.5-midbrain 0-midbrain 1.5-midbrain 0.71

1.38

1.25

1.25

0.88

2

*p b 0.05. a ALS-dementia case.

2

12

D. Irwin et al. / Journal of the Neurological Sciences 276 (2009) 9–13

There was no statistical difference in staining scores between ALS and control cases (Table 1). 3.4. Hippocampus Both control and ALS samples displayed consistent immunoreactivity to PGRN in the granule layer of the hippocampus (Fig. 4A). No differences between the groups were evident. 3.5. Cerebellum The Purkinje cell layer displayed consistent immunoreactivity in both ALS and control groups without statistical differences (Fig. 4B). 3.6. ALS-D case The ALS-D case had the most intense immunoreactivity compared to the other ALS cases and controls (Table 1). Vacuolization of motor

Fig. 4. Photomicrographs of PGRN immunostaining in control hippocampus and cerebellum tissue. A: Control hippocampus displaying PGRN reactive cells of region CA1 (60×). B: Control cerebellum (60×) displaying PGRN reactive purkinje cell.

tracts was more severe. The staining was more widespread than the other cases and included non-motor frontal areas (Fig. 3C). 4. Discussion

Fig. 3. Photomicrographs of PGRN immunostaining in ALS and control frontal cortex tissue. A. Control pyramidal cell displaying PGRN reactivity (60×). B. Betz cell from ALS case displaying punctate PGRN staining (60×). C. ALS-dementia frontal cortex displaying gliosis and intense PGRN reactivity (20×).

Through examination of post-mortem tissue of ALS patients, it appears that PGRN expression is increased in degenerating long motor tracts in the spinal cord and brainstem. Expression was seen within both the neurons and reactive glia. Both upper and lower motor neuron staining was variable, although many dystrophic neurons displayed intense reactivity. Extra motor-cortical staining was not enhanced except for the one ALS case with accompanying dementia. No differences between ALS and control cases were found in the frontal cortex, hippocampus, and cerebellum. Control hippocampal granule cells, purkinje cells, motor cortex pyramidal cells, and inferior olive nucleus neurons displayed moderate PGRN reactivity. In addition, variable lower motor neuron staining was evident in control spinal cord sections. TDP43 staining was present in ALS and control spinal cord sections, but was not as prominent as PGRN staining (data not included). The main finding from this study is that increased PGRN expression is associated with areas of neuronal cell loss in ALS. The results compliment previous work which demonstrated a fourhundred fold increase in PGRN mRNA expression in ALS spinal cord samples [18] by localizing the increased expression to neurons and glia directly involved in cell death. One hypothesis for PGRN expression in areas of neurodegeneration is that it is upregulated as a protective response to cell damage. Neurotrophic properties of PGRN would be important for this mechanism. Also of note, are PGRN's anti-inflammatory properties, which may aid in neuronal preservation. One study of hippocampal gene expression after blunt force trauma in mice found a delay of 24 h in PGRN mRNA expression [19], suggesting PGRN may function in chronic neuronal damage, such as neurodegeneration, rather than in acute insults [6].

