Age-related decline in hippocampal tyrosine phosphatase PTPRO is a mechanistic factor in chemotherapy-related cognitive impairment

Chemotherapy-related cognitive impairment (CRCI) or “chemo brain” is a devastating neurotoxic sequela of cancer-related treatments, especially for the elderly individuals. Here we show that PTPRO, a tyrosine phosphatase, is highly enriched in the hippocampus, and its level is tightly associated with neurocognitive function but declined significantly during aging. To understand the protective role of PTPRO in CRCI, a mouse model was generated by treating Ptpro–/– female mice with doxorubicin (DOX) because Ptpro–/– female mice are more vulnerable to DOX, showing cognitive impairments and neurodegeneration. By analyzing PTPRO substrates that are neurocognition-associated tyrosine kinases, we found that SRC and EPHA4 are highly phosphorylated/activated in the hippocampi of Ptpro–/– female mice, with increased sensitivity to DOX-induced CRCI. On the other hand, restoration of PTPRO in the hippocampal CA3 region significantly ameliorate CRCI in Ptpro–/– female mice. In addition, we found that the plant alkaloid berberine (BBR) is capable of ameliorating CRCI in aged female mice by upregulating hippocampal PTPRO. Mechanistically, BBR upregulates PTPRO by downregulating miR-25-3p, which directly targeted PTPRO. These findings collectively demonstrate the protective role of hippocampal PTPRO against CRCI.


Introduction
It has been well established that chemotherapy either alone or in combination with other cancer treatment modalities (including targeted reagents and immunotherapies) may induce cognitive dysfunctions that are JCI Insight 2023;8 (14):e166306 https://doi.org/10.1172/jci.insight.166306 to neuronal differentiation, synaptic plasticity, neuronal recognition, neurogenesis, neuronal generation, and dendritic formation (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.166306DS1). In addition, a different GEO database (GSE14938) indicated that PTPRO is highly expressed in the hippocampus in addition to the kidney (Supplemental Figure 2A). Reverse transcription-quantitative PCR (RT-qPCR) assays of different human and mouse tissues demonstrated that PTPRO is indeed highly expressed in hippocampi ( Figure 1A), whereas immunohistochemistry (IHC) revealed that hippocampal CA3 pyramidal neurons exhibits the strongest PTPRO staining ( Figure 1B). These data are also consistent with that in the Allen Brain Atlas (Figure 1, C and D, and Supplemental Figure 2B). Of note, the data extrapolated from the Brain EXPression Database (BrainEXP) and GEO showed that PTPRO levels in postnatal hippocampi negatively correlated with aging (Supplemental Figure 3, A-E), and these findings were also corroborated in mouse hippocampi by IHC, RT-qPCR, and immunoblotting ( Figure 2, A-C). In contrast, there was no detectable age-dependent change in the levels of mouse kidney PTPRO (Supplemental Figure 4, A and B). These findings altogether indicate that the expression level of hippocampal PTPRO declines with age, and suggest that PTPRO may play an indispensable role in neurocognitive-related functions.
Ptpro deficiency increases doxorubicin-induced CRCI in 3-month-old mice. Given that hippocampal PTPRO expression is tightly associated with neurocognitive-related functions and decreases with age, we hypothesized that the age-related decrease in hippocampal PTPRO might be a mechanistic factor for CRCI in elderly cancer patients. To understand the protective role of PTPRO in CRCI, we selected administration of doxorubicin (DOX), which is one of the most active agents for treatment of breast cancer, once a week for 4 weeks to induce a CRCI model in 3-month-old Ptpro +/+ and Ptpro -/female mice ( Figure 3A). DOX can cause severe cognitive impairment in patients through a variety of mechanisms. Notably, the hippocampus is the most likely brain region affected in DOX-induced CRCI (39)(40)(41).
