. 2023 May 17;111(10):1591-1608.e4.

doi: 10.1016/j.neuron.2023.02.020. Epub 2023 Mar 8.

Choroid plexus-targeted NKCC1 overexpression to treat post-hemorrhagic hydrocephalus

Cameron Sadegh¹, Huixin Xu², Jason Sutin³, Benoit Fatou², Suhasini Gupta², Aja Pragana², Milo Taylor⁴, Peter N Kalugin⁵, Miriam E Zawadzki⁶, Osama Alturkistani⁷, Frederick B Shipley⁸, Neil Dani², Ryann M Fame², Zainab Wurie², Pratik Talati⁹, Riana L Schleicher¹⁰, Eric M Klein¹¹, Yong Zhang¹², Michael J Holtzman¹², Christopher I Moore¹¹, Pei-Yi Lin³, Aman B Patel⁹, Benjamin C Warf¹³, W Taylor Kimberly¹⁰, Hanno Steen¹⁴, Mark L Andermann¹⁵, Maria K Lehtinen¹⁶

Affiliations

¹ Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA; Department of Neurosurgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.
² Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA.
³ Fetal-Neonatal Neuroimaging and Developmental Science Center, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
⁴ Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA; Harvard College, Harvard University, Cambridge, MA 02138, USA.
⁵ Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA; Harvard/MIT MD-PhD Program, Harvard Medical School, Boston, MA 02115, USA; Graduate Program in Neuroscience, Harvard Medical School, Boston, MA 02115, USA.
⁶ Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA; Harvard/MIT MD-PhD Program, Harvard Medical School, Boston, MA 02115, USA; Graduate Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA 02115, USA.
⁷ Cellular Imaging Core, Boston Children's Hospital, Boston, MA 02115, USA.
⁸ Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA; Graduate Program in Biophysics, Harvard University, Cambridge, MA 02138, USA.
⁹ Department of Neurosurgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.
¹⁰ Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.
¹¹ Carney Institute for Brain Science, Brown University, Providence, RI 02912, USA.
¹² Pulmonary and Critical Care Medicine, Department of Medicine, Washington University, St. Louis, MO, 63110, USA.
¹³ Department of Neurosurgery, Boston Children's Hospital, Boston, MA 02115, USA.
¹⁴ Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA; Precision Vaccines Program, Boston Children's Hospital, Boston, MA 02115, USA.
¹⁵ Graduate Program in Neuroscience, Harvard Medical School, Boston, MA 02115, USA; Graduate Program in Biophysics, Harvard University, Cambridge, MA 02138, USA; Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.
¹⁶ Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA; Graduate Program in Neuroscience, Harvard Medical School, Boston, MA 02115, USA; Graduate Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA 02115, USA; Graduate Program in Biophysics, Harvard University, Cambridge, MA 02138, USA. Electronic address: maria.lehtinen@childrens.harvard.edu.

PMID: 36893755
PMCID: PMC10198810
DOI: 10.1016/j.neuron.2023.02.020

Choroid plexus-targeted NKCC1 overexpression to treat post-hemorrhagic hydrocephalus

Cameron Sadegh et al. Neuron. 2023.

. 2023 May 17;111(10):1591-1608.e4.

doi: 10.1016/j.neuron.2023.02.020. Epub 2023 Mar 8.

Authors

Affiliations

¹ Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA; Department of Neurosurgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.
² Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA.
³ Fetal-Neonatal Neuroimaging and Developmental Science Center, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
⁴ Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA; Harvard College, Harvard University, Cambridge, MA 02138, USA.
⁵ Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA; Harvard/MIT MD-PhD Program, Harvard Medical School, Boston, MA 02115, USA; Graduate Program in Neuroscience, Harvard Medical School, Boston, MA 02115, USA.
⁶ Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA; Harvard/MIT MD-PhD Program, Harvard Medical School, Boston, MA 02115, USA; Graduate Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA 02115, USA.
⁷ Cellular Imaging Core, Boston Children's Hospital, Boston, MA 02115, USA.
⁸ Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA; Graduate Program in Biophysics, Harvard University, Cambridge, MA 02138, USA.
⁹ Department of Neurosurgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.
¹⁰ Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.
¹¹ Carney Institute for Brain Science, Brown University, Providence, RI 02912, USA.
¹² Pulmonary and Critical Care Medicine, Department of Medicine, Washington University, St. Louis, MO, 63110, USA.
¹³ Department of Neurosurgery, Boston Children's Hospital, Boston, MA 02115, USA.
¹⁴ Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA; Precision Vaccines Program, Boston Children's Hospital, Boston, MA 02115, USA.
¹⁵ Graduate Program in Neuroscience, Harvard Medical School, Boston, MA 02115, USA; Graduate Program in Biophysics, Harvard University, Cambridge, MA 02138, USA; Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.
¹⁶ Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA; Graduate Program in Neuroscience, Harvard Medical School, Boston, MA 02115, USA; Graduate Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA 02115, USA; Graduate Program in Biophysics, Harvard University, Cambridge, MA 02138, USA. Electronic address: maria.lehtinen@childrens.harvard.edu.

