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Normal Development of the Perineuronal Net in Humans; In Patients with and without Epilepsy
Accepted Manuscript Research Article Normal development of the Perineuronal net in humans; in patients with and without epilepsy Stephanie L. Rogers, Elyse Rankin-Gee, Rashmi M. Risbud, Brenda E. Porter, Eric D. Marsh PII: DOI: Reference:
S0306-4522(18)30389-0 https://doi.org/10.1016/j.neuroscience.2018.05.039 NSC 18476
To appear in:
Neuroscience
Received Date: Accepted Date:
11 May 2017 24 May 2018
Please cite this article as: S.L. Rogers, E. Rankin-Gee, R.M. Risbud, B.E. Porter, E.D. Marsh, Normal development of the Perineuronal net in humans; in patients with and without epilepsy, Neuroscience (2018), doi: https://doi.org/ 10.1016/j.neuroscience.2018.05.039
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Title: Normal development of the Perineuronal net in humans; in patients with and without epilepsy. Authors: Stephanie L. Rogers1, Elyse Rankin-Gee2, Rashmi M. Risbud1, Brenda E. Porter2, Eric D. Marsh1,3
Addresses: 1. Division of Child Neurology, Children’s Hospital of Philadelphia, Philadelphia, PA 19104. 2. Department of Neurology, Stanford University School of Medicine, Palo Alto CA, 94305 3. Departments of Pediatrics and Neurology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104.
Corresponding Author: Eric D. Marsh Abramson Research Building- Room 502E 3415 Civic Center Boulevard Children’s Hospital of Philadelphia Philadelphia PA 19104 Email: [email protected] Telephone: 215-590-5654 Fax Number: 215-590-3776
Running Title: Human Net Development
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Abstract The perineuronal net (PN), a highly organized extracellular matrix structure, is believed to play an important role in synaptic function, including maturation and stabilization. In addition to its role in restricting plasticity, alterations in the PN are implicated in disorders such as epilepsy and schizophrenia. However, the time course of PN development is not known in humans. Therefore we set out to document the developmental timeline of the PN formation in humans in 14 frontal and hippocampal specimens from donors aged 27 days to 31 years old. Using immunohistochemistry and western blotting, we demonstrate that the PN begins to form as early as the second month of life but does not reach its robust, mature appearance until around 8 years of age, though aggrecan cleavage products are observed prior to this. A similar developmental time course was observed in specimens from epilepsy patients. Our data suggests that aggrecan is present early in development but the structured PN develops throughout early childhood, similar to what has been observed in rodents. This timeline provides information for future pathological studies on the role of the PN in disease and an additional parallel between human and rodent development.
Key Words: Development, Epilepsy, Interneurons, Perineuronal Net
Introduction The perineuronal net (PN) is a specific component of the extracellular matrix visible as a highly organized, lattice-like structure around perisomatic synapses and proximal dendrites of particular subsets of neurons, including parvalbumin-positive interneurons and, to a lesser degree, pyramidal neurons (Hockfield et al. 1990; Bruckner et al. 1993; Celio and Blumcke 1994, Hartig et al. 1999, Giamanco and Matthews 2012). The PN is a unique extracellular matrix structure as it contains small quantities of several traditional extracellular matrix components (i.e. collagen, laminin and fibronectin) but is enriched in chondroitin sulfate proteoglycans and hyaluronan (Giamanco and Matthews, 2012). The chondroitin sulfate proteoglycans consist of neurocan, brevican, and aggrecan. Aggrecan (ACAN) is the lectican specific to the mature PNs (Matthews et al. 2002; Dino et al. 2006).
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The maturation of aggrecan-containing PNs is believed to play a critical role in the normal development of the brain, contributing to the regulation of synaptic plasticity and synaptic stabilization (Frischknecht et al. 2009). Perineuronal net development is activity dependent; decreased activity suppresses expression (Sur et al. 1988; Guimaraes et al.1990; Kind et al. 1995, 2013; Lander et al. 1997; McRae et al. 2007) while an increase augments expression (McRae et al. 2007). After a synapse is surrounded by the PN, little synaptic reorganization occurs (McRae et al. 2007), thus implicating the PN as a major regulator of plasticity. Further supporting its role in synapse stabilization, PN maturation has been demonstrated to coincide directly with the closure of the critical period in the visual system (Sur et al. 1988; Pizzorusso et al. 2002), the mouse barrel cortex (McRae et al. 2007), and memory consolidation in the amygdala (Gogolla et al. 2009). Further proof of the importance of net formation in the critical period was demonstrated by a series of experiments reactivating the critical period by degrading the PN (Pizzorusso et al. 2002, 2006; Gogolla et al. 2009). In addition to its role in normal development, the PN has also been implicated in various diseases, such as epilepsy, Alzheimer’s, schizophrenia, and autism. There have been studies focusing on the role of the PN in rodent epilepsy models, but not in humans. In rodents, McRae et al. (2012) demonstrated that the PN degrades after a prolonged seizure and predicts progression into spontaneous, chronic epilepsy in rodents. Net degradation also has biochemical implications, loss of net structures resulted in increased internal chloride concentration via ion influx into the ensheathed neuron (Glykys et al. 2014), which facilitates epileptiform activity (Dzhala et al. 2010). In addition to PN alteration in epilepsy, it has been hypothesized that the loss of the PN is related to the neuropathology of Alzheimer’s, schizophrenia and autism (for example see Hartig et al. 2001, Pantazopoulos et al. 2010; Leblanc and Fagollini 2011). In Alzheimer’s disease, the presence of perineuronal nets was associated with decreased Tau pathology, one of the hallmarks of AD (Hartig et al. 2001: Morawski et al. 2010, 2012). Autopsy specimens from patients with schizophrenia have shown degradation of the PN (Pantazopoulos et al. 2010; Berretta et al. 2012; Mauney et al. 2013). In addition, alterations of net structure have been hypothesized to play a role in autism spectrum disorder, as ASD has been conjectured to be a disorder of critical period dysregulation (Leblanc and Fagollini 2011; Berger et al. 2013).
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Despite the importance of the PNs in normal development, a comprehensive developmental timeline has not been reported in human tissue, though the post-natal development has been described in rodents (Bruckner et al. 2000; McCrae et al. 2010). Here we describe the developmental timeline of PN formation in human autopsy specimens and compare it to patients with epilepsy. The results of this study allows for direct comparison of pathologic specimens of different ages and give insight into normal development of the frontal cortex and hippocampus.
Experimental Procedures Cases: Human tissue was obtained from the University of Maryland NICHD brain tissue bank or the Stanford University School of Medicine after being deemed exempt by the Children’s Hospital of Philadelphia IRB. Tissue and de-identified patient data was collected with the approval of the Stanford University School of Medicine IRB. Control frozen and fixed samples from the middle-frontal gyrus and hippocampus of each donor between the ages of 27 days to 31 years with no known prior neurologic disorders were obtained. Thirty-two brain bank specimens were used in total (see Table 1). There were 6 epilepsy specimens were obtained during an epilepsy surgery resection, and specimens where frozen directly from the operating room. The location of the resected tissue varied by patient (see Table 2 for details of tissue location).
Identification of anatomical regions and applied nomenclature: Anatomical regions were identified by anti-neuronal nuclear protein (NeuN) marked sections adopting the nomenclature of brain regions from the Allen human brain atlas (Brain-map.org).
