# Presentation: Y2LTADAVHX5WL5RMXQUY2IWDDCOHER55

## Respecting patient confidentiality, some slides -- including patient photos -- have been removed from this presentation.Office of the Clinical Director, NICHD

## Infantile Neuronal Ceroid Lipofuscinosis (INCL): 

*Bench to Bedside*

-        Sondra W. Levin, M.D.

**Adjunct Scientist, Section on Developmental Genetics**
**Heritable Disorders Branch, NICHD**
**Chief, Genetics Section**
**Dept. of Pediatrics / Exceptional Family Member Program**
**Walter Reed Army Medical Center**
- Anil B. Mukherjee, MD, PhD
**Head, Section on Developmental Genetics**
**Heritable Disorders Branch, NICHD**

## History of NCL /Batten Disease

- Most common group of progressive neurodegenerative disorders in children
- Actually first described by Otto-Christian Stengel in Norway (published in 1826) – however, 4 sibs with juvenile NCL in family died and were buried before Stengel could do autopsies
- Multiple eponyms: Santavuori, Haltia, Jansky, Batten, Bielschowsky, Spielmeyer, Sjogren, Kufs

## First clinicopathological correlation by F.E. Batten, 1903 

- Defines “familial cerebromacular degeneration”
- Later on in 1914 separates this collection of diseases from Tay-Sachs

## Considered “geographically ubiquitous”

- General frequency 1 per 12,500 births
- Enriched gene frequency in Finland where infantile and juvenile forms are more prevalent
- All forms autosomal recessive except for rare instances of autosomal dominant mode of inheritance in some adults

##        Incidence of  Different types of NCL in the US

-     (Shriver Center Study, Waltham, Massachusetts: January, 1984 to December, 1986).
-         **LINCL** is most common (35%) with a mean age-of-
-           onset around 3 yrs.
-         **JNCL** is slightly less frequent (32%) with a mean
-           age-of-onset of 8 yrs;
-         **INCL** ranked third (23%) with a mean age-of-onset 11
-           months;
-         **Adult** form of the disease was the least common
-          (10%) with a mean age-of-onset 25 yrs.

## NEURONAL CEROID LIPOFUSCINOSIS (NCL)

**Clinical Subtype**

**Mutant Gene**

**Chromosomal Location**

**Gene Product**

**Infantile NCL ****	****        CLN 1                       1p32                      Palmitoyl-Protein ****	**
**     ****(INCL)****	****	****	****	****	****                       Thioesterase (PPT)**

**Classic Late Infantile    CLN 2                      11p15                     Tripeptidyl Peptidase **
**     ****(LINCL)****  **

**Juvenile NCL                CLN 3                      16p12                     438-aa Membrane**
**    ****(JNCL)****                                                                                      Protein**

**Adult NCL                      CLN 4                          ?                                     ?**
**(Kufs Disease)**

**Finish Variant                CLN 5                      13q22                     407-aa Membrane**
**     ****(LINCL)****                                                                                        Protein**

**Variant LINCL                CLN 6                      15q21                                 ?**

**Northern Epilepsy         CLN 8 (CLN 7?)         8p23                                 ?**

## Neuronal  Ceroid   Lipofuscinosis  (NCL)

**(Batten Disease)**

**    **_**Clinical Features**_**:**
**     ****Retinal Blindness**
**     ****Seizures**
**     ****Psychomotor deterioration **
**     ****Progressive Encephalopathy Leading to Death**
**    **_**Pathological Findings**_**:**
**  ****Marked Cortical Atrophy**
**  ****Lysosomal Accumulation of Auto-fluorescent  **
**     ****Lipopigments in Neurons and Other Cell Types**
**  ****Storage Material is Similar in Composition**
**     ****to the Ageing Pigments: Ceroid & Lipofuscin**