To determine PTPRO's neurocognitive role in the CRCI mouse model, we evaluated the performance of Ptpro +/+ and Ptpro -/female mice in the Y maze and the Morris water maze (MWM). During a 10-minute session of the Y-maze test, the Ptpro +/+ and Ptpro -/mice in the saline treatment group did not show any observable difference in the proportion of alternation. However, the Ptpro -/mice in the DOX treatment group decreased their alternation rate significantly compared with the age-matched Ptpro +/+ mice ( Figure 3B). In addition, DOX-treated Ptpro -/mice exhibited obvious defects in cognitive abilities, as measured by latency to reach the platform (Figure 3, C and D), distance traveled ( Figure 3E), time in quadrants ( Figure 3F), and the number of platform crossings ( Figure 3G) in the MWM test. It has been reported that blood pressure, cerebral hemodynamics, and the integrity of the BBB are closely related to brain/hippocampal function, and DOX can increase blood pressure, reduce CBF, and destroy the BBB, thus promoting cognitive impairment (42)(43)(44)(45). We monitored and evaluated these physiological indicators in DOX-induced CRCI at the end of the trial. As shown in Supplemental Figure 5, the Ptpro +/+ and Ptpro -/mice in either the saline-treated or DOX-treated group did not show any significant difference in their blood pressure, CBF, and BBB integrity, indicating that PTPRO plays a protective role against cognitive dysfunction in DOX-induced CRCI through mechanisms other than influencing these physiological indicators.
In addition, due to the CRCI research using tumor-bearing animals to mimic humans with newly diagnosed cancer is necessary to screen potential drug candidates against CRCI (46), we investigated the potential relevance of PTPRO to cognitive function in tumor-bearing mice. We transplanted tumors derived from MMTV-PyMT transgenic mice orthotopically into the mammary fat pads of Ptpro +/+ and Ptpro -/mice followed by DOX treatment to establish tumor-bearing mouse model of CRCI (Supplemental Figure 6A). DOX treatment significantly inhibited tumor growth (Supplemental Figure 6B). Tumor volumes in the saline-or DOX-treated group were comparable, suggesting that Ptpro deficiency in host mice does not affect tumor growth (Supplemental Figure 6B). These findings are consistent with previous studies showing that tumor-bearing mice displayed cognitive impairments compared with the normal mice (46) (Supplemental Figure 6, C-I). In addition, saline treatment did not affect cognitive function and hippocampal synaptic plasticity, as measured by MWM testing and long-term potentiation (LTP) (Supplemental Figure 6, C-I). On the other hand, cognitive abilities and hippocampal synaptic plasticity were significantly affected when the Ptpro -/tumor-bearing mice were treated with DOX (Supplemental Figure 6, C-I). These data collectively indicate that PTPRO has comparable neuroprotective roles in both healthy and tumor-bearing mice.
Ptpro deficiency reduces neuronal survival and neurogenesis and leads to neurodegeneration in DOX-induced CRCI. neuronal morphology and quantity of hippocampal CA3 regions in Ptpro +/+ and Ptpro -/female mice treated with DOX. As shown in Figure 4A, the neurons were obviously shrunken and weakly stained in the Ptpro -/mice treated with DOX, indicating diffusely deteriorated neurons and increased neuronal loss. The number of surviving CA3 neurons in the Ptpro -/mice treated with DOX was decreased compared with the wild-type RT-qPCR analysis of PTPRO and Ptpro mRNA in different human (n = 6 individuals per group with equal sex ratio) and mouse tissues (n = 6 mice per group with equal sex ratio), respectively. Results are representative of 3 independent experiments. Error bars: SEM. (B) Representative images of IHC staining of PTPRO in different human (left, n = 6 individuals per group with equal sex ratio) (Scale bars: 100 μm) and mouse (right, n = 6 mice per group with equal sex ratio) (Scale bars: 100 μm) tissues. High expression of PTPRO in the hippocampus (upper panel). The kidney (middle panel) and the testis (bottom panel) were used as controls, which express high and barely detectable levels of PTPRO, respectively. (C) The heatmap shows the expression of 4 PTPRO probes in different human brain regions. Gene expression is shown as individually normalized gene expression; red indicates high expression, and green indicates low expression. The red dashed box indicates the hippocampus. Images and data were derived from BrainSpan (http://www.brainspan.org/lcm/search?search_type=user_selections). (D) Representative in situ hybridization staining image (upper panel; coronal mouse brain sectional views) and the quantification of the region-specific expression of Ptpro in the mouse brain (bottom panel). Red arrows indicate the hippocampus. Images and data were obtained from Allen Mouse Brain Atlas (http://mouse.brain-map.org).  Figure 4B). TUNEL staining also showed increased apoptosis in the CA3 region of the Ptpro -/mice treated with DOX ( Figure 4, C and D). These data suggest that the Ptpro deletion leads to an increased susceptibility for hippocampal neuronal death in mice treated with DOX. Additionally, the number of Ki67 (proliferation marker) and doublecortin (DCX; an immature progenitor cell marker) double-labeled neurons (indicating proliferating immature neurons) in the subgranular zone (SGZ) of the dentate gyrus significantly decreased in the Ptpro -/mice treated with DOX ( Figure 4, E and F).