Immunohistochemistry: Immunofluorescence and immunohistochemistry was performed on fixed (1), frozen (2), and frozen-fixed (3) tissue. Methods for handling each type are presented. (1)The fixed tissue, except for the 27 day sample, was cryoprotected in 30% sucrose in phosphate buffer solution at 4C, then cut into 40 micron thick, free-floating sections and stored in 1X phosphate buffer solution with 0.2% sodium azide. The 27 day old tissue was cut in 30 micron thick slices directly onto glass slides and stored at -80 C due to the fragility of the tissue at this age. (2) The fresh-frozen tissue was first cut in the cryostat in 30 micron thick sections directly onto glass slides and stored at -80 C. Before processing, the slides were incubated at
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55 C for 15 minutes before being treated with 4% paraformaldehyde (PFA) in PBS at room temperature for 20 minutes. (3) The frozen-fixed tissue was fixed by incubating the frozen tissue in 4% PFA for 3 nights at 4 C. Frozen-fixed tissue blocks were then cryoprotected by placing the blocks in 30% sucrose solution until they sunk prior to cryosectioning in 40 micron thick, free-floating sections. Rationale for the multiple tissue processing steps is described below. Each tissue was stained using a number of antibodies directed against aggrecan and parvalbumin (See supplementary table 1). While multiple antibodies are reported to detect components of the perineuronal net (Brückner et al. 2008; Schmidt et al. 2010; Lendvai et al. 2012), only one (pan-aggrecan, Serotec, clone 7D4; Virgintino et al. 2009) consistently worked in our hands on post-mortem brain banked human tissue. CAT-315 and WFA did not stain the post-mortem tissue used in the study. Sections were first washed in a 1X PBS with 0.05% Tween (TBST) for five minutes, three times, at room temperature. The sections were next incubated for 30 minutes at room temperature in blocker A solution (2% BSA, 0.3% milk, 0.5% goat serum in PBST) to eliminate non-specific binding. After 3 washes in PBST, the primary antibodies were diluted in blocker A and incubated overnight at 4 C. The next day, the primary antibody was removed and sections were washed in PBST 3 times before being incubated for 2 hours at room temperature in secondary solution (1:400 dilution of Alexa Fluor (Abcam, Cambridge MA) secondary in a mixture of 1:1 Blocker A and PBST). Sections were washed 3 more times with PBST before being treated with Sudan Black B to reduce autofluorescence (Schnell et al. 1999). For parvalbumin staining in frozen-fixed tissue, Blocker B (10% BSA in PBS with 0.5% Triton X-100) replaced blocker A solution and PBST washes were substituted with 1X PBS containing 0.5% Triton X-100.
Tissue Imaging: All stained sections were imaged on either a Ziess Axioplan (Ziess Inc, Germany) or Leica DM6000B microscope (Leica Inc, Germany) or with images taken at 10X and 20X using a 10x tube for camera mount with final magnification at 100 or 200x. The camera was either a SPOT RT (1600x1200 pixels {1.92Mpixel} @ 11.8x8.9mm; Diagnostic Instruments) or Leica DFC360FX (1392 x 1040 pixels {1.4Mpixel}@9x6.7mm; Leica) digital camera. Images were imported into Photoshop and levels corrected all in parallel and pseudocolored. Multiple sections were taken from each age specimen and stained at different times.
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Multiple images from each age specimen were compared and representative images presented. SR and EM reviewed all images, with EM being ‘blind’ to the age of the subjects.
Protein Analysis: Tissue from fresh, frozen samples was homogenized on ice using a sonicator in RIPA buffer and protease inhibitor cocktail (Calbiochem). Protein extracts were quantified using a bicinchoninic acid (BCA) protein assay (Thermo Scientific). 25 µg of protein, 2.5 µL of 1M DTT and 12.5 µL of loading dye was added to each sample and sample volume was adjusted to 35 µL using 1X PBS. Samples were boiled at 90˚ C for ten minutes. Either a NuPage 4-12% Bis-Tris gel (Life Technologies, Grand Island NY) or 8% BisTris gel (poured in house) was used for electrophoresis prior to transfer to a nitrocellulose membrane (3 hour transfer at 0.23 Amps constant current). Precision Plus ProteinTM Kaleidoscope Standards (Life Technologies) were used to determine protein size. Membranes were incubated in 5% milk in low salt Tris-buffered saline with 0.1% Tween-20 (TBST) for one hour at room temperature to minimize nonspecific binding. Membranes were incubated overnight at 4˚ C in 5% milk solution in TBST and primary antibodies (See supplemental table 1). Horseradish peroxidase conjugated secondary antibodies (1:10000 in 5% milk; low salt TBST; Sigma) were incubated for one hour at room temperature with the blot. Immunoreactive proteins were visualized using the SuperSignal® resolving agents (Life Technologies).
Results To ensure that all post-mortem samples had cellular organization typical of the brain region of interest with an appropriate cell density, we stained all processed tissue with DAPI (4',6diamidino-2-phenylindole). Cell nuclei were present throughout the tissue at all ages (representative images from early ages (27 and 54 days respectively) in Fig. 1B-B’). Additionally, early developmental time points were co-stained with NeuN and DAPI to ensure that the cells present included differentiated neurons (Fig. 1A and A’). All sections displayed positive staining for both DAPI and NeuN further confirming that the tissue was relatively preserved at all ages (data not shown). All subsequent figures display the timeline of PV and the PN from the CA1/subiculum region in the hippocampus (Fig. 1C) and layer 3 in the middle prefrontal cortex (middle frontal gyrus; MFG) (Fig. 1D), but the findings were consistent across the cortex and hippocampus.
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The time course of PV expression in humans has been previously reported (Yew et al. 1997; Grateron et al. 2003; Fung et al. 2010), but the relationship of PV development to PN development has not been described. The PN surrounds a number of cell types throughout the brain, but in the cortex and hippocampus, the most well studied interaction is with parvalbuminpositive interneurons. Therefore we characterized the developmental timelines for PV and the PN concurrently during human development. We stained fixed tissue with PV antibody (Swant PV25) and observed PV expression as early as one month of life in both the prefrontal cortex and the hippocampus, although the early staining was sparse and of relatively low intensity (Fig. 2AB 27 days; insets highlight representative neuronal staining). At older ages, PV immunoflorescense became more prominent until around 2 years of age, at which time a mature pattern of staining exists. From 2 years to 20 years (our oldest sample tested for PV) there was no qualitative difference in the number or intensity of PV labeling in both frontal cortex and hippocampus (Fig. 2A and B- all ages). Interestingly, in these few sections, the hippocampal PV expression appeared stronger at postnatal day 27 than in the MFG, but reaches maturity at the same time as the MFG.
Once the PV developmental time course was established, we determined the maturation pattern of aggrecan, the core component of the PN. Several antibodies have been developed that detect the fully developed PN, each detecting a different component of the PN. The three best validated antibodies for detecting the PN are CAT-315, which binds an epitope of the Chondroitin sulfate proteoglycan aggrecan, WFA, a lectin which binds to the terminal N-acetylgalactosamine on the chondroitin sulfate proteoglycans sidechains of aggrecan, and aggrecan clone 7D4, which detects epitopes in the N-terminal G1-IGD-G2 region. Using fresh-frozen tissue we were able to identify aggrecan staining using the 7D4 antibody. The other aggrecan antibodies and tissue preparations did not give a detectible signal. The 7D4 staining, however, was reproducible over a number of staining runs and different sections from all samples and clearly detected the typical appearance of the PN (Fig. 3B; insets highlight morphology of perineuronal nets at different ages). Using the 7D4 aggrecan antibody PNs were not present in either the MFG or the hippocampus during the first month of life (Fig. 3A and C- 27d). By 2 months aggrecan begins to condense into perineuronal nets in the MFG but not the hippocampus, although the staining was hazy in the background with lightly labeled perineuronal nets surrounding very few cells (Fig. 3A 54d for
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MFG). At two years of age the intensity and number of aggrecan containing PNs increased in both the MFG and hippocampus. (Fig. 3A-C 2y 71d) Aggrecan staining continues to increase in number per 10x field and net quality until about age 8, at which time the PN appears to have reached maturity both in morphology and amount (Fig. 3A-C- labeled ages).