D. Irwin et al. / Journal of the Neurological Sciences 276 (2009) 9–13

An alternate hypothesis is that PGRN is an active participant in neuronal cell death in ALS and other neurodegenerative diseases. As previously described, PGRN cleavage products (granulins) formed by elastase activity have pro-inflammatory properties through IL-8 activation [4]. Perhaps an abnormal extracellular milieu in ALS could lead to increased levels of elastase relative to SLPI, and thus form a pro-inflammatory condition leading to neuronal cell damage. Alternatively, increased SLPI could lead to increased inhibition of PGRN trophic affects [20]. Another important result is that the pattern of PGRN expression in control tissue showed an association to highly metabolic neuronal areas. This suggests PGRN may be important in sustaining long axonal tracts and dendrite maintenance. Interestingly, motor neurons also have long axonal tracts which may require increased PGRN for survival, especially when experiencing metabolic stress, as in ALS. This is supported by a recent study which found PGRN and GRN E (a polypeptide product of PGRN) to promote both motor and cortical rat neuronal cell culture survival and neurite outgrowth in a dosedependant relationship [20]. PGRN's association with neuronal cell death is not unique to ALS. Despite PGRN haploinsufficiency in FTLD-U, increased reactivity has been described in affected frontal cortex areas in these patients [1]. In addition, increased PGRN immunoreactivity has also been seen within dystrophic neurons in Alzheimer's disease (AD) patients [1,9]. It is unknown if abnormalities in PGRN are directly responsible for some forms of ALS. In one study of PGRN mutation FTLD-U cases, little motor neuron pathology was observed [21], leading the authors to conclude PGRN deficiency is unlikely to cause motor neuron disease. A large screening study of the PGRN gene in familial and sporadic ALS and ALS– FTD found only two missense mutations with unclear significance [22]. Based on these results, it is doubtful that PGRN mutation can directly cause ALS, but there are several mechanisms for PGRN expression to modulate the degree of cognitive and motor symptoms in the ALS–FTD spectrum. This is best demonstrated by a recent largescale genetic screening study of ALS patients which found single nucleotide polymorphisms in the PGRN gene associated with shorter survival and younger age of onset [23]. In addition, although immunostaining techniques fail to demonstrate PGRN reactivity within the ubiquitinated inclusion bodies in FTLD-U and ALS [9,21], PGRN may interact with TDP-43, as granulins have been found to bind to other TAR-DNA-binding proteins [24]. Another recent study reports that a reduction of PGRN expression in vitro causes abnormal TDP-43 proteolysis and cytoplasmic redistribution through a caspasemediated process [25]. This finding shows an important link between PGRN deficiency in FTLD-U and TDP-43 accumulation into the shared inclusion body pathology seen in ALS and FTLD-U. Another potential mechanism for PGRN involvement in both ALS and FTLD-U is through interaction of growth factors. PGRN induces vascular endothelial growth factor (VEGF) production in breast cancer cells [5]. VEGF is regulated by angiogenin (ANG), another hypoxiainduced vascular growth factor found to be elevated in serum of ALS patients [26]. Loss-of-function mutations in ANG have been discovered in some cases of ALS [27]. Thus, a complex interaction between growth factors may play a role in ALS, ALS-D and FTLD-U. Perhaps relative imbalances in these factors could lead to variable expression of motor and/or cognitive symptoms. Talbot and Ansorge [28] suggest that with new discoveries, ALS and FTD may need to be reclassified into “ubiquitinopathies.” Reclassification of these two neurological diseases once considered entirely different clinical entities is remarkable. There is a definite clinical and pathologic link between ALS and FTD. The role of PGRN in this association is yet to be determined, but the association of PGRN deficiency and abnormal TDP-43 processing, and potential interaction with other growth factors implicated in ALS, serve as potential mechanisms. This research contributes to this rapidly changing area by providing further evidence of PGRN involvement in neuronal degeneration in ALS.