JCI
Loss of Ptpro results in the dysregulation of synaptic plasticity in DOX-induced CRCI. Synaptic plasticity is important to cognitive functions and its dysregulation is associated with many neuropsychiatric disorders, including cognitive dysfunctions such as AD (47)(48)(49). Golgi staining was performed, using hippocampal samples from DOX-treated Ptpro +/+ and Ptpro -/female mice, to examine the morphology of CA3 pyramidal neurons. In the hippocampal region ( Figure 5A), the total dendritic length and the numbers of primary dendrites of CA3 pyramidal cells in Ptpro -/mice were lower than in WT controls following DOX treatment ( Figure 5, B-D). Moreover, Sholl's analysis revealed markedly reduced dendritic branching of CA3 neurons in Ptpro -/mice compared with WT controls ( Figure 5E). Apical dendrites of Ptpro -/-CA3 pyramidal neurons displayed an approximately 50% decreased spine density compared with WT controls when treated with DOX ( Figure 5, F and G). Synaptophysin (Syp, an essential presynaptic vesicle membrane protein) and postsynaptic density protein 95 (PSD95, a postsynaptic scaffold protein) are closely related to hippocampal synaptic plasticity and cognitive function. Immunofluorescent staining revealed reduced Syp and PSD95 intensity in CA3 of the hippocampi in Ptpro -/mice compared with WT controls when treated with DOX ( Figure 5, H-J). These results were further validated by immunoblotting for Syp and PSD95 ( Figure 5K). Thus, the DOX-treated Ptpro -/mice, compared with WT controls, display more dramatically severe cognitive deficits that correlate with alterations in synaptic plasticity of CA3 hippocampal neurons.
Hippocampal Ptpro deficiency is associated with abnormal activation of SRC/EPHA4 in DOX-induced CRCI. PTPRO is a single-pass transmembrane protein with an extracellular domain containing 8 fibronectin type III-like domains and an intracellular protein tyrosine phosphatase domain (19). Given its well-defined function as a tyrosine phosphatase, we focused our attention on the 40 tyrosine kinases that have been reported to be associated with neurocognition. Of note, 3 of them -SRC, EPHA4, and EPHB2 -that are not only related to neurotoxicity but also serve as PTPRO substrates (Supplemental Figure 7 and Supplemental Table 3), and these kinases are highly expressed in both human and mouse hippocampi (Supplemental Figure 8). Furthermore, results from GSEA of human and mouse data sets revealed that activated SRC, EPHA4, and EPHB2 were closely associated with AD (Supplemental Figure 9). Consistently, these kinases in mice are positively correlated with neuronal death and negatively correlated with neurogenesis (Supplemental Figure 10).
To determine whether these kinases are indeed regulated by PTPRO during DOX-induced CRCI, we quantified the levels of phosphorylated forms of these enzymes when the mice were treated with DOX. The levels of phosphorylated SRC and EPHA4 were significantly elevated in the hippocampi of the Ptpro -/female mice when they were treated with DOX ( Figure 6). Since phosphorylation of EPHB2 did not appear to be affected by Ptpro deletion (Figure 6), we are inclined to conclude that PTPRO-repressed SRC and EPHA4 phosphorylation/activation is likely to protect CRCI in our mouse model.