After establishing the PV and aggrecan time courses independently, we wanted to study their colocalization during development. While we observed expression of aggrecan in structures with PN morphology in the MFG early in life and the hippocampus at a year, aggrecan and PV immunohistochemistry did not co-localize until 2 years of age in the MFG and hippocampus (Fig. 4). After this time-point, the pattern of maturation of the PN matches the independent maturation of aggrecan, reaching complete co-localization, as nearly all parvalbumin-positive interneurons were ensheathed by a mature appearing PN by age 8 in both the MFG and the hippocampus (Fig. 4).
Having established the developmental timeline for the PN in humans using immunohistochemistry, we attempted to confirm the immunohistochemistry by measuring PN components during development via western blots. The Serotec 7D4 aggrecan antibody detected a band at the appropriate molecular weight of 150-200kDa (Fig 5A,B) without chondroitinase treatment. The ~200kDa band matched the immunohistochemistry developmental increase in expression well in the cortex (Fig 5A) and partially in the hippocampus (Fig 5B). To further determine if net components were present during development, we probed for the neoepitope NITEGE, a known ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) aggrecan cleavage product, allowing for an indirect assessment of aggrecan secretion by determining the level of the cleavage product in the tissue. The NITEGE neo-epitope was present at all time points supporting production and ongoing degradation of aggrecan even when the PN, <1 year of age, appears poorly formed by immunohistochemistry (Fig 5C,D). Hyaluronan synthase (HAS), specifically HAS3, was also probed to ensure production of the PN at these time points, as HAS3 has an integral role in the formation of the PN. HAS3, a neuronal membrane bound protein, both synthesizes and acts as a receptor for hyaluronan, a major component of the extracellular matrix (Kwok et al. 2010). HAS3 expression is variable across development in our western blot data, but was present in all the samples (data
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not shown) suggesting PN formation can occur at all of the tested ages. Lastly, as a check for a known developmental increase in Gycerol 3-Phosphate dehydrogenase (GAPDH), we probed for the protein. GAPDH increased developmentally, as expected, (Fig 5E,F) where as the NITEGE and HAS3 have similar concentrations at all ages’ demonstrating the increase in ACAN was not due to a loading error. We sought to elucidate whether the PN is susceptible to degradation as a result of recurrent seizures. McRae et al. (2012) showed that after inducing status epilepticus in rats, the PN is degraded. Using fresh-frozen tissue samples obtained during epilepsy surgery, we studied the immuno-reactivity of the PN in epilepsy patients from childhood to young adulthood (see Table 2 for patient details and outcomes). Somewhat surprising, based on the rodent data, no perceptible difference in PN development was present in children and adults with epilepsy. Supporting our post mortem developmental time course, the PN appearance and numbers in the epilepsy patients matched the non-epilepsy patient profile. PNs were present in the first year of life and appeared to reach full maturity by adolescence (Fig. 6).
Discussion We have, for the first time, demonstrated the concurrent time course of PN and PV interneuron maturation in human prefrontal cortex and hippocampus from the neonatal period to adulthood. Our findings demonstrate that in humans medial frontal gyrus/prefrontal cortex and hippocampus, PV, a marker of a subset of GABAergic interneurons, is present at birth, although at low levels, and increases reaching peak levels at 2 years of age in both the MFG and the hippocampus that persists into adulthood. These data are consistent with the timeline of PV development as described by Fung et al. (2010). Aggrecan, a major component of the PN, appears by immunohistochemical staining slightly later than PV, appearing in the human MFG during the second month of life and the second year in the hippocampus. From 2 years of age into late childhood, the PN continues to condense around PV-positive interneurons until it reaches a mature appearance at 8 years of age. From 8 to 32 years of age both PN and PV immunoreactivity appears stable and co-labeled in both the MFG and hippocampus. Finally, our data suggests that the development of the PN was unaffected by recurrent seizures (epilepsy) in humans, though more cases should be examined.
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Our data corroborates the trend from Mauney et al. (2013), which showed that PNs increase in density through postnatal development, and the rate plateaus around the preadolescent period in humans. Our results are an aid to researchers performing translational research on rodents to understand human disorders as a set of defined milestones is typically used to determine equivalency measure between rodent and human brain development (Hagberg et al. 1997; Avishai-Eliner et al. 2002). Our data is the first to demonstrate that PN and PV developments can be used as a marker of a developmental time window of postnatal day (p)14 to p60 (McCrae et al. 2010) in rodents being roughly equivalent to ages 1 to 8 years in humans. In addition to providing the time course for PN development in humans, and equivalency between rodent and human developmental windows, this study suggests a timeline for plasticity and critical period closure in humans. The PN is believed to regulate synaptic plasticity, as without a physical barrier locking a synapse in place, synapses can be modified and shifted through the extracellular space. Due to its role in regulating plasticity, the formation of the PN is one marker for the closure of the critical period. If our timeline of PN development is matched (which we expect as MFG and hippocampus time courses are similar but one is neocortical the other archicortex) in visual or auditory cortices where defined critical periods exist one could hypothesize that the critical period closes by age 8 in humans. This has potential implications for rehabilitation therapies and the effects of treatments on the developing brain. The role of the PN in epilepsy has been raised by a series of epilepsy studies (McCrae et al. 2012; Rankin-Gee et al. 2015) using chemoconvulsant or electroconvulsant seizures. The etiology of the epilepsy in our small cohort is different than the acute epilepsy models in rodents. The closest parallel was one patient who had a history of prolonged episode of status epilepticus as the predisposition to the development of epilepsy. The etiology of the epilepsy and the semiology of the seizures may be important for the seizures to have an impact on the perineuronal nets. In addition, we are unable to conclude that the PNs are unaffected in the seizure onset zone as all the specimens were large and not carefully vetted for seizure onset versus secondary spread. It will be important in the future to analyze the PN in the seizure onset zone and in samples with a variety of epilepsy etiologies to determine if the PN is abnormal in human epilepsy. Studying human tissue provided unique challenges in performing these experiments. There are multiple limitations with human studies in general and our study in particular. First,
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controlling for the time from death to collecting specimens, the post-mortem delay (PMD) is an issue and differences between surgical collected samples and post-mortem samples need to be accounted for. In this study, to control for PMD as best as possible, our samples were selected with a longest post mortem delay time of 24 hours. The PMD can affect antigen binding, as previously shown for WFA staining (Morawski et al. 2012), and WFA staining did not produce detectable immunohistology in our human tissue samples. However, the aggrecan 7D4 clone has been shown to work independently of PMD (Morawski et al. 2012) and, in our hands, was the only antibody that efficiently stained human tissue, though it was affected by time in fixative. In fact, the amount of time in fixation impacted the visibility of antigen binding with both aggrecan and PV. The longer the tissue was fixed, the more robust parvalbumin staining became while aggrecan staining was diminished. This loss of aggrecan staining is likely due to crosslinking of the ECM by paraformaldehyde. A few groups have successfully performed immunohistochemistry with antibodies that were not successful in our study (Brückner et al. 2008; Schmidt et al. 2010; Lendvai et al. 2012). These studies all had direct control of obtaining autopsy specimens and therefore could control for fixation conditions and duration. A second limitation is obtaining sufficient number of samples from human brain tissue. This is an issue all researchers face, particularly in the very young ages that are the focus of this study. Since the developmental process appeared fairly linear in our sample time line we believe that this accurately represents what occurs in the typically developing population. In addition, the few epilepsy samples matched the developmental time line from the “control” tissue, lending further credence to the time line presented herein strongly suggesting that the time line proposed in this study is reliable and reproducible. A third issue was the quality of the tissue. The long PMD, the way tissue is handled in brain banks and the difficult of using human tissue results in cutting and freezing artifact in our tissue and decreases the success of some of the staining as stated above. While these are all issues that make this type of project challenging, the consistency of the results between the MFG and the Hippocampal tissue from the brain bank with the tissue from the epilepsy specimens all argues for the developmental time line presented. Future studies with more samples to corroborate and extend this study into other components of the PN, interneurons, and the inhibitory circuits in the brain should be performed.