PMID: 36893755
PMCID: PMC10198810
DOI: 10.1016/j.neuron.2023.02.020

Abstract

Post-hemorrhagic hydrocephalus (PHH) refers to a life-threatening accumulation of cerebrospinal fluid (CSF) that occurs following intraventricular hemorrhage (IVH). An incomplete understanding of this variably progressive condition has hampered the development of new therapies beyond serial neurosurgical interventions. Here, we show a key role for the bidirectional Na-K-Cl cotransporter, NKCC1, in the choroid plexus (ChP) to mitigate PHH. Mimicking IVH with intraventricular blood led to increased CSF [K⁺] and triggered cytosolic calcium activity in ChP epithelial cells, which was followed by NKCC1 activation. ChP-targeted adeno-associated viral (AAV)-NKCC1 prevented blood-induced ventriculomegaly and led to persistently increased CSF clearance capacity. These data demonstrate that intraventricular blood triggered a trans-choroidal, NKCC1-dependent CSF clearance mechanism. Inactive, phosphodeficient AAV-NKCC1-NT51 failed to mitigate ventriculomegaly. Excessive CSF [K⁺] fluctuations correlated with permanent shunting outcome in humans following hemorrhagic stroke, suggesting targeted gene therapy as a potential treatment to mitigate intracranial fluid accumulation following hemorrhage.

Keywords: Cerebrospinal fluid; NKCC1; adeno-associated virus; choroid plexus; epithelial cells; gene therapy; hydrocephalus; intraventricular hemorrhage; ventricles.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests M.K.L., R.M.F., C.S., and H.X. are co-inventors on a provisional patent application related to this manuscript.

Figures

**Figure 1.. Intraventricular blood leads to ventriculomegaly in mouse models of pediatric PHH.**
(A) Schematics of IVH models at E14.5 (modeling very preterm human infants of 12–20 gestational weeks (GW)) and at P4 (modeling preterm human infants of 24–32 GW). Age-matched blood from donors (litter a) was obtained and immediately delivered into the lateral ventricles of recipient embryos (litter b). Sterile PBS was used as control (litter c). Similar approach used at P4, where donor, recipients, and PBS controls were littermates. Schematic: grown mice with pediatric IVH have no overt changes in head shape, suggesting *compensated* ventriculomegaly. (B) Workflow for IVH induction and hydrocephalus evaluation at E14.5 and P4. (C) Representative T2-weighted MRI images (slice #16 of 20-slice series) showing ventriculomegaly in E14.5 and P4 IVH model (red arrow) vs PBS control (white arrow), imaged at P14 and P21, respectively. Scale bar = 5 mm. (D) Lateral ventricle volumes in E14.5 and P4 IVH models at P14 and P21, respectively. E14.5: PBS N = 9, IVH N = 11, *** p = 0.0008; P4: PBS N = 8, IVH N = 9, **** p < 0.0001. Welch’s two-tailed unpaired t-test. Data are mean ± SD. (E) Schematic depicting the infusion test and data from an adult control mouse for analysis using the Marmarou model of CSF dynamics. Inset shows representative magnified view of ICP waveforms, which oscillate with the expected frequency of cardiorespiratory pulsations. (**F-H**) Measurements from infusion test, including baseline ICP, compliance, and capacity for CSF clearance. Coefficients of compliance and clearance were normalized with respect to the average PBS control value on each acquisition date. E14.5: PBS N = 11, IVH N = 9; P4: PBS N = 4, IVH N = 4. (F) not significant; (G) * p = 0.0232 (if exclude the one outlier, * p = 0.0237), ** p = 0.0020; (H) * p = 0.0144, ** p = 0.001. All statistics in F-H were determined by Holm-Sidak method with multiple-comparison corrected. Each data point represents one mouse. (I) Activated (CD68⁺) brain microglia (Iba1⁺) were restricted to site of needle track 2 days following ICV (dotted lines, left panels). No noticeable microglial activation immediately following ICP infusion through a cannula compared to naïve mice (right panels).