##  NCL subtypes defined by age of onset and ultrastructural morphology  

## Ultrastructural Morphology

- Infantile NCL - GROD
- Classical Late-Infantile NCL - CVB
- Juvenile NCL - FPP

## INFANTILE Neuronal Ceroid Lipofuscinosis (INCL)

**      ****Caused by Lysosomal Palmitoyl-    **
**            ****Protein Thioesterase (PPT)   **
**            ****Deficiency**
**      ****Most severe among all the NCLs**
**      ****No effective treatment**

## CLINICAL COUSE

**    ****Normal At Birth**
**     ****Complete Retinal Blindness **
**         ****by 2 yrs of Age**
**     ****Ataxia & Seizures**
**     ****Flat EEG ~4 yrs of Age**
**     ****Death ~ 8-11 yrs of Age**

## INFANTILE NEURONAL CEROID LIPOFUSCINOSIS

**	****	****	****(INCL)**

**   ****Incidence: 1 in 100,000 births**

**   ****Due to the Lack of an Effective Treatment**
**      ****INCL is  an Uniformly Fatal Disease**

## CLEAVAGE OF THIOESTER LINKAGE BY 

**PALMITOYL-PROTEIN THIOESTERASE (PPT)**

## Proposed Mechanism of Pathogenesis in INCL

**Plasma Membrane**

**   ****Normal-PPT**

**PPT- ****Deficient**** (INCL)**

**Proposed Mechanism of Pathogenesis in INCL**

**Palmitoylated Protein**

**Endolysosome**

## SINCE THIOESTER LINKAGES ARE SUSCEPTIBLE

- <date/time>

**SINCE THIOESTER LINKAGES ARE SUSCEPTIBLE**
**TO NUCLEOPHILIC ATTACKS**

**We sought to determine whether drugs with**
**nucleophilic properties have therapeutic potential**
**for INCL**

## Some functional groups that

- <date/time>

**Some functional groups that**
**Act as nucleophiles within cells**

## [14C]Palmitoyl-CoA:

- [14C]Palmitoyl-CoA:

- A Model Thioester Substrate of PPT

- *

## Cleavage of Thioester Linkage by Nucleophilic Drugs

**14****C-****Palmitoyl CoA**

**14****C-****Palmitic Acid + CoA**

**14****C-****Palmitic**
**       ****Acid**

**  ****Phosphocysteamine            N-Acetylcysteine          Hydroxylamine**

**14****C-****Palmitoyl CoA**

**1        2        3        4        5  **

**Cleavage of Thioester Linkage by Nucleophilic Drugs**

**THIN LAYER CHROMATOGRAPHY**

** ****Drug**

## [35S]Cysteine-labeled Thioesters in Normal & INCL Cells

**[****35****S****]Cysteine-labeled Thioesters in ****Normal**** & ****INCL**** Cells**

**Normal   ****INCL**

## -  +        -   +        -   +         -   +       -   +

**Control**

- Phosphocysteamine

**Patients**

**I                II                 III             IV              V**

**Phosphocysteamine Mediates Degradation of Lipid-**
**Thioesters From Acylated Proteins in INCL Cells**

## Phosphocysteamine Mediates Depletion of Lysosomal Ceroids in INCL Cells

**Phosphocysteamine Mediates Depletion of Lysosomal Ceroids in INCL Cells**

**Fibroblasts:**

**Lymphoblasts:**

- 1 wk

- 3 wks

- Untreated

- 1 wk

- 3 wks

**Untreated**

## P-Cysteamine Inhibits Apoptosis in INCL Lymphoblasts 

**P-Cysteamine Inhibits Apoptosis in INCL Lymphoblasts **

**Normal**
**Control**

**    ****INCL**
**Untreated**

**   ****INCL**
**Treated**

- [TUNEL  Assay_]_

## CYSTAGON

**(Cysteamine bitartrate)**

**   ****Safely used for more than 20 yrs for the treatment of**
**      ****Cystinosis**
**   ****Readily gets into lysosomes**
**   ****Crosses the brain-blood barrier**