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Equally as challenging was performing the western blot studies. Not all antibodies worked as found in the literature, though the aggrecan western blots confirmed the immunohistochemical data and the direct protein measurements of the NITEGE and HAS3 support the presence of all the components necessary to form mature PN the issues (Virgintino et al. 2009). The difficulties of full length protein detection may be due to the vagaries of human tissue processing, the complexity of the tissue, or a complicated biology (either formation, maturation, or degradation). Our data does suggest the presence of mature aggrecan with the time line described by the immunohistochemistry from the normal and epileptic samples.
The perineuronal net plays a critical role in normal brain development by regulating synaptic plasticity and acting as a marker for the closure of the critical period. These data establish a timeline for PN development in humans. With a developmental timeline established for the PN in humans, a more accurate and in depth analysis of neurologic disorders that might affect the PN can be performed.
Acknowledgements: All tissue was purchased from the University of Maryland Brain and Tissue Bank, and without their kindness and cooperation, this project would not have been possible. We would also like to thank Dr. Rick Matthews for his gift of the CAT 315 antibody, critical reading of the manuscript, and his advice on working with human tissue, staining, and western analysis protocols. We would also like to thank Almedia McCoy for her assistance in generating the final images for this manuscript.
Funding: This work was funded by the Research and Cure of Refractory Epilepsy fund (EDM) and grants from the National Institutes of Health, National Institutes of Neurological Disease and Stroke: RO1 RO1NS082761(EDM) and RO1NS056222 (BEP).
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(2013) Developmental pattern of peineuronal nets in the human prefrontal cortex and their deficit in schizophrenia. Biol Psychiatry. 74(6): 427-435.
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Tables Table 1: Details of Human Tissue used Age
Sex
Frozen Control Tissue 0y 27d Male 0y 54d Male
Race
Post Mortem Delay
Cause of death
13 hrs 6 hrs 18 hrs
Asphyxia by suffocation Sudden unexplained death in infancy Respiratory failure
27 hrs
Asthma
17 hrs 12 hrs 12 hrs 16 hrs 26 hrs
Drowning Drowning Drowning Cardiac arrhythmia Cardiac arrhythmia
13 hrs 25 hrs 35 hrs 24 hrs 5 hrs 13 hrs 18 hrs
Asphyxia by suffocation Drowning Multisystem failure Respiratory insufficiency Cardiac arrhythmia Drowning Accident, multiple injuries
1y 159d
Male
2y 71d
Male
4y 258d 7y 272d 8y 286d 14y 60d 31y 229d Fixed Tissue 0y 27d 1y 262d 2y 195d 7y 276d 8y 2d 12y 297d 20y 50d
Male Male Male Male Male
Caucasian African American African American African American Caucasian Caucasian Caucasian Caucasian Caucasian
Male Male Male Male Male Male Male
Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian
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Table 2: Details of resected tissue from epilepsy surgery patients Age at surgery
Epilepsy Duration
Pathology
Resection location
Follow-up duration
Seizure Status (modified Engel)
1year 3 months 4 years
1 year 2 months 4 years
Right parietal
9 months
1 3
Unknown
Left frontal and temporal Right functional hemispherectomy
2 years
13 years
2 years
1
13 years
8 years
Right frontal
11 months
3
44 years
5 years
47 years
13 years
Tuberous Sclerosis Gliosisperinatal stroke Focal Cortical Dysplasia type IIB Gliosis- history of hemolytic uremic syndrome Ganglioglioma and mesial temporal sclerosis Mesial temporal sclerosis
Right temporal lobe 1 year
1
Left temporal lobe
1
1 month
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Figure Legends: Figure 1: Tissue quality assessment and location. Representative (A) middle frontal gyrus (MFG) and (A’) hippocampal tissue samples stained for NeuN at 27 and 54 days of age. (b) Samples from the same ages, displayed DAPI staining as well. Additionally, the organization typical of both the (C) hippocampus and (D) medial prefrontal cortex are present. Dashed boxes indicate the enlarged areas imaged in all subsequent figures; CA1/subiculum region in the hippocampus and layer 3 of the cortex in the MFG. White scale bar in A, A’, B, B’ is 75microns. Scale bar in D is 100 microns. B and C are 10x images stitched together to visual full brain region. DG- Dentate Gyrus. CA1- Cornu Ammonis 1, L- layer.
Figure 2: Developmental time course of parvalbumin expression in the mpc and hippocampus. Parvalbumin is expressed at 27 days in both the (A) Medial prefrontal cortex and (B) the hippocampus. Expression continues to increase until reaching maturity at approximately 2 years of age (ages stated in each image). White scale bars in all images are 75 microns
Figure 3: Aggrecan immunohistochemistry increases with age in middle frontal gyrus and hippocampus. Aggrecan condenses into perineuronal nets starting around 54 days after birth in the (A) MFG and by 2 years in the (C) hippocampus. All images at 10X magnification. Arrows highlight the few and light perineuronal nets in the young ages. However, the intensity of aggrecan immunohistochemistry in the perineuronal nets increases until approximately 8 years of age in the both regions at which time they appear to be fully mature (ages stated in each image). Insets are enlarged images of representative net quality over time (ages stated in each image). White scale bar is 75microns in all images.
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Figure 4: Co-localization of aggrecan and parvalbumin immunoreactivity during human development. Samples were co-labeled with aggrecan and parvalbumin antibodies in frozenfixed tissue. Parvalbumin, aggrecan, and the superimposed images are shown for both Medial Prefrontal cortex tissue (hippocampal tissue not shown). Co-localization of the perineuronal nets around parvalbumin positive interneurons does not begin until age 2 years (not shown), becomes more prominent by 4 years (A) and reaches full maturity with complete co-labeling by age 8 (CE). Insets highlight the increasing co-localization of the PV and aggrecan staining. White scale bar in all merged images is 75 microns
Figure 5: Western blot analysis of aggrecan, NITEGE, and GAPDH expression. A, C, and E are representative blots and quantification from (MFG). B,C, and F are representative blots and quantification from hippocampus. (A-B) Aggrecan (ACAN) detected by the 7D4 antibody is present at ~ 175kD and increases in intensity with age. (C-D) NITEGE detects an epitope of ADAMTS proteolysis of aggrecan also present in all samples (E-F) GAPDH a housekeeping enzyme known to increase in abundance with age also increases in intensity with age. ADAMTS- A Disintegrin And Metalloproteinase with Thrombospondin MotifS). NITEGE- is the Aggrecan monoclonal antibody to C-terminal neoepitope.
Figure 6: Aggrecan development in human epilepsy patient tissue. Development of the PN appears to follow a similar trajectory in epilepsy patients with the perineuronal nets present by (A) 1 year of age. Net expression continues to increase as observed in the amount and quality of the perineuronal nets in 4 year old (B) frontal lobe and (B’) hippocampus. (C and C’) Nets reach
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maturity in the frontal lobe by adolescence, as shown in two patients both aged 13. (D) Net maturation in the hippocampus appears to be unaltered in an older patient with epilepsy, aged 44 years. White scale bar in all image is 75 microns.