Region-specific restoration of PTPRO in the hippocampus of Ptpro -/mice rescues DOX-induced CRCI. We next asked whether hippocampal PTPRO plays an essential role of protection in CRCI. To assess whether ectopic  expressing Ptpro (LVPtpro) into the hippocampal CA3 neurons (PTPRO highly enriched region) of 3-month-old female mice followed by DOX treatment. Two weeks after virus injection, mice were treated with DOX once a week for 4 weeks, and behavioral tests were conducted to evaluate their spatial learning and memory abilities ( Figure 7A). Two weeks after virus injection, overexpression of PTPRO in the hippocampus CA3 region was confirmed by checking FLAG expression in hippocampal slices ( Figure 7B). Significantly rescued learning and memory abilities of the Ptpro -/mice injected with LVPtpro (Ptpro -/--LVPtpro mice) were observed both in the Y-maze test ( Figure 7C) and in the MWM test (Figure 7, D-H) when compared with Ptpro -/mice injected with LVCon. However, there was no significant change in cognitive ability between Ptpro +/+ mice injected with LVPtpro and LVCon (Figure 7, C-H). In addition, LV-mediated PTPRO restoration led to dephosphorylation/ inactivation of SRC and EPHA4 in the hippocampal CA3 region in both Ptpro +/+ and Ptpro -/mice ( Figure  7I). Consistent with the results in Supplemental Figure 5, region-specific restoration of hippocampal PTPRO did not affect blood pressure or CBF and BBB integrity in the CRCI mouse model (Supplemental Figure 11). Furthermore, specific ectopic overexpression of hippocampal PTPRO can effectively rescue neuronal survival, apoptosis, and neurogenesis in Ptpro -/-, but not Ptpro +/+ mice ( Figure 8).
PTPRO is expressed in both brain and kidney (18). In the kidney, PTPRO regulates the glomerular pressure/filtration rate by affecting podocyte structure and function, and PTPRO reduction is associated with worse outcome of the glomerulus (50). Since impaired kidney function is closely related to cognitive disorders (51), we further examined the relevance of kidney PTPRO to cognitive function in DOX-induced CRCI. We overexpressed PTPRO in the kidney by local injection of LVPtpro in both Ptpro +/+ and Ptpro -/mice (Supplemental Figure 12A). As shown in Supplemental Figure 12B, PTPRO levels increased in both Ptpro +/+ and Ptpro -/mice when LVPtpro but not LVCon was injected. However, kidney-expressed PTPRO had no detectable effect on cognitive function (Supplemental Figure 12, C-G). These results provide direct evidence showing that the DOX-induced cognitive dysfunctions in Ptpro -/mice can be largely ameliorated by region-specific restoration of hippocampal but not kidney PTPRO, supporting the essentially protecting role of hippocampal PTPRO in DOX-induced CRCI.
Region-specific restoration of PTPRO in the hippocampus of Ptpro -/mice reverses impairment of hippocampal synaptic plasticity in CRCI mice. Golgi staining was performed in Ptpro +/+ -LVCon, Ptpro +/+ -LVPtpro, Ptpro -/--LVCon, and Ptpro -/--LVPtpro female mice to observe dendrite morphogenesis in the hippocampal CA3 region ( Figure 9A). Compared with the CA3 neurons of Ptpro -/--LVCon mice, the CA3 neurons in Ptpro -/--LVPtpro mice exhibited enhanced dendritic growth and increased primary dendrites (Figure 9, B and C). Sholl's analysis revealed marked increases in the dendritic branching of CA3 neurons in Ptpro -/--LVPtpro mice compared with Ptpro -/--LVCon mice ( Figure 9D). The spine densities of CA3 pyramidal neurons in Ptpro -/--LVPtpro mice increased compared with those of Ptpro -/--LVCon mice ( Figure 9, E and F). Next, we performed LTP recording to evaluate hippocampal synaptic plasticity. Consistent with behavioral results, the degree of LTP at CA3-CA1 synapses elicited by high-frequency stimulation of Schaffer collaterals was significantly reduced in Ptpro -/mice compared with WT controls following DOX treatment ( Figure 9, G and H). LV-mediated PTPRO overexpression rescued impaired LTP in Ptpro -/mice ( Figure 9, G and H). Consistently, the relative fluorescence intensities and protein levels of Syp and PSD95 increased in Ptpro -/--LVPtpro mice when compared with Ptpro -/--LVCon mice (Figure 9, I-L). Consistent with the results in Figures 7 and 8, there was no significant difference in synaptic plasticity between Ptpro +/+ mice injected with LVPtpro or LVCon (Figure 9, A-K). It is likely that the level of hippocampal PTPRO in young mice is sufficiently high to effectively protect DOX-induced CRCI. These results together suggest that hippocampal PTPRO plays essential roles in regulating synaptic plasticity.