13

References [1] Baker M, Mackenzie IR, Pickering-Brown SM, Gass J, Rademakers R, Lindholm C, et al. Mutations in progranulin cause tau-negative frontotemporal dementia linked to chromosome 17. Nature 2006;442(7105):916–9. [2] Cruts M, Gijselinck I, van der Zee J, Engelborghs S, Wils H, Pirici D, et al. Null mutations in progranulin cause ubiquitin positive frontotemporal dementia linked to chromosome 17q21. Nature 2006;442(7105):920–4. [3] Gass J, Cannon A, Mackenzie IR, Boeve B, Baker M, Adamson J, et al. Mutations in progranulin are a major cause of ubiquitin-positive frontotemporal lobar degeneration. Hum Mol Genet 2006;15(20):2988–3001. [4] He Z, Bateman A. Progranulin (granulin epithelin precursor, PC-cell-derived growth factor, acrogranulin) mediates tissue repair and tumorigenesis. J Mol Med 2003;81:600–12. [5] Tangkeangsirisin W, Serrero G. PC cell-derived growth factor (PCDGF/GP88, progranulin) stimulates migration, invasiveness and VEGF expression in breast cancer cells. Carcinogenesis 2004;25(9):1587–92. [6] Ahmed Z, Mackenzie IRA, Hutton ML, Dickson DW. Progranulin in frontotemporal degeneration and neuroinflammation. J Neuroinflamm 2007;4:7. [7] Daniel R, He Z, Carmichael P, Halper J, Bateman A. Cellular localization of gene expression for progranulin. J Histochem Cytochem 2000;48(7):999–1009. [8] Snowden JS, Pickering-Brown SM, Mackenzie IR, Richardson AM, Varma A, Neary D, et al. Progranulin mutations associated with frontotemporal dementia and progressive non-fluent aphasia. Brain 2006;129:3091–102. [9] Mukherjee O, Pastor P, Cairns NJ, et al. HDDD2 is a familial frontotemporal lobar degeneration with ubiquitin-positive, tau negative inclusions caused by a missense mutation in the signal peptide of progranulin. Ann Neurol 2006;60: 314–22. [10] Irwin DJ, Lippa CF, Swearer JM. Cognition in ALS. Am J Alzhiemers Dis Other Demen 2007;22(4):300–12. [11] Lomen-Hoerth C, Murphy J, Langmore S, Kramer JH, Olney RK, Miller B. Are amyotrophic lateral sclerosis patients cognitively normal? Neurology 2003;60:1094–7. [12] Ringholz GM, Appel SH, Bradshaw M, Cooke NA, Mosnik DM, Schulz PE. Prevalence and patterns of cognitive impairment in sporadic ALS. Neurology 2005;65:586–90. [13] Massman PJ, Sims J, Cooke N, Haverkamp LJ, Appel V, Appel SH. Prevalence and correlates of neuropsychological deficits in amyotrophic lateral sclerosis. J Neurol Neurosurg Psychiatry 1996;61:450–5. [14] Neary D, Snowden JS, Mann DM, Northern B, Goulding PJ, Macdermott N. Frontal lobe dementia and motor neuron disease. J Neurol Neurosurg Psychiatry 1990;53:23–32. [15] Neumann M, Sampathu DM, Kwong LK, Traux AC, Micsenyi MC, Chou TT, et al. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 2006;314:130–3. [16] Arai T, Hasegawa M, Akiyama H, Ikeda K, Nonaka T, Mori H, et al. TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Biochem Biophys Res Commun 2006;351:602–11. [17] Nagarsheth MH, Viehman A, Lippa SM, Lippa CF. Notch-1 immunoexpression is increased in Alzheimer's and Pick's disease. J Neurol Sci 2006;244(1–2):111–6. [18] Malaspina A, Kaushik N, Belleroche J. Differential expression of 14 genes in amyotrophic lateral sclerosis spinal cord using gridded cDNA arrays. J Neurochemistry 2001;77:132–45. [19] Matzilevich DA, Rall JM, Moore AN, Grill RJ, Dash PK. High-density microarray analysis of hippocampal gene expression following experimental brain injury. J Neurosci Res 2002;67:646–63. [20] Van Damme P, Van Hoecke AV, Lambrechts D, Vanacker P, Bogaert E, van Swieten J, et al. Progranulin functions as a neurotrophic factor to regulate neurite outgrowth and enhance neuronal survival. J Cell Biol 2008;181(1):37–41. [21] Mackenzie IR, Baker M, Pickering-Brown S, Hsiung GY, Lindholm C, Dwosh E, et al. The neuropathology of frontotemporal lobar degeneration caused by mutations in the progranulin gene. Brain 2006;129:3081–90. [22] Schymick JC, Yang Y, Andersen PM, Vonsattel JP, Greenway M, Momeni P, et al. Progranulin mutations and amyotrophic lateral sclerosis–frontotemporal dementia phenotypes. J Neurol Neurosurg Psychiatry 2007;78:754–6. [23] Sleegers K, Brouwers N, Maurer-Stroh S, van Es MA, Van Damme P, van Vught PW, et al. Progranulin genetic variability contributes to amyotrophic lateral sclerosis. Neurology 2008;71(4):253–9. [24] Hoque M, Young TM, Lee CG, Serrero G, Mathews MB, Pe’ery T. The growth factor granulin interacts with cyclin T1 and modulates P-TEFb-dependant transcription. Mol Cell Biol 2003;23(5):1688–702. [25] Zhang YJ, Xu Y, Dickey CA, Buratti E, Baralle F, Bailey R, et al. Progranulin mediates caspase-dependant cleavage of TAR DNA binding protein-43. J Neurosci 2007;27 (39):10530–4. [26] Cronin S, Greenway MJ, Ennis S, Kieran D, Green A, Prehn JH, et al. Elevated serum angiogenin levels in ALS. Neurology 2006;67(10):1833–6. [27] Greenway MJ, Andersen PM, Russ C, Ennis S, Cashman S, Donaghy C, et al. ANG mutations segregate with familial and ‘sporadic’ amyotrophic lateral sclerosis. Nat Genet 2006;38(4):411–3. [28] Talbot K., Ansorge O. Recent advances in the genetics of amyotrophic lateral sclerosis and frontotemporal dementia: common pathways in neurodegenerative disease. Hum Mol Genet 20.