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Highlights: This is the first presentation of the human developmental timeline of the Perineuronal Net. The Perineuronal Net begins to form in the second month of life and reaches its mature appearance around 8 years of age. Perineuronal Net development is not altered in a few patients with intractable epilepsy.
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S0306-4522(18)30389-0 https://doi.org/10.1016/j.neuroscience.2018.05.039 NSC 18476
To appear in:
Neuroscience
Received Date: Accepted Date:
11 May 2017 24 May 2018
Please cite this article as: S.L. Rogers, E. Rankin-Gee, R.M. Risbud, B.E. Porter, E.D. Marsh, Normal development of the Perineuronal net in humans; in patients with and without epilepsy, Neuroscience (2018), doi: https://doi.org/ 10.1016/j.neuroscience.2018.05.039
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title: Normal development of the Perineuronal net in humans; in patients with and without epilepsy. Authors: Stephanie L. Rogers1, Elyse Rankin-Gee2, Rashmi M. Risbud1, Brenda E. Porter2, Eric D. Marsh1,3
Addresses: 1. Division of Child Neurology, Children’s Hospital of Philadelphia, Philadelphia, PA 19104. 2. Department of Neurology, Stanford University School of Medicine, Palo Alto CA, 94305 3. Departments of Pediatrics and Neurology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104.
Corresponding Author: Eric D. Marsh Abramson Research Building- Room 502E 3415 Civic Center Boulevard Children’s Hospital of Philadelphia Philadelphia PA 19104 Email: [email protected] Telephone: 215-590-5654 Fax Number: 215-590-3776
Running Title: Human Net Development
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Abstract The perineuronal net (PN), a highly organized extracellular matrix structure, is believed to play an important role in synaptic function, including maturation and stabilization. In addition to its role in restricting plasticity, alterations in the PN are implicated in disorders such as epilepsy and schizophrenia. However, the time course of PN development is not known in humans. Therefore we set out to document the developmental timeline of the PN formation in humans in 14 frontal and hippocampal specimens from donors aged 27 days to 31 years old. Using immunohistochemistry and western blotting, we demonstrate that the PN begins to form as early as the second month of life but does not reach its robust, mature appearance until around 8 years of age, though aggrecan cleavage products are observed prior to this. A similar developmental time course was observed in specimens from epilepsy patients. Our data suggests that aggrecan is present early in development but the structured PN develops throughout early childhood, similar to what has been observed in rodents. This timeline provides information for future pathological studies on the role of the PN in disease and an additional parallel between human and rodent development.
Key Words: Development, Epilepsy, Interneurons, Perineuronal Net
Introduction The perineuronal net (PN) is a specific component of the extracellular matrix visible as a highly organized, lattice-like structure around perisomatic synapses and proximal dendrites of particular subsets of neurons, including parvalbumin-positive interneurons and, to a lesser degree, pyramidal neurons (Hockfield et al. 1990; Bruckner et al. 1993; Celio and Blumcke 1994, Hartig et al. 1999, Giamanco and Matthews 2012). The PN is a unique extracellular matrix structure as it contains small quantities of several traditional extracellular matrix components (i.e. collagen, laminin and fibronectin) but is enriched in chondroitin sulfate proteoglycans and hyaluronan (Giamanco and Matthews, 2012). The chondroitin sulfate proteoglycans consist of neurocan, brevican, and aggrecan. Aggrecan (ACAN) is the lectican specific to the mature PNs (Matthews et al. 2002; Dino et al. 2006).
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The maturation of aggrecan-containing PNs is believed to play a critical role in the normal development of the brain, contributing to the regulation of synaptic plasticity and synaptic stabilization (Frischknecht et al. 2009). Perineuronal net development is activity dependent; decreased activity suppresses expression (Sur et al. 1988; Guimaraes et al.1990; Kind et al. 1995, 2013; Lander et al. 1997; McRae et al. 2007) while an increase augments expression (McRae et al. 2007). After a synapse is surrounded by the PN, little synaptic reorganization occurs (McRae et al. 2007), thus implicating the PN as a major regulator of plasticity. Further supporting its role in synapse stabilization, PN maturation has been demonstrated to coincide directly with the closure of the critical period in the visual system (Sur et al. 1988; Pizzorusso et al. 2002), the mouse barrel cortex (McRae et al. 2007), and memory consolidation in the amygdala (Gogolla et al. 2009). Further proof of the importance of net formation in the critical period was demonstrated by a series of experiments reactivating the critical period by degrading the PN (Pizzorusso et al. 2002, 2006; Gogolla et al. 2009). In addition to its role in normal development, the PN has also been implicated in various diseases, such as epilepsy, Alzheimer’s, schizophrenia, and autism. There have been studies focusing on the role of the PN in rodent epilepsy models, but not in humans. In rodents, McRae et al. (2012) demonstrated that the PN degrades after a prolonged seizure and predicts progression into spontaneous, chronic epilepsy in rodents. Net degradation also has biochemical implications, loss of net structures resulted in increased internal chloride concentration via ion influx into the ensheathed neuron (Glykys et al. 2014), which facilitates epileptiform activity (Dzhala et al. 2010). In addition to PN alteration in epilepsy, it has been hypothesized that the loss of the PN is related to the neuropathology of Alzheimer’s, schizophrenia and autism (for example see Hartig et al. 2001, Pantazopoulos et al. 2010; Leblanc and Fagollini 2011). In Alzheimer’s disease, the presence of perineuronal nets was associated with decreased Tau pathology, one of the hallmarks of AD (Hartig et al. 2001: Morawski et al. 2010, 2012). Autopsy specimens from patients with schizophrenia have shown degradation of the PN (Pantazopoulos et al. 2010; Berretta et al. 2012; Mauney et al. 2013). In addition, alterations of net structure have been hypothesized to play a role in autism spectrum disorder, as ASD has been conjectured to be a disorder of critical period dysregulation (Leblanc and Fagollini 2011; Berger et al. 2013).
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Despite the importance of the PNs in normal development, a comprehensive developmental timeline has not been reported in human tissue, though the post-natal development has been described in rodents (Bruckner et al. 2000; McCrae et al. 2010). Here we describe the developmental timeline of PN formation in human autopsy specimens and compare it to patients with epilepsy. The results of this study allows for direct comparison of pathologic specimens of different ages and give insight into normal development of the frontal cortex and hippocampus.
Experimental Procedures Cases: Human tissue was obtained from the University of Maryland NICHD brain tissue bank or the Stanford University School of Medicine after being deemed exempt by the Children’s Hospital of Philadelphia IRB. Tissue and de-identified patient data was collected with the approval of the Stanford University School of Medicine IRB. Control frozen and fixed samples from the middle-frontal gyrus and hippocampus of each donor between the ages of 27 days to 31 years with no known prior neurologic disorders were obtained. Thirty-two brain bank specimens were used in total (see Table 1). There were 6 epilepsy specimens were obtained during an epilepsy surgery resection, and specimens where frozen directly from the operating room. The location of the resected tissue varied by patient (see Table 2 for details of tissue location).
Identification of anatomical regions and applied nomenclature: Anatomical regions were identified by anti-neuronal nuclear protein (NeuN) marked sections adopting the nomenclature of brain regions from the Allen human brain atlas (Brain-map.org).