BBR prevents DOX-induced cognitive dysfunction by upregulating hippocampal PTPRO in aged female mice. We next sought to test the interference strategy to experimentally prevent CRCI in the mouse model. The plant alkaloid BBR has been reported for its BBB permeability and neuroprotective effect, as well as its potent modulation of tyrosine kinases (30,32,52). Given that CRCI is particularly frequent in elderly cancer patient populations and PTPRO expression in the hippocampus declines with age (Figure 2), the aged WT female mice (18 months old) were pretreated with BBR (or corn oil) for 4 weeks, followed by exposure to DOX (or saline) injection ( Figure 10A), and followed by assays of cognitive-behavioral performance. It appeared that BBR had little effect on the behavior of mice when they were not exposed to DOX (Figure  10, B-G). However, BBR effectively reduced DOX-induced cognitive behavioral dysfunctions (Figure 10, B-G, and Supplemental Figure 13, A and B). Consistent with results from both preclinical and clinical studies indicating that BBR can reduce hypertension, protect BBB integrity, and improve CBF (53-55), we found that BBR plays a protective role against DOX-induced BBB damage, systolic and diastolic blood pressure elevation, and CBF reduction (Supplemental Figure 14). These results suggest multiple mechanisms exist in BBR's protection of DOX-induced cognitive dysfunction in aged mice. Of note, BBR had no influence on the body weight of the animals, suggesting it had no obvious adverse effect on the mice (Supplemental Figure 13C).
Based on their similarly protective effect on DOX-induced dysfunction, we speculated that the protective effects of BBR on cognition may be through the PTPRO signaling pathway. Immunoblotting assays indicate that BBR upregulated PTPRO and downregulated the phosphorylation of SRC and EPHA4 in mouse hippocampi (Supplemental Figure 13D). Furthermore, we also noticed that DOX treatment did not  Figure 13D). More interestingly, BBR is capable of counteracting DOX-induced phosphorylation of SRC and EPHA4 (Supplemental Figure 13D). These findings suggest that BBR plays an important neuroprotective role against DOX-induced cognitive dysfunctions in aged mice.
BBR upregulates hippocampal PTPRO by downregulating miR-25-3p. Next, we wanted to explore the mechanism in BBR-regulated PTPRO expression in vitro. We found that both protein and mRNA levels of PTPRO were upregulated in dose-and time-dependent manners when the mouse hippocampal cell line HT-22 was treated with BBR (0, 12.5, 25, and 50 μM) (Figure 11, A and B). Multiple lines of evidence implied that BBR could function through targeting different miRNAs (56). We conducted bioinformatics analysis (http:// www.targetscan.org/vert72/) and identified 3 BBR-downregulated miRNAs -miR-25-3p, miR-93-5p, and miR-106b-5p -that also potentially interacted with the 3′-UTR of PTPRO ( Figure 11C and Supplemental Figure 15). To experimentally determine whether any of these miRNAs were involved in BBR-mediated PTPRO upregulation, we estimated the effect of BBR on the levels of these miRNAs. We found that miR-25-3p, but not the other 2 miRNAs, was dramatically downregulated by BBR in time-and dose-dependent manners (Figure 11, D-F). To determine whether miR-25-3p played any role in BBR-upregulated PTPRO,  Figure 11G shows that miR-25-3p is capable of downregulating not only the mRNA and protein of PTPRO at the basal level but also the BBR-upregulated PTPRO. To demonstrate that miR-25-3p downregulates PTPRO by directly interacting with the 3′-UTR of the Ptpro mRNA, we first constructed a luciferase reporter harboring the 3′-UTR of Ptpro with either WT or mutant miR-25-3p binding sites and estimated the effect of miR-25-3p using a luciferase assay. When the HT-22 cells were transiently transfected with miR-25-3p mimics with the reporter plasmids, the luciferase activity was  only inhibited when the reporter was composed of the WT but not the mutant binding site ( Figure 11H). These data suggest that BBR could upregulate PTPRO expression through decreasing miR-25-3p, which directly targets PTPRO.