**Figure 2.. The ChP responds rapidly to ventricular blood exposure.**
(A) Schematic depicting ChP explant imaging in response to blood exposure. (B) Two-photon calcium imaging of ChP explant (*FoxJ1-Cre::GCaMP6f* mice) shows increased response following application of plasma. Scale bar = 100 μm. Top: 10 seconds prior to plasma. Bottom: 0.25 sec after plasma exposure. (C) Two-photon calcium activity of epithelial cells shown in (B), sorted by peak activity within 25 seconds following application of plasma. Black arrow indicates onset of plasma. Red dashed line indicates cutoff of non-responder cells (responder cells were defined as those that exceeded the cutoff of 6 standard deviations or more above the pre-stimulation mean within 25 seconds of stimulus exposure), indicating that 91% (987 out of 1080) of ChP epithelial cells are sensitive to plasma. (D) Epifluorescence calcium activity of entire ChP explant following addition of plasma (red) and aCSF (blue). Shaded color zones indicate standard deviation (n = 7 mice for plasma, 3 for aCSF). (E) ChP calcium activity showing peak intensity. (**F-H**) IVH increased total ChP NKCC1 and pNKCC1 after 48 hrs. (G) E14.5: PBS N = 5, IVH N = 10, ** p = 0.0012; P4: PBS N = 6, IVH N = 5, * p = 0.02852. (H) E14.5: PBS N = 5, IVH N = 10, * p = 0.0104; P4: PBS N = 6, IVH N = 5, * p = 0.0272. Welch’s two-tailed unpaired t-test. Data presented as mean ± SD. (I) CSF [K⁺] increased 2 days after IVH and returned to baseline 4 days after IVH, * p = 0.0448. N = 5 for each condition, Welch’s two-tailed unpaired t-test. Data presented as mean ± SD. (J) CSF osmolarity remained unchanged by IVH. N = 5 for each condition except for uninjected Day 21, where N = 3. Each data point in Figure 2 represents one mouse, except for CSF data where each data point represents one biological replicate, and 2–3 mice were pooled in each biological replicate to reach sufficient CSF volume for performing the assay.

**Figure 3.. AAV-NKCC1 delivered ICV shows tropism for ChP epithelial cells and mitigates IVH-induced ventriculomegaly in mouse models of pediatric PHH**
(A) AAV-NKCC1 localizes to apical CSF surface of ChP epithelial cells. Scale = 100 μm. (B) HA signal observed in the LV ChP. Scale = 250 μm. (**C-E**) Increased pNKCC1 (quantified in D) and total NKCC1 (quantified in E) levels 2 days following AAV-NKCC1 ICV delivery to P4 pups. NKCC1 overexpression does not persist long term. Welch’s two-tailed unpaired t-test. Data presented as mean ± SD. (F) Timeline of AAV-NKCC1 treatment following IVH, with AAV-GFP as control. (**G-I**) Representative T2-MRI images and quantification of ventricular volume in IVH mice treated with AAV-GFP (G, grey arrows showing ventricles) vs AAV-NKCC1 (H, light purple arrows showing ventricles). Scale = 2 mm. IVH+GFP N = 13, IVH+NKCC1 N = 11, * p = 0.0104 (If exclude the one outlier, * p = 0.0044). Welch’s two-tailed unpaired t-test. Data presented as mean ± SD. For comparison with values from unpretreated mice, see Fig. 1D. (**J-K**) baseline ICP and capacity for CSF clearance. IVH+GFP N = 4, IVH+NKCC1 N = 4, * p = 0.015. Welch’s two-tailed unpaired t-test. Data presented as mean ± SD. (L) CSF [K⁺] was reduced by AAV-NKCC1 treatment 3 days following IVH. IVH+GFP N = 3, IVH+NKCC1 N = 6, ** p = 0.0035. Welch’s two-tailed unpaired t-test. Data presented as mean ± SD. Each data point represents one mouse, except for CSF data in Q where each data point represents one biological replicate, and 2–3 mice were pooled in each biological replicate to reach sufficient CSF volume for performing assay.