Immunohistochemistry: Immunofluorescence and immunohistochemistry was performed on fixed (1), frozen (2), and frozen-fixed (3) tissue. Methods for handling each type are presented. (1)The fixed tissue, except for the 27 day sample, was cryoprotected in 30% sucrose in phosphate buffer solution at 4C, then cut into 40 micron thick, free-floating sections and stored in 1X phosphate buffer solution with 0.2% sodium azide. The 27 day old tissue was cut in 30 micron thick slices directly onto glass slides and stored at -80 C due to the fragility of the tissue at this age. (2) The fresh-frozen tissue was first cut in the cryostat in 30 micron thick sections directly onto glass slides and stored at -80 C. Before processing, the slides were incubated at
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55 C for 15 minutes before being treated with 4% paraformaldehyde (PFA) in PBS at room temperature for 20 minutes. (3) The frozen-fixed tissue was fixed by incubating the frozen tissue in 4% PFA for 3 nights at 4 C. Frozen-fixed tissue blocks were then cryoprotected by placing the blocks in 30% sucrose solution until they sunk prior to cryosectioning in 40 micron thick, free-floating sections. Rationale for the multiple tissue processing steps is described below. Each tissue was stained using a number of antibodies directed against aggrecan and parvalbumin (See supplementary table 1). While multiple antibodies are reported to detect components of the perineuronal net (Brückner et al. 2008; Schmidt et al. 2010; Lendvai et al. 2012), only one (pan-aggrecan, Serotec, clone 7D4; Virgintino et al. 2009) consistently worked in our hands on post-mortem brain banked human tissue. CAT-315 and WFA did not stain the post-mortem tissue used in the study. Sections were first washed in a 1X PBS with 0.05% Tween (TBST) for five minutes, three times, at room temperature. The sections were next incubated for 30 minutes at room temperature in blocker A solution (2% BSA, 0.3% milk, 0.5% goat serum in PBST) to eliminate non-specific binding. After 3 washes in PBST, the primary antibodies were diluted in blocker A and incubated overnight at 4 C. The next day, the primary antibody was removed and sections were washed in PBST 3 times before being incubated for 2 hours at room temperature in secondary solution (1:400 dilution of Alexa Fluor (Abcam, Cambridge MA) secondary in a mixture of 1:1 Blocker A and PBST). Sections were washed 3 more times with PBST before being treated with Sudan Black B to reduce autofluorescence (Schnell et al. 1999). For parvalbumin staining in frozen-fixed tissue, Blocker B (10% BSA in PBS with 0.5% Triton X-100) replaced blocker A solution and PBST washes were substituted with 1X PBS containing 0.5% Triton X-100.
Tissue Imaging: All stained sections were imaged on either a Ziess Axioplan (Ziess Inc, Germany) or Leica DM6000B microscope (Leica Inc, Germany) or with images taken at 10X and 20X using a 10x tube for camera mount with final magnification at 100 or 200x. The camera was either a SPOT RT (1600x1200 pixels {1.92Mpixel} @ 11.8x8.9mm; Diagnostic Instruments) or Leica DFC360FX (1392 x 1040 pixels {1.4Mpixel}@9x6.7mm; Leica) digital camera. Images were imported into Photoshop and levels corrected all in parallel and pseudocolored. Multiple sections were taken from each age specimen and stained at different times.
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Multiple images from each age specimen were compared and representative images presented. SR and EM reviewed all images, with EM being ‘blind’ to the age of the subjects.
Protein Analysis: Tissue from fresh, frozen samples was homogenized on ice using a sonicator in RIPA buffer and protease inhibitor cocktail (Calbiochem). Protein extracts were quantified using a bicinchoninic acid (BCA) protein assay (Thermo Scientific). 25 µg of protein, 2.5 µL of 1M DTT and 12.5 µL of loading dye was added to each sample and sample volume was adjusted to 35 µL using 1X PBS. Samples were boiled at 90˚ C for ten minutes. Either a NuPage 4-12% Bis-Tris gel (Life Technologies, Grand Island NY) or 8% BisTris gel (poured in house) was used for electrophoresis prior to transfer to a nitrocellulose membrane (3 hour transfer at 0.23 Amps constant current). Precision Plus ProteinTM Kaleidoscope Standards (Life Technologies) were used to determine protein size. Membranes were incubated in 5% milk in low salt Tris-buffered saline with 0.1% Tween-20 (TBST) for one hour at room temperature to minimize nonspecific binding. Membranes were incubated overnight at 4˚ C in 5% milk solution in TBST and primary antibodies (See supplemental table 1). Horseradish peroxidase conjugated secondary antibodies (1:10000 in 5% milk; low salt TBST; Sigma) were incubated for one hour at room temperature with the blot. Immunoreactive proteins were visualized using the SuperSignal® resolving agents (Life Technologies).
Results To ensure that all post-mortem samples had cellular organization typical of the brain region of interest with an appropriate cell density, we stained all processed tissue with DAPI (4',6diamidino-2-phenylindole). Cell nuclei were present throughout the tissue at all ages (representative images from early ages (27 and 54 days respectively) in Fig. 1B-B’). Additionally, early developmental time points were co-stained with NeuN and DAPI to ensure that the cells present included differentiated neurons (Fig. 1A and A’). All sections displayed positive staining for both DAPI and NeuN further confirming that the tissue was relatively preserved at all ages (data not shown). All subsequent figures display the timeline of PV and the PN from the CA1/subiculum region in the hippocampus (Fig. 1C) and layer 3 in the middle prefrontal cortex (middle frontal gyrus; MFG) (Fig. 1D), but the findings were consistent across the cortex and hippocampus.
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The time course of PV expression in humans has been previously reported (Yew et al. 1997; Grateron et al. 2003; Fung et al. 2010), but the relationship of PV development to PN development has not been described. The PN surrounds a number of cell types throughout the brain, but in the cortex and hippocampus, the most well studied interaction is with parvalbuminpositive interneurons. Therefore we characterized the developmental timelines for PV and the PN concurrently during human development. We stained fixed tissue with PV antibody (Swant PV25) and observed PV expression as early as one month of life in both the prefrontal cortex and the hippocampus, although the early staining was sparse and of relatively low intensity (Fig. 2AB 27 days; insets highlight representative neuronal staining). At older ages, PV immunoflorescense became more prominent until around 2 years of age, at which time a mature pattern of staining exists. From 2 years to 20 years (our oldest sample tested for PV) there was no qualitative difference in the number or intensity of PV labeling in both frontal cortex and hippocampus (Fig. 2A and B- all ages). Interestingly, in these few sections, the hippocampal PV expression appeared stronger at postnatal day 27 than in the MFG, but reaches maturity at the same time as the MFG.
Once the PV developmental time course was established, we determined the maturation pattern of aggrecan, the core component of the PN. Several antibodies have been developed that detect the fully developed PN, each detecting a different component of the PN. The three best validated antibodies for detecting the PN are CAT-315, which binds an epitope of the Chondroitin sulfate proteoglycan aggrecan, WFA, a lectin which binds to the terminal N-acetylgalactosamine on the chondroitin sulfate proteoglycans sidechains of aggrecan, and aggrecan clone 7D4, which detects epitopes in the N-terminal G1-IGD-G2 region. Using fresh-frozen tissue we were able to identify aggrecan staining using the 7D4 antibody. The other aggrecan antibodies and tissue preparations did not give a detectible signal. The 7D4 staining, however, was reproducible over a number of staining runs and different sections from all samples and clearly detected the typical appearance of the PN (Fig. 3B; insets highlight morphology of perineuronal nets at different ages). Using the 7D4 aggrecan antibody PNs were not present in either the MFG or the hippocampus during the first month of life (Fig. 3A and C- 27d). By 2 months aggrecan begins to condense into perineuronal nets in the MFG but not the hippocampus, although the staining was hazy in the background with lightly labeled perineuronal nets surrounding very few cells (Fig. 3A 54d for
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MFG). At two years of age the intensity and number of aggrecan containing PNs increased in both the MFG and hippocampus. (Fig. 3A-C 2y 71d) Aggrecan staining continues to increase in number per 10x field and net quality until about age 8, at which time the PN appears to have reached maturity both in morphology and amount (Fig. 3A-C- labeled ages).