Discussion
In view of the fact that CRCI is more frequent in the elderly cancer patient population, CRCI-associated cognitive dysfunction may add to the burden of preexisting age-related performance decline, and therefore the management of older cancer populations is of growing concern. However, there were few studies focusing on older cancer patients with CRCI, aging-associated CRCI model systems, and the underling mechanisms. In this study, we show a substantial enrichment of the tyrosine phosphatase PTPRO in the hippocampus and the age-related decline of hippocampal PTPRO. To establish a CRCI animal model that mimics an elderly cancer patient population with preexisting PTPRO downregulation, Ptpro -/female mice were treated with DOX. Ptpro deletion results in severe cognitive phenotypes of CRCI, while site-specific restoration of PTPRO in the hippocampal CA3 region of Ptpro -/female mice significantly reduced CRCI. Furthermore, Ptpro deficiency was associated with abnormal activation of hippocampal SRC/EPHA4 when the mice were treated with DOX. The plant-derived BBR can ameliorate CRCI in aged female mice by upregulating hippocampal PTPRO. Mechanistically, BBR upregulates PTPRO by downregulating miR-25-3p and subsequently reducing miR-25-3p-mediated PTPRO degradation.
Compared with most reported mechanistic studies of CRCI using non-genetically modified rodent models treated with chemotherapeutic reagents (46), our study utilized gene-deleted mice to define the underlying genetic factors of CRCI. PTPRO is abundantly expressed in both brain and kidney (18). We found that levels of hippocampal PTPRO are negatively correlated with aging and found no detectable age-dependent change in kidney PTPRO. Thus, we conclude that PTPRO in the hippocampus but not the kidney is an important susceptibility factor to chemotherapy in the elderly. Meanwhile, region-specific restoration of kidney PTPRO in Ptpro -/mice by local infection with lentivirus did not affect cognitive function in DOX-induced CRCI, further indicating that kidney PTPRO is irrelevant to cognition. From this perspective, our conventional knockout mice can be considered as largely equivalent to hippocampal PTPRO knockout, and this notion is further supported by results showing that hippocampal CA3-specific restoration of PTPRO largely rescued the DOX-induced CRCI in Ptpro -/female mice. Moreover, given that CRCI is highly prevalent in women with breast cancer and females are more vulnerable to CRCI (1, 57), we deliberately focused on the ameliorative effects of PTPRO on DOX-induced CRCI in female mice. However, whether PTPRO has similar neuroprotective and neurorestorative effects in male mice needs to be validated further.
Many cancer-related factors and their signaling pathways are deregulated in neurocognitive abnormalities such as AD and dementia (10)(11)(12)(13). The tumor suppressor PTPRO is highly enriched in the hippocampi of both humans and mouse, while reduced levels of PTPRO were found in the hippocampi of AD patients (Supplemental Figure 16, A and B). Severe cognitive dysfunctions induced by chemotherapeutic reagent occur in Ptpro -/female mice, and these in vivo data unambiguously demonstrated that the hippocampal PTPRO played an indispensable role in protecting against CRCI. Based on the fact that the PTPRO substrates SRC and EPHA4 are involved in the development of AD, cognitive deficiency, and neuronal differentiation (58)(59)(60)(61), it is conceivable that reduced levels of PTPRO lead to phosphorylation/activation of SRC and EPHA4, and therefore the PTPRO/SRC/EPHA4 axis plays what we believe is a previously unrecognized role in CRCI and possibly other cognition-related disorders. Since PTPRO is a tyrosine phosphatase with a broad spectrum of substrates, it would be interesting to test whether other PTPRO-regulated enzymes also participate in PTPRO-mediated functions in CRCI.