**Figure 4.. Inactive NKCC1 fails to mitigate ventriculomegaly in IVH mice.**
(A) Alignment of sequences in the regulatory domain of WT and NKCC1_NT51 shows residues with silent inactivating mutations in NKCC1_NT51. (B) ChP apical localization of both WT and NKCC1_NT51, identified by HA and FLAG, respectively. Scale = 25 μm. (C) P5 pups expressing AAV-NKCC1_NT51 had lower pNKCC1 level than littermates expressing AAV-NKCC1_WT. (**D-E**) Lower pNKCC1 (D) and lower pNKCC1/NKCC1 ratio (E) by AAV-NKCC1_NT51. AAV-NKCC1_WT N=3, AAV-NKCC1_NT51 N=4. Welch’s two-tailed unpaired t-test. Data presented as mean ± SD. (**F-G**) Representative T2-MRI images and quantification of ventricular volume in IVH mice treated with AAV-NKCC1_WT (purple arrows denote ventricles) or AAV-NKCC1_NT51 (black arrows denote ventricles). Scale = 2 mm. IVH+AAV-NKCC1_WT, N = 13; IVH+AAV-NKCC1_NT51 N = 11, *** p = 0.0001. Welch’s two-tailed unpaired t-test. Data presented as mean ± SD. (H) CSF [K⁺] was higher in IVH mice treated with AAV-NKCC1-NT51 compared to mice treated with wild-type AAV-NKCC1 3 days following IVH. Wild-type AAV-NKCC1: N=3 (pooled across 10 pups); AAV-NKCC1-NT51: N=6 (pooled across 16 pups), * p = 0.0112. Welch’s two-tailed unpaired t-test. Data presented as mean ± SD.

**Figure 5.. Adult mouse model of PHH mirrors key aspects of pediatric PHH and responds to ChP-NKCC1 therapy.**
(A) Schematic depicting devices and surgical implantation for *in vivo* ChP live-imaging with two-photon microscopy. (**B-C**) Calcium responses to serum in ChP epithelial cells (*FoxJ1-Cre::GCaMP6f* mice) *in vivo*. Scale bar = 100 μm. (**D-E**) Representative fluorescence intensity trace with serum infusion and quantification of peak intensity. aCSF N = 4, serum N = 3. * p = 0.0318. Welch’s two-tailed unpaired t-test. Data presented as mean ± SD. (F) Schematic depicting adult IVH serial infusion test post-IVH. (G) Adult CSF clearance capacity post-IVH measured by infusion test. Naïve: N = 3, 3 days: N = 5, 21 days: N = 5, 46 days: N = 6. ** p = 0.0091, ** p = 0.0061. Welch’s two-tailed unpaired t-test. Data presented as mean ± SD. (H) Schematic depicting the therapeutic approach in adult PHH model and the time-course of serial MRI. (I) Representative T2-MRI images showing rapid reversal of ventriculomegaly in adult PHH model by ChP-NKCC1 overexpression. AAV-GFP was used as control. Scale = 5 mm. (J) Relative ventricular volume in adult PHH mice with AAV-NKCC1 treatment or control. For each mouse, the ventricular volumes on different days were normalized to the value of Day 2 post-IVH, immediately prior to AAV transduction. N = 4, * p < 0.05 (Day 3: p = 0.0138; Day 6: p = 0.0386; Day 13: p = 0.0152). Data analyzed by multiple t-test and corrected for multiple comparisons using Holm-Sidak method. Each data point represents one mouse.