After establishing the PV and aggrecan time courses independently, we wanted to study their colocalization during development. While we observed expression of aggrecan in structures with PN morphology in the MFG early in life and the hippocampus at a year, aggrecan and PV immunohistochemistry did not co-localize until 2 years of age in the MFG and hippocampus (Fig. 4). After this time-point, the pattern of maturation of the PN matches the independent maturation of aggrecan, reaching complete co-localization, as nearly all parvalbumin-positive interneurons were ensheathed by a mature appearing PN by age 8 in both the MFG and the hippocampus (Fig. 4).
Having established the developmental timeline for the PN in humans using immunohistochemistry, we attempted to confirm the immunohistochemistry by measuring PN components during development via western blots. The Serotec 7D4 aggrecan antibody detected a band at the appropriate molecular weight of 150-200kDa (Fig 5A,B) without chondroitinase treatment. The ~200kDa band matched the immunohistochemistry developmental increase in expression well in the cortex (Fig 5A) and partially in the hippocampus (Fig 5B). To further determine if net components were present during development, we probed for the neoepitope NITEGE, a known ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) aggrecan cleavage product, allowing for an indirect assessment of aggrecan secretion by determining the level of the cleavage product in the tissue. The NITEGE neo-epitope was present at all time points supporting production and ongoing degradation of aggrecan even when the PN, <1 year of age, appears poorly formed by immunohistochemistry (Fig 5C,D). Hyaluronan synthase (HAS), specifically HAS3, was also probed to ensure production of the PN at these time points, as HAS3 has an integral role in the formation of the PN. HAS3, a neuronal membrane bound protein, both synthesizes and acts as a receptor for hyaluronan, a major component of the extracellular matrix (Kwok et al. 2010). HAS3 expression is variable across development in our western blot data, but was present in all the samples (data
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not shown) suggesting PN formation can occur at all of the tested ages. Lastly, as a check for a known developmental increase in Gycerol 3-Phosphate dehydrogenase (GAPDH), we probed for the protein. GAPDH increased developmentally, as expected, (Fig 5E,F) where as the NITEGE and HAS3 have similar concentrations at all ages’ demonstrating the increase in ACAN was not due to a loading error. We sought to elucidate whether the PN is susceptible to degradation as a result of recurrent seizures. McRae et al. (2012) showed that after inducing status epilepticus in rats, the PN is degraded. Using fresh-frozen tissue samples obtained during epilepsy surgery, we studied the immuno-reactivity of the PN in epilepsy patients from childhood to young adulthood (see Table 2 for patient details and outcomes). Somewhat surprising, based on the rodent data, no perceptible difference in PN development was present in children and adults with epilepsy. Supporting our post mortem developmental time course, the PN appearance and numbers in the epilepsy patients matched the non-epilepsy patient profile. PNs were present in the first year of life and appeared to reach full maturity by adolescence (Fig. 6).
Discussion We have, for the first time, demonstrated the concurrent time course of PN and PV interneuron maturation in human prefrontal cortex and hippocampus from the neonatal period to adulthood. Our findings demonstrate that in humans medial frontal gyrus/prefrontal cortex and hippocampus, PV, a marker of a subset of GABAergic interneurons, is present at birth, although at low levels, and increases reaching peak levels at 2 years of age in both the MFG and the hippocampus that persists into adulthood. These data are consistent with the timeline of PV development as described by Fung et al. (2010). Aggrecan, a major component of the PN, appears by immunohistochemical staining slightly later than PV, appearing in the human MFG during the second month of life and the second year in the hippocampus. From 2 years of age into late childhood, the PN continues to condense around PV-positive interneurons until it reaches a mature appearance at 8 years of age. From 8 to 32 years of age both PN and PV immunoreactivity appears stable and co-labeled in both the MFG and hippocampus. Finally, our data suggests that the development of the PN was unaffected by recurrent seizures (epilepsy) in humans, though more cases should be examined.
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Our data corroborates the trend from Mauney et al. (2013), which showed that PNs increase in density through postnatal development, and the rate plateaus around the preadolescent period in humans. Our results are an aid to researchers performing translational research on rodents to understand human disorders as a set of defined milestones is typically used to determine equivalency measure between rodent and human brain development (Hagberg et al. 1997; Avishai-Eliner et al. 2002). Our data is the first to demonstrate that PN and PV developments can be used as a marker of a developmental time window of postnatal day (p)14 to p60 (McCrae et al. 2010) in rodents being roughly equivalent to ages 1 to 8 years in humans. In addition to providing the time course for PN development in humans, and equivalency between rodent and human developmental windows, this study suggests a timeline for plasticity and critical period closure in humans. The PN is believed to regulate synaptic plasticity, as without a physical barrier locking a synapse in place, synapses can be modified and shifted through the extracellular space. Due to its role in regulating plasticity, the formation of the PN is one marker for the closure of the critical period. If our timeline of PN development is matched (which we expect as MFG and hippocampus time courses are similar but one is neocortical the other archicortex) in visual or auditory cortices where defined critical periods exist one could hypothesize that the critical period closes by age 8 in humans. This has potential implications for rehabilitation therapies and the effects of treatments on the developing brain. The role of the PN in epilepsy has been raised by a series of epilepsy studies (McCrae et al. 2012; Rankin-Gee et al. 2015) using chemoconvulsant or electroconvulsant seizures. The etiology of the epilepsy in our small cohort is different than the acute epilepsy models in rodents. The closest parallel was one patient who had a history of prolonged episode of status epilepticus as the predisposition to the development of epilepsy. The etiology of the epilepsy and the semiology of the seizures may be important for the seizures to have an impact on the perineuronal nets. In addition, we are unable to conclude that the PNs are unaffected in the seizure onset zone as all the specimens were large and not carefully vetted for seizure onset versus secondary spread. It will be important in the future to analyze the PN in the seizure onset zone and in samples with a variety of epilepsy etiologies to determine if the PN is abnormal in human epilepsy. Studying human tissue provided unique challenges in performing these experiments. There are multiple limitations with human studies in general and our study in particular. First,
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controlling for the time from death to collecting specimens, the post-mortem delay (PMD) is an issue and differences between surgical collected samples and post-mortem samples need to be accounted for. In this study, to control for PMD as best as possible, our samples were selected with a longest post mortem delay time of 24 hours. The PMD can affect antigen binding, as previously shown for WFA staining (Morawski et al. 2012), and WFA staining did not produce detectable immunohistology in our human tissue samples. However, the aggrecan 7D4 clone has been shown to work independently of PMD (Morawski et al. 2012) and, in our hands, was the only antibody that efficiently stained human tissue, though it was affected by time in fixative. In fact, the amount of time in fixation impacted the visibility of antigen binding with both aggrecan and PV. The longer the tissue was fixed, the more robust parvalbumin staining became while aggrecan staining was diminished. This loss of aggrecan staining is likely due to crosslinking of the ECM by paraformaldehyde. A few groups have successfully performed immunohistochemistry with antibodies that were not successful in our study (Brückner et al. 2008; Schmidt et al. 2010; Lendvai et al. 2012). These studies all had direct control of obtaining autopsy specimens and therefore could control for fixation conditions and duration. A second limitation is obtaining sufficient number of samples from human brain tissue. This is an issue all researchers face, particularly in the very young ages that are the focus of this study. Since the developmental process appeared fairly linear in our sample time line we believe that this accurately represents what occurs in the typically developing population. In addition, the few epilepsy samples matched the developmental time line from the “control” tissue, lending further credence to the time line presented herein strongly suggesting that the time line proposed in this study is reliable and reproducible. A third issue was the quality of the tissue. The long PMD, the way tissue is handled in brain banks and the difficult of using human tissue results in cutting and freezing artifact in our tissue and decreases the success of some of the staining as stated above. While these are all issues that make this type of project challenging, the consistency of the results between the MFG and the Hippocampal tissue from the brain bank with the tissue from the epilepsy specimens all argues for the developmental time line presented. Future studies with more samples to corroborate and extend this study into other components of the PN, interneurons, and the inhibitory circuits in the brain should be performed.