There are limited studies on CRCI prevention and/or treatment, particularly those focusing on pathophysiological mechanisms (2,4,35,62). Repurposing existing drugs to prevent CRCI is likely to be cost effective in terms of time and money. We demonstrated here that BBR could effectively alleviate CRCI-related cognitive deficits in aged female mice. We also showed that BBR downregulated miR-25-3p, which directly interacts with and could downregulate PTPRO in vitro. In addition to alleviating the CRCI phenotypes, BBR might modify the trajectory of CRCI at least in a subgroup of elderly cancer patients with preexisting hippocampal CA3 sections. n = 5 per group. (L) Immunoblotting of Syp and PSD95 in the hippocampi of mice. n = 3 per group. These results are representative of 3 independent experiments. Error bars: SEM. NS, not significant; *P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA followed by a Tukey-Kramer post hoc test (B, C, F, H, J, and K)  PTPRO downregulation. Given its high permeability across the BBB and tolerability, BBR could be promising as a protective reagent against CRCI in aged patients, and therefore clinical investigation of BBR for CRCI prevention and treatment will be worthwhile.
One of the striking but previously undocumented findings in our study is the age-dependent downregulation of hippocampal PTPRO evidenced by IHC, RT-qPCR, and immunoblotting in mice as well as the results from bioinformatic analyses of age-dependent expression of hippocampal PTPRO in a variety of species. In addition, it has been reported that PTPRO downregulation can be related to viral infections (63), sleep deprivation (64), systemic inflammation (65), alcohol addiction (66), corticosterone levels (67), anxiety (68), unpredictable chronic mild stress (69), prenatal stress (70), and high-fat diet (Supplemental Figure 17). However, whether exposure to these adverse reagents could exacerbate age-dependent hippocampal PTPRO downregulation is unknown. We and others have found that in many cancer types PTPRO downregulation can be partially attributed to promoter methylation (71)(72)(73)(74)(75)(76)(77)(78)(79). It would be interesting to determine whether age-dependent downregulation of hippocampal PTPRO is also mediated by promoter hypermethylation.
In summary, using DOX-treated Ptpro -/female mice to mimic elderly cancer patients with preexisting PTPRO downregulation, we demonstrated that age-decreased PTPRO is an important determining factor  Table 6.
of CRCI. In protecting against CRCI, hippocampal PTPRO acts as a "brake" to slow down the deterioration of cognitive function, while the age-associated reduction in hippocampal PTPRO is analogous to loss of the brake and consequently increased susceptibility to CRCI ( Figure 10H). On the other hand, BBR ameliorates CRCI-related cognitive dysfunction in aged female mice by upregulating PTPRO ( Figure  10H). Therefore, BBR and any reagents possessing similar activities could become promising candidates for CRCI prevention or treatment. Considering the age-related decrease in hippocampal PTPRO, upregulating PTPRO could be a plausible strategy to prevent CRCI in older patients.

Methods
Human specimens. Human specimens were collected from 3 females and 3 males with a median age of 40.5 years (34 to 55) who underwent forensic autopsy between 2012 and 2014 in the Forensic Identification Center of Shantou University (FICSU). Tissue samples included kidney, hippocampus, cerebrum, cerebellum, liver, heart, lung, trachea, testis, ovary, and lymph nodes.
Surgery and intracranial injection. Surgeries were carried out as described previously (82). The young mice were anesthetized (Avertin, 13 μL/g, i.p.) and placed in an SA-100 stereotactic instrument (RWD Life Science). A small craniotomy hole was made using a dental drill (OmniDrill35, WPI). A glass cannula filled with virus solution was lowered to the CA3 region (AP, -2.1 mm; ML, ± 2.3 mm; DV, -2.4 mm) and the virus solution (1.0 μL/injection) was injected using a nanoliter injector (NANOLITER 2010, WPI) system at a rate of 0.1 μL per minute sequentially into each side of the hippocampus. VSVG-Lenti-hSyn-Ptpro-3×Flag (LVPtpro, viral titer: 1.10 × 10 10 GC/mL) and VSVG-Lenti-hSyn-EGFP (LVCon, viral titer: 1.37 × 10 9 GC/mL) were generated and packaged by Shanghai Taitool Bioscience Co., Ltd. The injection cannula was slowly withdrawn 5 minutes after the virus infusion. The scalp was then sealed and injected mice were monitored as they recovered from anesthesia. Behavioral experiments or electrophysiological recordings were performed at least 14 days after virus injection. Virus infection was examined at 2 weeks after virus injection.