**Figure 6.. Adult human CSF shows positive correlation between post-SAH / IVH ionic disturbance and shunted outcome.**
(A) Study design. (B) Representative CT images from patients with resolved (#1) vs. shunted (#2) hydrocephalus. (**C-D**) CSF osmolarity decreased in some CSF samples regardless of disease course. Shaded area shows 95% normal values. Blue: resolved, N = 18 (7 patients, 2–3 timepoints per patients). Red: shunted, N = 21 (8 patients, 2–3 timepoints per patient). (**E-F**) Absolute CSF [K⁺] was not altered but relative CSF [K⁺] to total osmolarity was higher in individuals requiring permanent shunting vs. individuals who recovered and had initial ventricular catheter removed. Blue: resolved, N = 18 (7 patients, 2–3 timepoints per patients). Red: shunted, N = 21 (8 patients, 2–3 timepoints per patient). * p = 0.016. Welch’s two-tailed unpaired t-test. All data presented as mean ± SD. Each data point represents one CSF sample collected from one patient at a single timepoint.

**Figure 7.. ChP compensatory actions in PHH.**
(A) Two scenarios of ChP activities following IVH. *Upper panels*: The ChP responds to blood with acute calcium activation. After 48 hrs, CSF-[K⁺] increases. With elevated CSF-[K⁺], NKCC1 activation leads to net flux of ions and osmotically obliged water movement from CSF into ChP, resulting in compensated PHH characterized by mild ventriculomegaly and enhanced CSF clearance capacity. *Lower panels:* AAV-mediated overexpression of NKCC1 enhances the total amount of Na⁺-K⁺−2Cl⁻ cotransport, preventing ventriculomegaly. (B) Mouse and human developmental timelines.

See this image and copyright information in PMC

Comment in

Boosting NKCC1 in the choroid plexus: From CSF clearance to a potential therapy for posthemorrhagic hydrocephalus.
Jeong I, Jurisch-Yaksi N. Jeong I, et al. Neuron. 2023 May 17;111(10):1521-1523. doi: 10.1016/j.neuron.2023.04.014. Neuron. 2023. PMID: 37201502

References

1. Kulkarni AV, Riva-Cambrin J, Butler J, Browd SR, Drake JM, Holubkov R, Kestle JR, Limbrick DD, Simon TD, Tamber MS, et al. (2013). Outcomes of CSF shunting in children: comparison of Hydrocephalus Clinical Research Network cohort with historical controls: clinical article. J Neurosurg Pediatr 12, 334–338. 10.3171/2013.7.PEDS12637. - DOI - PubMed
1. Robinson S (2012). Neonatal posthemorrhagic hydrocephalus from prematurity: pathophysiology and current treatment concepts. J Neurosurg Pediatr 9, 242–258. 10.3171/2011.12.PEDS11136. - DOI - PMC - PubMed
1. Kahle KT, Kulkarni AV, Limbrick DD Jr., and Warf BC (2016). Hydrocephalus in children. Lancet 387, 788–799. 10.1016/S0140-6736(15)60694-8. - DOI - PubMed
1. Warf BC (2005). Comparison of endoscopic third ventriculostomy alone and combined with choroid plexus cauterization in infants younger than 1 year of age: a prospective study in 550 African children. J Neurosurg 103, 475–481. 10.3171/ped.2005.103.6.0475. - DOI - PubMed
1. Kulkarni AV, Schiff SJ, Mbabazi-Kabachelor E, Mugamba J, Ssenyonga P, Donnelly R, Levenbach J, Monga V, Peterson M, MacDonald M, et al. (2017). Endoscopic Treatment versus Shunting for Infant Hydrocephalus in Uganda. N Engl J Med 377, 2456–2464. 10.1056/NEJMoa1707568. - DOI - PMC - PubMed

Publication types

Actions
Actions

MeSH terms

Actions
Actions
Actions
Actions
Actions

Grants and funding

LinkOut - more resources

Full Text Sources
Other Literature Sources
- The Lens - Patent Citations Database
Medical
- MedlinePlus Health Information
Molecular Biology Databases
- Mouse Genome Informatics (MGI)
- NIAID Data Ecosystem - Find datasets on Infectious and Immune-mediated Diseases
Research Materials
- Addgene Non-profit plasmid repository

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

Choroid plexus-targeted NKCC1 overexpression to treat post-hemorrhagic hydrocephalus

Affiliations

Choroid plexus-targeted NKCC1 overexpression to treat post-hemorrhagic hydrocephalus

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

Comment in

References

Publication types

MeSH terms

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Medical

Molecular Biology Databases

Research Materials