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Equally as challenging was performing the western blot studies. Not all antibodies worked as found in the literature, though the aggrecan western blots confirmed the immunohistochemical data and the direct protein measurements of the NITEGE and HAS3 support the presence of all the components necessary to form mature PN the issues (Virgintino et al. 2009). The difficulties of full length protein detection may be due to the vagaries of human tissue processing, the complexity of the tissue, or a complicated biology (either formation, maturation, or degradation). Our data does suggest the presence of mature aggrecan with the time line described by the immunohistochemistry from the normal and epileptic samples.
The perineuronal net plays a critical role in normal brain development by regulating synaptic plasticity and acting as a marker for the closure of the critical period. These data establish a timeline for PN development in humans. With a developmental timeline established for the PN in humans, a more accurate and in depth analysis of neurologic disorders that might affect the PN can be performed.
Acknowledgements: All tissue was purchased from the University of Maryland Brain and Tissue Bank, and without their kindness and cooperation, this project would not have been possible. We would also like to thank Dr. Rick Matthews for his gift of the CAT 315 antibody, critical reading of the manuscript, and his advice on working with human tissue, staining, and western analysis protocols. We would also like to thank Almedia McCoy for her assistance in generating the final images for this manuscript.
Funding: This work was funded by the Research and Cure of Refractory Epilepsy fund (EDM) and grants from the National Institutes of Health, National Institutes of Neurological Disease and Stroke: RO1 RO1NS082761(EDM) and RO1NS056222 (BEP).
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Tables Table 1: Details of Human Tissue used Age
Sex
Frozen Control Tissue 0y 27d Male 0y 54d Male
Race
Post Mortem Delay
Cause of death
13 hrs 6 hrs 18 hrs
Asphyxia by suffocation Sudden unexplained death in infancy Respiratory failure
27 hrs
Asthma
17 hrs 12 hrs 12 hrs 16 hrs 26 hrs
Drowning Drowning Drowning Cardiac arrhythmia Cardiac arrhythmia
13 hrs 25 hrs 35 hrs 24 hrs 5 hrs 13 hrs 18 hrs
Asphyxia by suffocation Drowning Multisystem failure Respiratory insufficiency Cardiac arrhythmia Drowning Accident, multiple injuries
1y 159d
Male
2y 71d
Male
4y 258d 7y 272d 8y 286d 14y 60d 31y 229d Fixed Tissue 0y 27d 1y 262d 2y 195d 7y 276d 8y 2d 12y 297d 20y 50d
Male Male Male Male Male
Caucasian African American African American African American Caucasian Caucasian Caucasian Caucasian Caucasian
Male Male Male Male Male Male Male
Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian
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Table 2: Details of resected tissue from epilepsy surgery patients Age at surgery
Epilepsy Duration
Pathology
Resection location
Follow-up duration
Seizure Status (modified Engel)
1year 3 months 4 years
1 year 2 months 4 years
Right parietal
9 months
1 3
Unknown
Left frontal and temporal Right functional hemispherectomy
2 years
13 years
2 years
1
13 years
8 years
Right frontal
11 months
3
44 years
5 years
47 years
13 years
Tuberous Sclerosis Gliosisperinatal stroke Focal Cortical Dysplasia type IIB Gliosis- history of hemolytic uremic syndrome Ganglioglioma and mesial temporal sclerosis Mesial temporal sclerosis
Right temporal lobe 1 year
1
Left temporal lobe
1
1 month
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Figure Legends: Figure 1: Tissue quality assessment and location. Representative (A) middle frontal gyrus (MFG) and (A’) hippocampal tissue samples stained for NeuN at 27 and 54 days of age. (b) Samples from the same ages, displayed DAPI staining as well. Additionally, the organization typical of both the (C) hippocampus and (D) medial prefrontal cortex are present. Dashed boxes indicate the enlarged areas imaged in all subsequent figures; CA1/subiculum region in the hippocampus and layer 3 of the cortex in the MFG. White scale bar in A, A’, B, B’ is 75microns. Scale bar in D is 100 microns. B and C are 10x images stitched together to visual full brain region. DG- Dentate Gyrus. CA1- Cornu Ammonis 1, L- layer.
Figure 2: Developmental time course of parvalbumin expression in the mpc and hippocampus. Parvalbumin is expressed at 27 days in both the (A) Medial prefrontal cortex and (B) the hippocampus. Expression continues to increase until reaching maturity at approximately 2 years of age (ages stated in each image). White scale bars in all images are 75 microns
Figure 3: Aggrecan immunohistochemistry increases with age in middle frontal gyrus and hippocampus. Aggrecan condenses into perineuronal nets starting around 54 days after birth in the (A) MFG and by 2 years in the (C) hippocampus. All images at 10X magnification. Arrows highlight the few and light perineuronal nets in the young ages. However, the intensity of aggrecan immunohistochemistry in the perineuronal nets increases until approximately 8 years of age in the both regions at which time they appear to be fully mature (ages stated in each image). Insets are enlarged images of representative net quality over time (ages stated in each image). White scale bar is 75microns in all images.
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Figure 4: Co-localization of aggrecan and parvalbumin immunoreactivity during human development. Samples were co-labeled with aggrecan and parvalbumin antibodies in frozenfixed tissue. Parvalbumin, aggrecan, and the superimposed images are shown for both Medial Prefrontal cortex tissue (hippocampal tissue not shown). Co-localization of the perineuronal nets around parvalbumin positive interneurons does not begin until age 2 years (not shown), becomes more prominent by 4 years (A) and reaches full maturity with complete co-labeling by age 8 (CE). Insets highlight the increasing co-localization of the PV and aggrecan staining. White scale bar in all merged images is 75 microns
Figure 5: Western blot analysis of aggrecan, NITEGE, and GAPDH expression. A, C, and E are representative blots and quantification from (MFG). B,C, and F are representative blots and quantification from hippocampus. (A-B) Aggrecan (ACAN) detected by the 7D4 antibody is present at ~ 175kD and increases in intensity with age. (C-D) NITEGE detects an epitope of ADAMTS proteolysis of aggrecan also present in all samples (E-F) GAPDH a housekeeping enzyme known to increase in abundance with age also increases in intensity with age. ADAMTS- A Disintegrin And Metalloproteinase with Thrombospondin MotifS). NITEGE- is the Aggrecan monoclonal antibody to C-terminal neoepitope.
Figure 6: Aggrecan development in human epilepsy patient tissue. Development of the PN appears to follow a similar trajectory in epilepsy patients with the perineuronal nets present by (A) 1 year of age. Net expression continues to increase as observed in the amount and quality of the perineuronal nets in 4 year old (B) frontal lobe and (B’) hippocampus. (C and C’) Nets reach
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maturity in the frontal lobe by adolescence, as shown in two patients both aged 13. (D) Net maturation in the hippocampus appears to be unaltered in an older patient with epilepsy, aged 44 years. White scale bar in all image is 75 microns.
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Highlights: This is the first presentation of the human developmental timeline of the Perineuronal Net. The Perineuronal Net begins to form in the second month of life and reaches its mature appearance around 8 years of age. Perineuronal Net development is not altered in a few patients with intractable epilepsy.
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