DOX treatment. DOX treatment dosage and schedule were established in previous studies (83). DOX (2 mg/kg, Sigma-Aldrich) was dissolved in sterile normal saline and injected i.p. once per week for 4 consecutive weeks.
BBR treatment. BBR was purchased from MedChemExpress. The BBR dosage was determined based on previous studies (33,84). The aged mice were randomly divided into 4 groups (n = 13 per group): corn oil/saline, BBR/saline, corn oil/DOX, or BBR/DOX. For BBR treatment, the mice were treated with BBR (50 mg/kg in corn oil) by oral gavage, 5 times a week for 4 continuous weeks. At the end of the study, 5 mice of each group were euthanized and the brain tissues were collected for Nissl staining. Three mice of each group were also collected for immunoblotting analysis. Y maze. This test was performed as described previously (85). The Y maze was a 3-arm (each 30 cm long, 8 cm wide, and 15 cm in height) maze with equal angles between all arms. The 3 identical arms were randomly designated as start arm, novel arm, and another arm. The percentage of triads in which all 3 arms are represented was recorded as an alternation to estimate short-term memory of the last arms entered. An alternation is defined as a visit to all 3 arms without reentry (ABC, ACB, BAC, BCA, CAB, or CBA). The total number of arm entries was used as a measure for locomotor activity, while the spontaneous alternation percentage (SAP) was used as a measure of spatial working memory. To calculate the SAP, the total number of alternations (i.e., every time a mouse explored the 3 arms consecutively) was divided by the total possible alternations (i.e., the number of arm entries minus 2) and multiplied by 100.
MWM. Spatial memory abilities of mice were examined in the MWM. The test was conducted in a circular tank (150 cm in diameter and 50 cm in depth) with a 10-cm diameter central round platform hidden 1 cm below the surface of the water that was maintained at 24°C. The pool was divided arbitrarily into 4 quadrants labeled N-S-E-W. Each mouse was given 4 swimming trials per day for 5 days. The start position was randomized among the 4 quadrants (N-S-E-W) for each trial. Each trial lasted until the animal found the platform or for a maximal observation period of 60 seconds, and the animals that failed to find the platform within 60 seconds were guided by the experimenter to the platform. Mice remained on the platform for 10 seconds before being removed to the home cage. On the sixth day, a probe trial without the platform was performed in order to measure the retention of spatial memory. For each trial, the time required to locate the hidden platform (escape latency), distance traveled (path length), percentage time in quadrant, and number of crosses were recorded using an EthoVision video tracking system.
Golgi staining. For Golgi-Cox impregnation of neurons, the FD Rapid GolgiStain kit (FD Neuro-Technologies) was used according to the manufacturer's protocol. The brains were cut in sections of 150 μm thickness using a vibratome. Hippocampal sections were collected on a 0.3% gelatin solution, dried at room temperature, dehydrated in alcohol, and cleared with xylene. Finally, they were mounted on 0.3% gelatinized slides. Bright-field images were taken on a Cytation 5 multi-mode plate reader (BioTek). Dendrites were traced, and their lengths were measured using the Fiji plugin Simple Neurite Tracer. For Sholl's analysis, we used NeuronStudio to plot proximal complexity and branching of apical and basal dendritic domains in hippocampal CA3 pyramidal neurons.
Immunoblotting. Immunoblotting was performed as described previously (80,81). Briefly, the CA3 tissues were homogenized and proteins were extracted using RIPA lysis buffer (Millipore). Protein concentrations were quantified by the BCA method. Protein samples were resolved by SDS-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membrane (Millipore). The membranes were immersed in blocking buffer (5% skim milk in PBS) for 1 hour at room temperature and incubated