## BENCH-TO-BEDSIDE PROTOCOL

- <date/time>

**BENCH-TO-BEDSIDE PROTOCOL**

**PILOT STUDY:**
- To Determine Whether Cystagon is an Effective Treatment for INCL
- Duration of the Protocol: 4 years
- Total Number of Patients: 5
- Age limit: 6 months to 3 years
- Evaluation Criteria: ERG, EEG, MRI, and EM of WBCs
- Number of Patients Admitted to date: 2

##    NATURAL HISTORY OF THE MOST SEVERE INCLs   

_**Age**_**	****	**_**PPT Mutations**_**	****             **_**Disease Course**_

** ****0--11 months             ----------------------------                      Normal**
**	****                 ****	****        ****	**

** ****11--15 months           ----------------------------                      Mild Visual Deficit**
**	****	****                    ****	****                 ****Abnormal ERG**
**	****                                                                          ****Mild Irritability**
**	****	****	****	****	****  **

** ****15M--2 Years               --------------------------                       Retinal blindness**
**                                                                                            ****Grossly Abnormal**
**                                                                                            ****ERG & EEG; Moderate**
**	****	****	****	****                  ****Cortical Atrophy**

**   ****2.5--4Years                  --------------------------                        Total Retinal Blindness**
**                                                                                               ****Marked Brain Atrophy**
**	****	****	****	****                     ****Frequent Seizures**
**	****	****	****	****                     ****Isoelectric EEG**

**  ****4--11 Years                 -------------------------                        Vegetative State**
**                                                                                             ****and Death**

**L10X, R151X, R164X, E184K**
**W296X,R122W, c.169insA**

## Pedigree

- S.P.’s mother is Finnish and her father is from Estonia - ? consanguinity, in that Estonia and Finland were once united and root language is the same from these two regions
- Prenatal diagnosis of S.P.’s brother done by Dr. Santavuori’s lab in Finland and fetus was determined to be unaffected

## CLINICAL  OBSERVATIONS 

-   **Markedly Less  Irritable**
**  ****No Seizures**
**  ****Increased Alertness**
**  ****Efforts to Move Head and to Rollover**
**  ****Increased Pupillary Response to Light **
**  ****Increased Ability to Focus on Objects**

## SUMMARY

-     INCL is the most severe form of NCL
-      Caused by PPT-deficiency
-      Phosphocysteamine appears to mediate
-          depletion of lysosomal ceroids in INCL cells
-      Preliminary results of a pilot study with
-          Cystagon show stabilization or  slight
-          improvement in some parameters in an
-          INCL patient

## ACKNOWLEDGEMENTS

- <date/time>

**ACKNOWLEDGEMENTS**

**Section on Developmental**
**Genetics**
**Anil Mukherjee**
**Gary Zhang**
**(Jean DeB. Butler)**

_**Collaborators**_** **
**Zena Quezado**
**Rafael Caruso**
**James Sidbury**
**Andrea Gropman**
**Kenneth Fischbeck**
**Nicholas Patronas**
**Carol Frattali**
**Scott Paul**
**Monique Perry**
**Sandra Hofmann**
**Krystyna Wisniewski**
**Pirkko Santavuori**

## From the Bench-to-Bedside 

**PILOT STUDY:**
- Drug: Cystagon (Cysteamine bitartrate)
- Duration of the Study: 4 years
- Total No. Patients to be Treated: 5
- PPT-Mutations:**L10X, R151X, R164X, E184K**
**                                    ****W296X, R122W, c.169insA**
- Age Limit: 6 months to 3 years
- Evaluation : ERG, VEP, MRI & EM
- No. Patients Admitted to Date:   2

## Developmental Expression of PPT  in Brain & Retina

**Developmental Expression of PPT  in Brain & Retina**
**                      ****(Immuno-localization)**

**Brain**

**Eye**

**12d                   14d                   16d                   NB**

**12d                   14d                   16d                   NB**